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Enhancing the photovoltaic performance of biogenic solar cells with synthetic biology Einarsson, Sean 2020

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ENHANCING THE PHOTOVOLTAIC PERFORMANCE OF BIOGENIC SOLAR CELLS WITH SYNTHETIC BIOLOGY  by  Sean Einarsson  BASc, The University of British Columbia, 2016  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE  in  THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemical and Biological Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) September 2020  © Sean Einarsson, 2020   ii   The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Enhancing the Photovoltaic Performance of Biogenic Solar Cells with Synthetic Biology  submitted by Sean Einarsson in partial fulfillment of the requirements for the degree of Master of Applied Science in Chemical and Biological Engineering  Examining Committee: Vikramaditya Yadav, Chemical and Biological Engineering Supervisor  Susan Baldwin, Chemical and Biological Engineering Supervisory Committee Member  Scott Dunbar, Mining Engineering Supervisory Committee Member  iii  Abstract  The development of organic photosensitive materials has opened up a breadth of new areas for advancement in photovoltaics and dye-sensitized solar cells (DSSCs). The approach of organic DSSCs is to use photo-excitable dyes over a conductive nanoparticle layer in the presence of an electrolyte to create a working electrode. There has been a large emphasis on the improvement of organic DSSCs in recent years, and there have been significant increases in their photovoltaic efficiencies. However, the fabrication process and extraction of the dyes involves complicated and costly methods that require the use of toxic chemicals and a tightly controlled clean-room environment. To alleviate these issues, a novel approach was developed that uses genetically engineered bacteria capable of producing lycopene, a photo-excitable dye, internally. Preliminary research of these genetically engineered cells implemented in organic solar cell production shows promising results, but significant improvements must be made in order to be comparable to conventional solar cells. The thesis focuses on improving the conductivity of the genetically engineered bacteria capable of synthesizing lycopene. The approach is to use the electroconductive properties found in another bacterial species, Shewanella Oneidensis MR-1 (SO MR-1), to increase the photovoltaic properties of the system. The conductive ability of SO MR-1 arises from its bacterial nanowires which are capable of extracellular electron transfer. The experimental methods include identifying the genes that are responsible for bacterial nanowires formation in SO MR-1, extracting and cloning the identified genes into the lycopene producing bacteria, verifying the expression of the bacterial nanowire genes, evaluating the photovoltaic characteristics, and comparing the measurements of the systems with and without bacterial nanowires.  iv  The results show successful implementation of the genes responsible for bacterial nanowire formation into the lycopene producing bacteria, but the expression level analysis revealed ambiguous results which could be addressed with more precise methods. The photovoltaic analysis had some issues with short-circuiting, which made it difficult to draw any significant conclusions. Although the main objective of the thesis might need to be further investigated, several integral objectives were achieved, which can be used as a stepping stone in future research.   v  Lay Summary  The key goals of the thesis are to increase the conductivity, efficiency, and overall photovoltaic performance of a specific type of organic solar cell that generates electricity by using genetically engineered bacteria that produce a photo-excitable dye. The process involves cloning genes into the dye producing bacteria that are responsible for electron transfer and nanowire formation from the conductive bacterial species, Shewanella Oneidensis MR-1. After cloning, the bacteria can be used for organic solar cell fabrication, and performance of the solar cells with and without nanowires can be compared. The research could have a significant impact on solar energy implementation, as these organic photovoltaic materials can be manufactured economically and sustainably. They also have the unique trait of working as efficiently under low light when compared to bright light. Therefore, the potential for solar energy implementation could be increased in areas that were previously considered to not be feasible.     vi  Preface  The thesis consists of five chapters. The research was conducted by Sean Einarsson under the direct supervision of Dr. Vikramaditya G. Yadav in the Department of Chemical and Biological Engineering at The University of British Columbia. The literature review, experimental design, experimental procedures, and data analysis were done by Sean Einarsson while being overseen by Vikramaditya Yadav.   Chapter 2 All SnapGene simulations prior to the molecular cloning experiments were conducted by Sean Einarsson. The experimental procedures and verification steps in the construction of the pET-28a(+)_SO5 plasmid including: deoxyribonucleic acid extractions, polymerase chain reactions, Gibson Assembly reactions, transformations, colony polymerase chain reactions, and Sanger Sequencing setup were conducted by Sean Einarsson and undergraduate research student Steven Zhou, with additional guidance from fellow master’s student and lab member, Amir Kashani, and PhD candidate and lab member, Carmen Balyl. The Sanger Sequencing analysis was carried out by genomics services company, GENEWIZ.   Chapter 3 The pAC-Lyc plasmid was gifted by current PhD candidate and former lab member Adhiti Raghavan who had previously conducted her own research using the pAC-Lyc plasmid. The pET-28a(+)_SO5 and pAC-Lyc co-transformation into BL21(DE3), and colony polymerase chain reaction verification were conducted by Sean Einarsson and Melissa Lehner, an undergraduate vii  research student completing her undergraduate thesis. The subsequent research experiments, including the following: generating a protein standard curve, conducting a Bradford assay, and expression analysis with protein gel electrophoresis were carried out by Sean Einarsson and Melissa Lehner, with the aid of fellow master’s student and lab member, Benson Chang, visiting scientist, Dr. Hong Ting Law, and undergraduate research student, Kevin Salim.    Chapter 4 The biogenic solar cell fabrication was previously developed by former post-doctoral fellow and lab member, Dr. Sarvesh Srivastava. The novel approach of biogenic solar cell fabrication using genetically engineered bacteria that have the pET-28a(+)_SO5 and pAC-Lyc plasmids was conducted by Sean Einarsson and Melissa Lehner. Specifically, the solar cell fabrication included bacterial culturing, cell extraction, titanium dioxide coating, ultraviolet-visible spectroscopy, and electrode construction. The solar simulator setup was conducted by David Dvorak, a staff member of Dr. Berlinguette’s lab group at The University of British Columbia, who is an instrumentation manager that has expertise in operating a solar simulator. Data analysis of the solar simulator results was carried out by Sean Einarsson and Melissa Lehner.   viii  Table of Contents  Abstract ......................................................................................................................................... iii Lay Summary .................................................................................................................................v Preface ........................................................................................................................................... vi Table of Contents ....................................................................................................................... viii List of Tables ............................................................................................................................... xii List of Figures ............................................................................................................................. xiv List of Abbreviations ................................................................................................................ xvii Glossary ...................................................................................................................................... xix Acknowledgements .................................................................................................................... xxi Dedication .................................................................................................................................. xxii Chapter 1: Introduction ................................................................................................................1 1.1 Current State of Biogenic Solar Cells ............................................................................. 1 1.1.1 Biogenic Solar Cell Components ................................................................................ 2 1.1.2 Lycopene Synthesis Genes ......................................................................................... 3 1.1.3 Nanoparticle Deposition ............................................................................................. 4 1.1.4 Performance ................................................................................................................ 6 1.2 Bacterial Nanowires ........................................................................................................ 8 1.2.1 Shewanella Oneidensis MR-1 ..................................................................................... 9 1.2.1.1 CymA ................................................................................................................ 12 1.2.1.2 OmcA & MtrC .................................................................................................. 13 1.2.1.3 MtrA & MtrB .................................................................................................... 13 ix  1.3 Research Goals.............................................................................................................. 14 1.4 Thesis Overview ........................................................................................................... 14 Chapter 2: Molecular Cloning ....................................................................................................16 2.1 Introduction ................................................................................................................... 16 2.2 Materials and Methods .................................................................................................. 19 2.2.1 Plasmid and Template DNA ..................................................................................... 19 2.2.2 Polymerase Chain Reaction & Gel Electrophoresis ................................................. 19 2.2.3 DNA Assembly and Transformation ........................................................................ 20 2.2.4 Verification of Inserted DNA ................................................................................... 21 2.2.5 Snapgene Simulation ................................................................................................ 23 2.3 Results and Discussions ................................................................................................ 24 2.3.1 CymA Insert .............................................................................................................. 24 2.3.2 OmcA Insert .............................................................................................................. 28 2.3.3 MtrC Insert ................................................................................................................ 32 2.3.4 MtrA & MtrB Insert .................................................................................................. 36 Chapter 3: Protein Expression ...................................................................................................41 3.1 Introduction ................................................................................................................... 41 3.2 Materials and Methods .................................................................................................. 42 3.2.1 Two Plasmid Co-Transformation ............................................................................. 42 3.2.2 Culture Conditions .................................................................................................... 43 3.2.3 Protein Standard Curve ............................................................................................. 44 3.2.4 Bradford Assay ......................................................................................................... 45 3.2.5 Protein Gel ................................................................................................................ 47 x  3.3 Results and Discussion ................................................................................................. 48 3.3.1 Mtr Pathway & Lycopene Synthesis Pathway Co-Transformation .......................... 48 3.3.2 Mtr-Pathway & Lycopene Synthesis Pathway Protein Expression .......................... 51 Chapter 4: Biogenic Solar Cells ..................................................................................................54 4.1 Introduction ................................................................................................................... 54 4.2 Materials and Methods .................................................................................................. 55 4.2.1 Culture Conditions .................................................................................................... 55 4.2.2 Cell Extraction .......................................................................................................... 55 4.2.3 Titanium Dioxide Coating ........................................................................................ 56 4.2.4 Ultraviolet-Visible Spectroscopy .............................................................................. 56 4.2.5 Solar Cell Fabrication ............................................................................................... 57 4.2.6 Solar Simulator Setup ............................................................................................... 58 4.3 Results and Discussion ................................................................................................. 58 4.3.1 Ultraviolet-Visible Spectroscopy Analysis ............................................................... 58 4.3.2 Photovoltaic Characterization ................................................................................... 60 Chapter 5: Conclusion and Future Work ..................................................................................63 5.1 Conclusion .................................................................................................................... 63 5.2 Future Work .................................................................................................................. 65 5.2.1 Molecular Cloning and Biogenic Solar Cell Fabrication .......................................... 65 5.2.2 Real-time PCR and Electron Microscopy ................................................................. 66 5.2.3 Viability of the Cells and Solid-State Solar Cell Integration .................................... 67 5.2.4 Shewanella Oneidensis MR-1 Genetic Engineering ................................................. 68 Bibliography .................................................................................................................................70 xi  Appendices ....................................................................................................................................76 Appendix A Mtr Pathway Protein Sequences ........................................................................... 76 A.1 CymA ........................................................................................................................ 76 A.2 OmcA ........................................................................................................................ 76 A.3 MtrC .......................................................................................................................... 77 A.4 MtrA .......................................................................................................................... 77 A.5 MtrB .......................................................................................................................... 78 Appendix B PCR Reaction Setup ............................................................................................. 79 B.1 CymA Insert Thermal Cycling Conditions ............................................................... 79 B.2 OmcA Insert Thermal Cycling Conditions ............................................................... 82 B.3 MtrC Insert Thermal Cycling Conditions ................................................................. 85 B.4 MtrA & MtrB Insert Thermal Cycling Conditions ................................................... 88 B.5 Mtr Pathway Co-Transformation Thermal Cycling Conditions ............................... 91 Appendix C Sanger Sequencing ............................................................................................... 92 C.1 Verification for CymA Insert .................................................................................... 92 C.2 Verification for OmcA Insert .................................................................................... 93 C.3 Verification for MtrC Insert ...................................................................................... 94 C.4 Verification for MtrA & MtrB Insert ........................................................................ 95  xii  List of Tables  Table B.1: PCR primers and thermal cycling conditions for the linearization of the pET-28a(+) vector............................................................................................................................................. 79 Table B.2: PCR primers and thermal cycling conditions for the linearization of the CymA gene insert .............................................................................................................................................. 80 Table B.3: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO1 Gibson Assembly ............................................................................................. 81 Table B.4: PCR primers and thermal cycling conditions for the linearization of the pET-28a(+)_SO1 vector ........................................................................................................................ 82 Table B.5: PCR primers and thermal cycling conditions for the linearization of the OmcA gene insert .............................................................................................................................................. 83 Table B.6: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO2 Gibson Assembly ............................................................................................. 84 Table B.7: PCR primers and thermal cycling conditions for the linearization of the pET-28a(+)_SO2 vector ........................................................................................................................ 85 Table B.8: PCR primers and thermal cycling conditions for the linearization of the MtrC gene insert .............................................................................................................................................. 86 Table B.9: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO3 Gibson Assembly ............................................................................................. 87 Table B.10: PCR primers and thermal cycling conditions for the linearization of the pET-28a(+)_SO3 vector ........................................................................................................................ 88 xiii  Table B.11: PCR primers and thermal cycling conditions for the linearization of the MtrA & MtrB gene insert ..................................................................................................................................... 89 Table B.12: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO5 Gibson Assembly ............................................................................................. 90 Table B.13: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO5 plasmid co-transformation ............................................................................... 91 Table C.1: Nucleotide BLAST of GENEWIZ Sanger Sequencing vs SnapGene simulation for CymA Insert ................................................................................................................................... 92 Table C.2: Nucleotide BLAST of GENEWIZ Sanger Sequencing vs SnapGene simulation for OmcA Insert .................................................................................................................................. 93 Table C.3: Nucleotide BLAST of GENEWIZ Sanger Sequencing vs SnapGene simulation for MtrC Insert .................................................................................................................................... 94 Table C.4: Nucleotide BLAST of GENEWIZ Sanger Sequencing vs SnapGene simulation for MtrA & MtrB Insert ...................................................................................................................... 95  xiv  List of Figures  Figure 1.1: Schematic of the major steps involved in designing a biogenic solar cell. a) genetic engineering and insertion of genes for lycopene expression in E. coli bacteria. b) encapsulation and binding of TiO2 nanoparticles facilitated by tryptophan. c) biogenic solar cell layout and graphical simplification of electron transfer (Srivastava, 2018). .................................................... 2 Figure 1.2: Lycopene synthesis genes in the pAC-Lyc plasmid ..................................................... 4 Figure 1.3: UV-vis spectroscopy of TiO2 deposition (Srivastava, 2018). ...................................... 6 Figure 1.4: Biogenic solar cell electrochemical measurements characterizing a) current-voltage curves; b) time dependent open-circuit photovoltage response; c) cyclic voltammetry curves (Srivastava, 2018). .......................................................................................................................... 7 Figure 1.5: Scanning electron microscope image of wild type SO MR-1 grown under limited electron acceptor conditions (Gorby, 2006). ................................................................................. 10 Figure 1.6: Structural model for Shewanella Oneidensis MR-1 nanowires and the proteins responsible for extracellular electron transfer (Pirbadian, 2014). ................................................. 11 Figure 2.1: Stepwise representation of the Gibson Assembly cloning process (Gibson, 2009). .. 17 Figure 2.2: Gibson Assembly schematic representation of one insert added to a plasmid (Gibson Assembly Cloning). ...................................................................................................................... 18 Figure 2.3: Linearization steps and assembly of CymA into pET-28a(+); a) linearization of the pET-28a(+) vector - lane 1 (farthest left): 1kb Plus Opti-DNA Marker, lanes 2, 3: linearized pET-28a(+) vector; b) linearization of CymA gene insert - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized CymA gene fragment; c) SnapGene simulation of pET-28a(+)_SO1 assembly .................................................................................... 25 xv  Figure 2.4: Colony PCR verification for the CymA insert; a) linearization of the pET-28a(+)_SO1 plasmid - lane 1 (farthest left) and lane 4: 1kb Plus Opti-DNA Marker, lanes 2, 3, 5, 6 (farthest right): linearized pET-28a(+)_SO1 plasmid - each lane was from a separate colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO1 plasmid ................................................. 27 Figure 2.5: Linearization steps and assembly of OmcA into pET-28a(+)_SO1; a) linearization of the pET-28a(+)_SO1 vector - lane 1 (farthest left): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5 (farthest right): linearized pET-28a(+)_SO1 vector; b) linearization of OmcA gene insert - lane 1 (farthest left): 1kb Plus Opti-DNA Marker, lanes 2, 3 (farthest right): linearized OmcA gene fragment; c) SnapGene simulation of pET-28a(+)_SO2 assembly .............................................. 29 Figure 2.6: Colony PCR verification for the OmcA insert; a) linearization of the pET-28a(+)_SO2 plasmid - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO2 plasmid - each lane was from a separate colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO2 plasmid ................................................. 31 Figure 2.7: Linearization steps and assembly of MtrC into pET-28a(+)_SO2; a) linearization of the pET-28a(+)_SO2 vector - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO2 vector; b) linearization of MtrC gene insert - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized MtrC gene fragment; c) SnapGene simulation of pET-28a(+)_SO3 assembly ........... 33 Figure 2.8: Colony PCR verification for the MtrC insert; a) linearization of the pET-28a(+)_SO3 plasmid - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO3 plasmid - each lane was from a separate colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO3 plasmid ................................................. 35 xvi  Figure 2.9: Linearization steps and assembly of MtrA & MtrB into pET-28a(+)_SO3; a) linearization of pET-28a(+)_SO3 vector - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO3 vector; b) linearization of MtrA & MtrB gene insert - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized MtrA and MtrB gene fragment; c) SnapGene simulation of pET-28a(+)_SO5 assembly ................................................................................................................... 37 Figure 2.10: Colony PCR verification for the MtrA & MtrB insert; a) linearization of the pET-28a(+)_SO5 plasmid - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO5 plasmid - each lane was from a separate colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO5 plasmid ................................ 39 Figure 3.1: Colony PCR verification for the pET-28a(+)_SO5 plasmid into BL21(DE3); a) linearization of the pET-28a(+)_SO5 plasmid - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO5 plasmid - lanes 1 and 2 were from the same colony, lanes 3 and 4 were from the same colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO5 plasmid .............................................................................. 49 Figure 3.2: Bacterial culture grown overnight from the pET28a(+)-Mtr plasmid and pAC-Lyc plasmid co-transformation ............................................................................................................ 50 Figure 3.3: BSA standard curve measured with absorbance at 595 nm ....................................... 51 Figure 3.4: Protein gel depicting the insoluble and soluble protein expression of BL21(DE3) with and without cloned DNA .............................................................................................................. 52 Figure 4.1: Ultraviolet-visible spectroscopy analysis of BL21(DE3)-Lyc cells with and without the addition of TiO2 ............................................................................................................................ 59 Figure 4.2: Current-voltage curve of BL21(DE3)-Lyc biogenic solar cell .................................. 60 xvii  List of Abbreviations  bp BSA DNA DSSC E. coli EET FTO IPTG ISC ISC-Direct I-V JSC LB MEP η PCR RNA SEM SO MR-1 TEM Base pairs Bovine serum albumin Deoxyribonucleic acid Dye sensitized solar cell Escherichia coli Extracellular electron transfer Fluorine-doped tin oxide Isopropyl β-d-1-thiogalactopyranoside  Short-circuit current Direct short-circuit current Current-voltage Short-circuit current density Luria Bertani Non-mevalonate pathway Total external efficiency Polymerase chain reaction Ribonucleic acid Scanning electron microscopy Shewanella Oneidensis MR-1 Transmission electron microscopy xviii  TiO2 UV-vis VOC VOC-Direct YT Titanium dioxide Ultraviolet-visible Open-circuit potential Direct open-circuit potential Yeast extract-tryptone  xix  Glossary  BL21(DE3)-Lyc  BL21(DE3)-Lyc@TiO2   BL21(DE3)-Mtr  BL21(DE3)-Mtr-Lyc   BL21(DE3)-pET-28a(+)  pAC-Lyc  pET-28a(+)_SO1  pET-28a(+)_SO2    Refers to BL21(DE3) bacteria transformed with the lycopene synthesis plasmid, pAC-Lyc. Refers to BL21(DE3) bacteria transformed with the lycopene synthesis plasmid, pAC-Lyc, and coated with titanium dioxide nanoparticles. Refers to BL21(DE3) bacteria transformed with the Mtr pathway plasmid, pET-28a(+)_SO5. Refers to BL21(DE3) bacteria co-transformed with the Mtr pathway plasmid, pET-28a(+)_SO5, and the lycopene synthesis plasmid, pAC-Lyc. Refers to BL21(DE3) bacteria transformed with the pET-28a(+) plasmid. Refers to the genes responsible for lycopene synthesis, crtE, crtB, and crtI, inserted into the pAC vector plasmid.   Refers to one gene of the Mtr pathway, CymA, from Shewanella Oneidensis MR-1 inserted into the pET-28a(+) vector plasmid. Refers to two genes of the Mtr pathway, CymA, and OmcA from Shewanella Oneidensis MR-1 inserted into the pET-28a(+) vector plasmid. xx  pET-28a(+)_SO3   pET-28a(+)_SO5 Refers to three genes of the Mtr pathway, CymA, OmcA, and MtrC from Shewanella Oneidensis MR-1 inserted into the pET-28a(+) vector plasmid. Refers to all five genes of the Mtr pathway, CymA, OmcA, MtrC, MtrA, and MtrB from Shewanella Oneidensis MR-1 inserted into the pET-28a(+) vector plasmid.  xxi  Acknowledgements  I would like to express my deepest gratitude to my research supervisor, Dr. Vikramaditya G. Yadav. He was an invaluable resource and a wealth of knowledge that helped guide my research. Having him in my corner gave me confidence and clear direction as I designed research questions, solved problems, and analyzed data. Through his supervision, I was able to develop a unique skillset that I will have for the rest of my life as I pursue my academic and professional goals. I can not thank him enough for everything he has done for me, and I am blessed to have had the opportunity to work with him and his first-class team.        I would also like to thank my fellow colleagues from the BioFoundry for their continuous support and exemplary insights. I would like to give special thanks to Amir Kashani for training me in the lab, and helping me with my molecular cloning experiments. I would also like to thank Carmen Balyl for always being open to sharing her experiences with me and helping me find solutions to my problems. To my undergraduate research students, Steven Zhou and Melissa Lehner, I thank the two of you for your extraordinary work ethic and willingness to go above and beyond what was needed.  Finally, I would like to express my appreciation for my family who have supported me financially, mentally, and emotionally throughout my years of education. I’m grateful to have them as the foundation of my support system, and words can not describe how lucky I am to have them in my life. xxii  Dedication    I dedicate my thesis to my beloved family, Norm, Tessie, and Greg Einarsson, my Aunt, Lita Roldan, my best friend, Josh Gould, and my partner, Chelsea Harkins, who have given me an immense amount of love, support, and encouragement throughout the whole process. I would also like to acknowledge my teammates, training staff, physiotherapists, assistant coaches, and head coach, Mike Mosher, who allowed me to play the sport I love throughout my time pursuing my undergraduate and master’s degree at The University of British Columbia as a member of the varsity soccer team. I would not have been able to come this far without all the amazing people in my life, and for that, I am grateful and I thank you.  1  Chapter 1: Introduction  1.1 Current State of Biogenic Solar Cells Biogenic photovoltaic materials are a class of organic compounds that offer a simple and inexpensive way to harness solar energy and ultimately produce electricity. The term biogenic refers to a substance produced by living organisms. First of its kind biogenic photovoltaic materials have been developed, consisting of genetically engineered Escherichia coli (E. coli) bacteria that have been engineered to overproduce photo-excitable dyes that are capable of absorbing light energy (Srivastava, 2018). These genetically engineered E. coli cells have been coated with conductive nanoparticles, specifically titanium dioxide (TiO2), in order to facilitate the transfer of electrons between the bacteria and an electrochemical cell.  Research towards the novel, biogenic, electrically conductive bacteria is important because it offers an improved alternative to other organic photovoltaic materials. A common type of organic solar cells used today are dye-sensitized solar cells (DSSCs). DSSCs also use photo-excitable-dyes; however, these dyes require an extraction step that requires toxic chemicals before being coated with titanium dioxide, and also needs to be manufactured in a clean room environment. The genetically engineered E. coli is favorable because they do not require the use of toxic chemicals, are less expensive, and are easier to manufacture since they do not need to be prepared in a clean room environment.  The biogenic solar cells also offer great benefits to Canada as well as other countries facing challenges with implementing solar energy technologies due to persistent overcast skies. The main advantage of these biogenic solar cells over nonorganic photovoltaic materials is the capability to 2  work as efficiently under dim light conditions as bright light conditions (Srivastava, 2018). For inorganic solar cells to produce energy efficiency they need to be under bright, direct sunlight conditions. Therefore, rather than seeing these biogenic photovoltaic materials as a replacement for nonorganic solar cells, they can be used in conjunction to one another to improve energy production capabilities under all light conditions. Thus, making solar energy more economical in areas where it was not previously conceivable.        1.1.1 Biogenic Solar Cell Components The making of the first of its kind biogenic solar cell started with the development of the biogenic material. A schematic representation is shown in Figure 1.1 (Srivastava, 2018).    Figure 1.1: Schematic of the major steps involved in designing a biogenic solar cell. a) genetic engineering and insertion of genes for lycopene expression in E. coli bacteria. b) encapsulation and binding of TiO2 nanoparticles facilitated by tryptophan. c) biogenic solar cell layout and graphical simplification of electron transfer (Srivastava, 2018).    3  The biogenic material consists of a porous mesh of E. coli BL21(DE3) cells enclosed with TiO2. The bacteria contain a genetically engineered plasmid that includes genes encoding for the production of lycopene. Lycopene is responsible for giving tomatoes their orange-red color, as it is a natural carotenoid pigment. In the photovoltaic system, lycopene acts as a photosensitive dye. Tryptophan is used to facilitate the deposition of TiO2 onto the cells by creating a core-shell morphology as a result of a tryptophan-mediate supramolecular interface. Once coated with TiO2, a thin layer of the biogenic material can be transferred onto a conductive glass surface, such as fluorine-doped tin oxide (FTO), along with an electrolyte solution, effectively making a biogenic solar cell, as shown in Figure 1.1c.    1.1.2 Lycopene Synthesis Genes Lycopene has several properties that make it favorable in photovoltaic and photocatalytic applications. Lycopene is a highly stable redox pigment, and can efficiently absorb light in the 380-520 nm range and mediate electron transfer (Richhariya, 2017; Zhuang, 2015). For the use in biogenic solar cells, the genomic sequence responsible for lycopene production was cloned from the bacterial species Erwinia herbicola. The genes responsible for lycopene synthesis are crtE, crtB, and crtI. The plasmid containing the lycopene synthesis genes was named pAC-Lyc. A representation of the genes and the plasmid are shown in Figure 1.2. For the lycopene biosynthetic pathway, transcription levels are controlled by a constitutive promoter. However, the rate of lycopene production can be significantly enhanced using another plasmid that is used to express increased copies of the rate-limiting genes of the non-mevalonate pathway (MEP) (Yang, 2014). Transcription levels of the additional genes of the MEP pathway are regulated by a trc promoter that can be induced with isopropyl β-d-1-thiogalactopyranoside (IPTG). With the enhancement of  4   Figure 1.2: Lycopene synthesis genes in the pAC-Lyc plasmid  the expressions levels for the rate-limiting genes in the MEP pathway and induction with IPTG, lycopene has been produced, in E. coli cells BL21(DE3), at yields and titers that are comparable to the best-in-class strains (Ajikumar, 2010; Yoon, 2007).  1.1.3 Nanoparticle Deposition Semi-conductive nanoparticles, such as TiO2, are practical in most solar-energy conversion applications due to their ability to entrap and transfer photoinduced electrons (Yan, 2019). However, the use of TiO2 in organic DSSCs has been minimal because of the lack of covalent linker groups that are fundamental in the binding process with semiconductors (Shinde, 2017). Accordingly, the challenge of coating the bacterial cells with TiO2 can be addressed with the use 5  of tryptophan. Tryptophan can help with the binding process by creating a conductive supramolecular bilayer. The creation of the bilayer makes a much more stable environment for the TiO2 to coat the cells. Using tryptophan to help with the binding of TiO2 is a different approach when compared to the classic technique of DSSC fabrication that coats the semi-conductive material with the dye or photosensitizer. The process by which E. coli cells are encapsulated with TiO2 begins with the bacterial cells being mixed with a tryptophan solution. Tryptophan begins to create a polymerical layer around the cells. Hydrogen bonding between the negatively charged cellular surface and the indole group of tryptophan and responsible for creation of the polymerical layer (Srivastava, 2018). Afterwards, TiO2 nanoparticles are added to the mixture. Once added to the mixture, the metallic species is reduced and bound by tryptophan’s active carboxyl groups resulting in a core-shell structure.  The binding of TiO2 nanoparticles to the E. coli cells can be confirmed by ultraviolet-visible (UV-vis) spectroscopy, as shown in Figure 1.3 (Srivastava, 2018). The absorption maxima of pure lycopene occur at 450, 475, and 505 nm while the absorption maxima of E. coli cells with the lycopene producing plasmid occur at 450, 485, and 595 nm (Srivastava, 2018). TiO2 nanoparticles do not show any absorption maxima in the visible region, however the deposition of TiO2 onto the genetically engineered E. coli cells shows an upward shift in the UV-vis spectrum. The increase in absorbance can be attributed to the interfacial displacement that materializes during the formation of the bilayer, thus confirming the encapsulation of TiO2 nanoparticles around the bacterial cells. Additional evidence of proper coating is further justified by the time-dependent UV-vis scans which show an increase in absorbance over time after the addition of the TiO2 nanoparticles to the solution of E. coli cells and tryptophan cells.   6   Figure 1.3: UV-vis spectroscopy of TiO2 deposition (Srivastava, 2018).  1.1.4 Performance To measure the system’s photovoltaic properties, after coating the genetically engineered lycopene producing E. coli cells with TiO2 nanoparticles, a thin layer of the composite was coated onto FTO glass creating the anode of the solar cell. A graphite cathode and an iodide/triiodide electrolyte between the electrodes were used to complete the makeup of the solar cell. A solar simulator was used to characterize the photovoltaic properties of the cells. The solar simulator was calibrated to simulate outdoor sunlight conditions. The standards used in conventional photovoltaic experimentation were followed (Ito, 2007). The air mass coefficient was set to 1.5, a standard simulated sunlight active surface area of 0.25 cm2 was accurately controlled, and spectral-mismatch corrected sunlamps were continuously calibrated. The results obtained from the solar simulator are shown in Figure 1.4 (Srivastava, 2018). 7  The open-circuit potential (VOC), short-circuit current (ISC), and corresponding short-circuit current density (JSC), were measured to be 0.289 V, 0.19 mA, and 0.686 mA cm-2, respectively. These measurements were derived from Figure 1.4a. The conversion of incident sunlight to usable electricity, or total external efficiency (η), was measured to be 0.057%. The total external efficiency, should not be confused with quantum or internal efficiencies, which characterize different measurements and are typically higher. To confirm the photovoltaic effect of the biogenic material, a time-dependent (ON/OFF) illumination of the solar cell was conducted, as shown in Figure 1.4b. The AM 1.5 illumination was repeatedly switched on and off every 30 seconds. The system showed significant variation when the light was illuminated versus when it was not. Over an interval of 5 minutes, a net potential difference of roughly 0.2 V was observed with minimal    Figure 1.4: Biogenic solar cell electrochemical measurements characterizing a) current-voltage curves; b) time dependent open-circuit photovoltage response; c) cyclic voltammetry curves (Srivastava, 2018).  8  decay – confirming the material’s photovoltaic effect. To further justify the material’s photovoltaic effect, cyclic voltammetry curves were generated, as shown in Figure 1.4c. When the light was on, a current flow of roughly 5 μA, or 0.02 mA cm-2, was measured. When the light was off, a capacitive-like voltammogram was observed (Fattah, 2016). From the photovoltaic characterization, the biogenic solar cells are fairly stable in the operating range of -0.1 to 0.2V, which is also the working potential range, since there is no indication of any reduction-oxidation peaks. Together, these findings confirm that lycopene producing genetically engineered E. coli cells coated with TiO2 nanoparticles are a suitable material for the anode of a DSSC, with photovoltaic effects that can be measured and definitively characterized (Srivastava, 2018).  1.2 Bacterial Nanowires Bacteria that have the capability of extracellular electron transfer (EET), called exoelectrogens, have garnered more interest in recent years for their applications in scientific fields such as bioenergy production, bioelectronics, and bioremediation (Sure, 2016). One type of EET is through bacterial nanowires. Bacterial nanowires have the capability to power themselves and transfer electricity to a multitude of solid surfaces through electrically conductive appendages (Ilshadsabah and Suchithra, 2019). In addition, these conductive appendages can also facilitate electron transfer between adjacent bacteria. These distinct features can also help with biofilm formation, which has shown to improve the electron transport in microbial fuel cells (Malvankar and Lovley, 2014). There are many types of bacteria that have bacterial nanowires, but one of the most studied and most notable is the metal reducing bacteria Shewanella Oneidensis MR-1 (SO MR-1).  9  1.2.1 Shewanella Oneidensis MR-1 SO MR-1 is a gram-negative proteobacteria that is typically rod shaped, having a length of 2-3 μm and a diameter of 0.4-0.7 μm. The SO MR-1 bacterium is of interest in biogenic photovoltaic applications due to its well established electroconductive properties, most notably the ability to reduce metal ions and electrodes in renewable energy devices (Bretschger, 2007; Pirbadian, 2014). The bacterial strain SO MR-1 is a facultative anaerobe that has the ability to respire using a multitude of different organic and inorganic compounds (Myers & Nealson, 1988; Venkateswaran, 1999). The MR-1 strain indicates “manganese reducing”, however the strain is also able to reduce heavy metals.   SO MR-1 has shown the ability to generate electrically conductive pilus-like bacterial nanowires as a direct response to electron-acceptor limitation (Gorby, 2006). Since SO MR-1 is a facultative anaerobe, oxygen is the terminal electron acceptor in cellular respiration under aerobic conditions. However, under anaerobic conditions, organic or inorganic compounds are used as the terminal electron acceptor in cellular respiration, and the bacterial nanowires are used to carry out the process. The bacterial nanowires of SO MR-1 are unlike other types of bacterial nanowires since they are made up of outer membrane vesicle chains that elongate and extend from the cell (Pirbadian, 2014). An image of the bacterial nanowires of SO MR-1 grown under limited electron acceptor conditions is shown in Figure 1.5 (Gorby, 2006). The outer membrane vesicle chains are made up of different cytochromes, periplasmic components, and outer membrane components, whereas other types of bacterial nanowires, such as pili or flagella, are mostly homopolymers of a  10   Figure 1.5: Scanning electron microscope image of wild type SO MR-1 grown under limited electron acceptor conditions (Gorby, 2006).   single subunit type. These outer membrane vesicle chains are crucial to the makeup of the conductive nanowires in SO MR-1 since it has been hypothesized that long-range electron transfer is conducted through an intricate cytochrome network called the electron hopping model (Strycharz-Glaven, 2012; Tender, 2011). The electron hopping model has been extensively investigated and there are multiple experimental and modelling studies that help confirm its theory (El-Naggar, 2010; Gorby, 2006; Leung, 2013; Pirbadian & El-Naggar, 2012; Polizzi, 2012). Since SO MR-1 has the cellular machinery capable of EET, it became a thought to use these attributes in biogenic solar cells. The idea was to genetically engineer E. coli cells capable of producing electrically conductive bacterial nanowires while also being able to produce lycopene. 11  The hypothesis was that the addition and overexpression of bacterial nanowires would allow for a greater network and surface area for electron transfer to occur, resulting in a greater conductivity and efficiency of the biogenic solar cells. In order to genetically engineer the conductive nanowires into E. coli cells, the first step was to identify which genes were the essential for EET and the structure of the nanowires. In SO MR-1, five primary component proteins were identified to be responsible for EET in the species: CymA, OmcA, MtrA, MtrB and MtrC (Shi, 2007). These 5 proteins are the components of the Mtr pathway, and are responsible for the reduction of metals and electrodes (Beliaev, 1998; Beliaev, 2001; Bretschger, 2007; Gorby 2006). The interrelation of components of the Mtr pathway and the mechanism for EET are shown in Figure 1.6 (Pirbadian, 2014).    Figure 1.6: Structural model for Shewanella Oneidensis MR-1 nanowires and the proteins responsible for extracellular electron transfer (Pirbadian, 2014). 12  Present models indicate that electron transfer in SO MR-1 is achieved by electrons from carbon source oxidation being passed through the menaquinone pool to CymA, an inner membrane-anchored c-type cytochrome (Gralnick, 2006; Myers, 1997). The electrons are then transferred from CymA to MtrA, a periplasmic c-type cytochrome. Next, the electrons are then transferred to OmcA and MtrC, which are both outer membrane anchored c-type cytochromes that interact with an integral outer membrane scaffolding protein, MtrB (Myers, 2002; Myers, 2004, Ross, 2007). The outer membrane cytochromes, OmcA and MtrC, can then reduce different substrates, such as iron oxides and electrodes (Borloo, 2007; Bretschger, 2007; Firer-Sherwood, 2008; Myers, 2001; Shi, 2007).   1.2.1.1 CymA The CymA gene found in SO MR-1 encodes for a membrane-associated periplasmic c-type cytochrome. CymA has a mass of 21 kDa and a 187 amino acid length. The amino acid sequence can be found in Appendix A, Section A.1. The tetraheme cytochrome protein, CymA, belongs to the NapC/Nir family and helps facilitate direct electron transfer from menaquinol, a cytoplasmic membrane-bound enzyme, to various oxidoreductases located in the periplasm.  In SO MR-1, CymA is essential for respiration and electron transfer from the inner membrane to redox proteins in the outer membrane, such as MtrA (Vellingiri, 2019). Furthermore, the overexpression of CymA has shown to improve the bioelectrochemical performance in microbial fuel cells (MFCs), through the ability of the protein to distribute respiratory electrons and balances redox states (Vellingiri, 2019). Since CymA showed the ability to improve the efficiency of MFCs, the metabolic engineering and overexpression of CymA in photovoltaic applications was considered to play an integral role in increasing the performance of biogenic solar cells.  13  1.2.1.2 OmcA & MtrC In SO MR-1, the OmcA and MtrC genes both encode for an outer membrane-associated c-type decaheme cytochromes. OmcA has a mass of 83 kDa and an amino acid length of 735, whereas MtrC has a mass of 75 kDa and an amino acid length of 671. The amino acid sequences of OmcA, and MtrC can be found in Appendix A, Section A.2, and Section A.3, respectively. OmcA and MtrC are two outer membrane cytochromes involved in the Mtr respiratory pathway and play an essential role in EET (Kasai, 2015). Both OmcA and MtrC contain 10 putative heme-binding sites and a lipoprotein consensus sequence, which are imperative in the transfer of electrons to extracellular electron acceptors (Shi, 2006). It has been suggested that the outer membrane cytochromes facilitate EET through direct and indirect pathways. The direct pathway consists of electrons being transferred from the outer membrane cytochromes to solid metals. The indirect pathway consists of electrons being transferred from the outer membrane cytochromes using secreted electron-shuttle compounds, such as flavins, in order to reach distant solid metals (Kasai, 2015). Furthermore, there are studies that have shown some functional differences between OmcA and MtrC. OmcA has shown a stronger ability in the attachment of cells to solid surfaces, whereas MtrC has shown to have a more prominent role in electron transfer to electrodes. However, studies have shown that both are able to facilitate EET to solid iron oxides (Kasai, 2015).   1.2.1.3 MtrA & MtrB Other important genes required from SO MR-1 for EET are MtrA and MtrB. The MtrA gene encodes for a decaheme c-type cytochrome periplasmic electron carrier, whereas the MtrB gene encodes for a non-heme containing integral outer membrane protein. MtrA and MtrB have masses of 32 kDa, and 76 kDa, respectively, and amino acid lengths of 333, and 697, respectively. The 14  amino acid sequences of MtrA and MtrB can be found in Appendix A, Section A.4, and Section A.5, respectively. MtrA helps to facilitate electron transfer from the periplasmic CymA cytochrome, to the outer membrane decaheme cytochromes OmcA and MtrC (Coursolle, 2010), whereas MtrB is essential for proper localization of MtrA, OmcA, and MtrC (Pitts, 2003). More specifically, electrons from the menaquinone pool are transferred to the periplasm through CymA, which can then be oxidized by the periplasmic face of MtrA before ultimately being transported through the outer membrane by interacting with MtrB, and exit through OmcA and MtrC (Pitts, 2003).  1.3 Research Goals The overarching goal of the research and experiments is to investigate how the presence of bacterial nanowires affect the conductivity and efficiency of the biogenic solar cells. To be achieved, the overarching goal must be broken down into several smaller objectives. The first objective is to successfully clone the genes from the Mtr pathway into a different bacterial species that is well-suited for protein expression. Second, the expression levels of the proteins in the cloned bacterial species need to be analyzed to ensure that the genes of the Mtr pathway are being adequately transcribed and translated. Finally, biogenic solar cells need to be assembled using the cloned bacteria, and the photovoltaic properties need to be measured and analyzed in order to evaluate and compare the performance of solar cells with and without the Mtr pathway genes.   1.4 Thesis Overview Chapter 1 has presented a brief introduction to the recent progress and principles of biogenic solar cells, and how they can potentially be improved by harnessing the electroconductive properties of 15  bacterial nanowires. Chapter 2 outlines the molecular cloning and genetic engineering of the genes of the Mtr pathway into a vector plasmid. Chapter 3 describes and validates the molecular expression of the Mtr pathway proteins and lycopene synthesis proteins. Chapter 4 illustrate the assembly, mechanisms, and performance of the biogenic solar cells with the genetically engineered bacteria expressing the Mtr pathway and lycopene synthesis pathway. Chapter 5 concludes the research findings and describes future work for further biogenic solar cell improvement.   16  Chapter 2: Molecular Cloning  2.1  Introduction To produce a species that could harness the electroconductive properties shown in SO MR-1, the genes of the Mtr pathway had to be cloned into another bacterial species so that the genes could be sufficiently expressed. Molecular cloning involves a series of experimental methods that are used to assemble recombinant deoxyribonucleic acid (DNA) molecules into a vector, and to direct their replication within a host organism. For these experiments, the recombinant DNA molecules of interest were the genes of the Mtr pathway, and the vector was the pET-28a(+) plasmid. The host organism for the molecular cloning experiments was chosen to be Trans1-T1 phage chemically competent cells purchased from Civic Bioscience. These cells were chosen due to their high transformation efficiency, and their fast-growing nature which allows colonies to be visible in 8-9 hours. Once all 5 of the genes of the Mtr pathway were assembled into one plasmid, the plasmid could then be transformed into another bacterial strain that was better suited for protein expression.   The methods involved in cloning require several steps. First, targeted DNA fragments, referred to as inserts, are isolated. Second, the inserts are ligated into the appropriate cloning vector, creating recombinant DNA molecules, such as plasmids. Third, the recombinant DNA molecules are transformed into the desired host, typically bacteria. Finally, the host cells are selected and screened to ensure that the desired recombinant DNA molecules have been successfully transformed into the species.  17  The specific type of molecular cloning method used to insert the genes of the Mtr pathway into the host Trans1-T1 cells was Gibson Assembly. A schematic representation of the Gibson Assembly process is shown in Figure 2.1 (Gibson, 2009).   The first step is to linearize the vector and insert, by polymerase chain reactions (PCR), with primers that create complementary overlapping sequences at each end of the fragments that are to be joined. The complementary sequences typically have a 20-40 base pair overlapping sequence with a melting temperature over 50ºC. After the vector and insert have been linearized, the vector and insert are placed in the same tube along with Gibson Assembly master mix and are incubated at 50ºC for 15 minutes.    Figure 2.1: Stepwise representation of the Gibson Assembly cloning process (Gibson, 2009).   18  The Gibson Assembly master mix has three enzymes: T5 exonuclease, DNA polymerase, and DNA ligase. During the reaction, the T5 exonuclease acts first by chewing back the 5’ end of each of the DNA fragments, exposing the complementary overlapping sequence. The two fragments anneal to one another since the melting temperature of the overlapping sequences are designed to be greater than 50ºC. DNA polymerase then closes the gaps on each of the fragments that were left by the T5 exonuclease. Finally, DNA ligase acts and joins the two fragments together. An additional diagram showing one insert being assembled into a plasmid is shown, as an example, in Figure 2.2 (Gibson Assembly Cloning). After completion of the reaction, the assembled DNA can immediately be transformed into the host cell, and grown overnight. The bacterial colonies grown overnight can then be screened by verification steps such as colony PCR and Sanger Sequencing to see if the transformation was successful. Once the bacteria have been screened and verified, the molecular cloning can be deemed to be successfully completed.       Figure 2.2: Gibson Assembly schematic representation of one insert added to a plasmid (Gibson Assembly Cloning). 19  2.2 Materials and Methods 2.2.1 Plasmid and Template DNA The plasmid chosen to be the vector for the cloning protocol was pET-28a(+) due to its low copy number and compatibility with the lycopene synthesizing plasmid, pAC-Lyc. The pET-28a(+) plasmid is used for bacterial expression with a high expression level, has bacterial resistance to kanamycin, and has a T7 promoter. To obtain an adequate amount of pET-28a(+) for the necessary experiments, E. coli BL21(DE3) containing the pET-28a(+) plasmid from a glycerol stock was grown overnight in Luria Bertani (LB) media with kanamycin antibiotics in an incubator at a temperature of 37ºC and shaken at 250 rpm. After the bacterial culture was grown overnight, a Thermo Scientific, GeneJET Plasmid Miniprep Kit was used to extract the plasmid DNA. The pET-28a(+) plasmid DNA was stored at -20ºC.  To obtain template DNA from SO MR-1, the bacteria was also taken from a glycerol stock and grown overnight in LB media at 37ºC and shaken at 250 rpm. After growing overnight, the genomic DNA was extracted using an Invitrogen, PureLink Genomic DNA Mini Kit. The SO MR-1 genomic DNA was stored at -20ºC. for later use.   2.2.2 Polymerase Chain Reaction & Gel Electrophoresis All PCR reactions used to linearize and amplify bacterial DNA were conducted using TransStart FastPfu Fly DNA Polymerase. In addition, one to three replicate reactions were conducted simultaneously for each PCR reaction, in case there were any unsuccessful reactions. The reaction components and thermal cycling conditions that were used for each of the reactions are outlined in Appendix B. After amplification, the samples that used plasmid DNA as the template were 20  digested for 90 minutes at 37ºC with a restriction enzyme, DpnI, in order to cleave any remaining template plasmid DNA. After digestion, the enzyme was heat inactivated by incubating at 80ºC for 20 minutes. DpnI digestion was not necessary for PCR reactions that used SO MR-1 genomic DNA as the template. Next, the lengths of the amplified DNA strands were verified using gel electrophoresis. All gel electrophoresis samples were applied a voltage of 140V for 30-40 minutes with an appropriate DNA ladder before being fluoresced under ultraviolet light. If the band of DNA corresponded with the correct area of the DNA ladder, the bands containing DNA were excised and the DNA was extracted using a Thermo Scientific, GeneJET Gel Extraction Kit. The successful linearized fragments were stored at -20ºC for future use. It can be noted that some gel electrophoresis results showed faint bands at the bottom of the gel, or in areas that were not anticipated. These faint bands can be from the primers, template DNA remaining within the system, or primers falling off the template DNA during the extension phase, resulting in incomplete, smaller DNA fragments being replicated.   2.2.3 DNA Assembly and Transformation Gibson Assembly was used to assemble all DNA fragments. Vectors and inserts were linearized using PCR, and the PCR primers were designed in a way so that the vector and insert fragments could be amplified with 20-40 base pair overlapping sequences. All primers used in each experiment, their nucleotide sequences, and their melting temperatures, are shown in Appendix B. There is a range of 20-40 base pairs to ensure that a melting temperature of roughly 60ºC for each of the overlapping sequences. After PCR linearization, gel electrophoresis, and gel extraction, the Gibson Assembly cloning reaction was setup. All Gibson Assembly reactions were carried out using a Civic Bioscience, pEASY-UNI Seamless Cloning and DNA Assembly Kit. A volume of 21  10 μL was used for all reactions. The vector:insert ratio was 1:2, within the range of 0.01-0.025pmols. Typically, 0.012pmols of the vector was used, and 0.024 pmol of the insert was used. 5 μL of the 2x assembly mix was added to the reaction, which contained the T5 exonuclease, DNA polymerase, and DNA ligase. Enough double-distilled water was added to the reaction so that the final reaction volume was 10 μL. Next, the reaction tube was gently mixed and incubated at 50ºC for 15 minutes. After incubation, the mixture was ready for DNA transformation. Civic Bioscience, Trans1-T1 Phage Resistance Chemically Competent Cells were used for each transformation for all of the Gibson Assembly reactions, due to the higher transformation efficiency. A tube of the competent cells was transferred from the -80ºC freezer, and was thawed on ice for 20 minutes. After thawing, 25 μL of the competent cells were placed in the fresh microcentrifuge tube on ice and 1 μL of the Gibson Assembly reaction mixture was transferred to the competent cells. The competent cells and DNA mixture was gently flicked to help mix the DNA, and was then left to incubate on ice for 30 minutes. Once incubation was complete, the mixture was heat-shocked in a water bath at 42ºC for 30 seconds and immediately placed on ice for 2 minutes. 450 μL of LB media was added to the reaction mixture, and was placed in an incubator at 37ºC and 250 rpm for 1 hour. LB-agar plates containing kanamycin antibiotics were setup and pre-warmed at 37ºC. After incubation for 1 hour, 100 μL of the competent cells were spread onto the plate and incubated overnight at 37ºC.  2.2.4 Verification of Inserted DNA To ensure that the DNA fragments were assembled correctly, colony PCR and Sanger Sequencing were used as verification steps. The reaction setup for colony PCR included the bacterial colonies grown overnight on an LB-agar plate used as the template DNA, DNA primers, and Froggabio 2X 22  Taq FroggaMix. Froggabio 2X Taq FroggaMix is a mixture that has the appropriate quantities of deoxynucleoside triphosphates, DNA polymerase enzymes, and DNA polymerase buffer, which are all required for PCR amplification reactions. A 20 μL reaction volume was used for colony PCR. Several colonies were picked from each plate, and each colony was used as the template DNA in its own microcentrifuge tube. The reaction components for each of the colony PCRs is shown in Appendix B, along with the thermal cycling conditions. After the reaction had concluded, gel electrophoresis was used to verify that the correct DNA length was present based on the DNA primers that were used. If the DNA length in the gel was in the expected area, the colony that was used was picked again and grown in LB media overnight with kanamycin antibiotics. After being grown overnight, a plasmid extraction was done using a Thermo Scientific, GeneJET Plasmid Miniprep Kit. The plasmid DNA was stored at -20ºC, and the next verification step, Sanger Sequencing, was to be conducted.  Sanger Sequencing is a method that is used to determine the nucleotide sequence of a DNA sample. All Sanger Sequencing verification steps were carried out by a genomics services company, GENEWIZ. The GENEWIZ universal primer, GENEWIZ T7-TERM, was used for all experiments because each time a new gene insert was added, the overlapping sequence of the two assembled fragments was positioned slightly upstream of the T7 terminator sequence. Therefore, if the gene insert was added successfully, the overlapping sequence and the inserted gene fragment would be amplified by the GENEWIZ T7-TERM primer, and the nucleotide sequence of the overlapping sequence and the inserted gene fragment would show on the Sanger Sequencing results, thus, verifying that the two fragments had attached. It is worth noting that Sanger Sequencing was not used to obtain the nucleotide sequence of the entire plasmid, just the area of 23  interest, explained above. After Sanger Sequencing was completed, GENEWIZ uploaded the nucleotide sequence to their website. The files were downloaded and opened with SnapGene and compared to the known DNA sequences of the vector and insert DNA fragments. A DNA BLAST of the nucleotide sequences of the SnapGene simulation of the assembled plasmid and the Sanger Sequencing results was conducted to compare the alignment of the two sequences. All DNA BLAST results are shown in Appendix C. If there was above a 99% match between the Sanger Sequencing results and the DNA sequence of the inserted gene, the assembly was shown to be successful.    2.2.5 Snapgene Simulation SnapGene is a computer software program that is used to visualize DNA sequences, plan experimental procedures, and document DNA cloning and PCR experiments. The SnapGene file for the pET-28a(+) plasmid was downloaded from the SnapGene website, and the DNA sequences for all SO MR-1 genes were gathered and uploaded to SnapGene from the National Center for Biotechnology Information gene database. Once the pET-28a(+) vector sequence and SO MR-1 gene insert sequences were uploaded, SnapGene actions such as PCR and Gibson Assembly were used to simulate the reactions before conducting lab experiments. For PCR experiments, directional DNA primers can be added to SnapGene DNA files, and the results from a PCR reaction with the selected primers can be visualized. Being able to visualize the results is essential to ensure that the correct section of DNA is being amplified, and it is also essential for setting up the thermal cycling conditions, since it is required to know the length of the region being amplified. For Gibson Assembly experiments in SnapGene, DNA primers are added to the vector and insert DNA fragments, and the DNA templates are linearized. Once linearized, the overlapping regions 24  at each end of the DNA fragments are visually aligned with one another. SnapGene shows the number of base pairs attached for each of the overlapping regions, as well as the melting temperature for each of the regions. SnapGene then shows a visual representation of the assembled vector and insert sequence in a circular DNA plasmid. The results from Sanger Sequencing were matched with the results from the simulated SnapGene experiments. If there was a match between the two sequences, the experiments were deemed to be successfully verified.       2.3 Results and Discussions 2.3.1 CymA Insert The first Gibson Assembly reaction consisted of inserting the CymA gene fragment into the pET-28a(+) vector. BL21(DE3) bacteria containing the pET-28a(+) plasmid, and SO MR-1 were grown, in LB media, in separate flasks overnight in an incubator at 37ºC and 250 rpm. DNA extractions were conducted on each of the overnight cultures, and used as template DNA for the PCR reactions. The gel electrophoresis results from the linearization of each of the fragments is shown in Figure 2.3, and the thermal cycling conditions are shown in Appendix B, Section B1.  For the SnapGene simulation, linearization of the pET-28a(+) vector with the primers specified in Appendix B, Section B1 yields a DNA length of 5221 base pairs (bp). As seen in Figure 2.3a, there is a bright band just above the 5k DNA ladder marker. Additionally, the SnapGene simulation of the linearization of the CymA gene yields a DNA length of 591 bp, and there is a bright band present in between the 500 bp and 750 bp DNA ladder markers in Figure 2.3b. The gel electrophoresis images suggest that both of the PCR reactions were successful. Figure 2.3c shows the SnapGene simulation of the Gibson Assembly reaction of the two linearized fragments. It can be noted that the plasmid was named pET-28a(+)_SO1 to signify that pET-28a(+) was the initial 25  a  b                             c   Figure 2.3: Linearization steps and assembly of CymA into pET-28a(+); a) linearization of the pET-28a(+) vector - lane 1 (farthest left): 1kb Plus Opti-DNA Marker, lanes 2, 3: linearized pET-28a(+) vector; b) linearization of CymA gene insert - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized CymA gene fragment; c) SnapGene simulation of pET-28a(+)_SO1 assembly 26  vector, and 1 gene from the SO MR-1 species had been added to the vector. To verify that the two fragments were correctly assembled, colony PCR was done on the transformed cells. The results of the colony PCR are shown in Figure 2.4. Four colonies were analyzed, and each colony had its own PCR reaction. Primers that attached to the f1 origin of replication and the LacI gene of pET-28a(+) were chosen, since they encompass the entirety of the inserted fragment, as shown in Figure 2.4b. The gel electrophoresis results of the colony PCR are shown in Figure 2.4a, and the thermal cycling conditions are shown in Appendix B, Section B1. The SnapGene simulation, shown in Figure 2.4b, of the colony PCR yields a DNA length of 1443 bp, and there is a bright band present slightly below the 1.5k bp DNA ladder markers in Figure 2.4a. Therefore, the colony PCR verification step has been successfully completed. Next, the colony from the second lane was picked and grown in LB media with kanamycin antibiotics overnight, in an incubator at 37ºC and 250 rpm. A plasmid extraction was conducted on the overnight culture and the plasmid was sent to GENEWIZ for Sanger Sequencing. The nucleotide sequence of the Sanger Sequencing is shown in Appendix C, Section C1, and the sequence was compared with the nucleotide sequence from the SnapGene simulation. There is a greater than 99% match between the two sequences, so the second verification step had been deemed to be successful.      27           a                        b   Figure 2.4: Colony PCR verification for the CymA insert; a) linearization of the pET-28a(+)_SO1 plasmid - lane 1 (farthest left) and lane 4: 1kb Plus Opti-DNA Marker, lanes 2, 3, 5, 6 (farthest right): linearized pET-28a(+)_SO1 plasmid - each lane was from a separate colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO1 plasmid 28  2.3.2 OmcA Insert For the second Gibson Assembly, the previously assembled plasmid, pET-28a(+)_SO1, became the vector, and the OmcA gene from SO MR-1 became the insert. The pET-28a(+)_SO1 vector, and SO MR-1 genomic DNA were linearized by PCR and the gel electrophoresis results are shown in Figure 2.5, and the thermal cycling conditions are shown in Appendix B, Section B2. From the primers used for the PCR reactions, as shown in Appendix B, Section B2, the SnapGene simulations show a DNA length of 5786 bp for the linearized pET-28a(+)_SO1 vector, and 2430 bp for the linearized OmcA gene. It can be noted that the OmcA gene itself is 2208 bp, however the forward primer was designed to anneal a couple of hundred base pairs prior to the start of the OmcA gene to ensure that a ribosome binding site was also part of the assembly – this is the case for all inserted genes, except for CymA since a ribosome binding site was already present on the pET-28a(+) vector. As shown in Figure 2.5a and Figure 2.5b, there are bright bands just below the 6k DNA ladder marker, and the 2.5k DNA ladder marker, respectively. The comparisons to the DNA ladder suggest that both of the PCR reactions were successful. The Gibson Assembly of the two linearized fragments is shown in Figure 2.5c. The plasmid was named pET-28a(+)_SO2 to signify that that there were now 2 genes from the Shewanella Oneidensis MR-1 species added to the pET-28a(+) vector plasmid.   29  a      b                            c   Figure 2.5: Linearization steps and assembly of OmcA into pET-28a(+)_SO1; a) linearization of the pET-28a(+)_SO1 vector - lane 1 (farthest left): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5 (farthest right): linearized pET-28a(+)_SO1 vector; b) linearization of OmcA gene insert - lane 1 (farthest left): 1kb Plus Opti-DNA Marker, lanes 2, 3 (farthest right): linearized OmcA gene fragment; c) SnapGene simulation of pET-28a(+)_SO2 assembly 30  Again, to verify assembly of the two fragments, colony PCR was conducted after transformation and overnight growth of the cells. The results of the colony PCR are shown in Figure 2.6. Four colonies were analyzed, and each colony had its own PCR reaction. The same primers that attach on the f1 origin of replication and the LacI gene of pET-28a(+) were used as they encompass the entirety of the inserted OmcA gene fragment, as shown in Figure 2.6b. From the SnapGene simulation shown in Figure 2.6b, the DNA length of the colony PCR shows a length of 3838 bp. The gel electrophoresis results of the colony PCR are shown in Figure 2.6a, and the results show that there is a bright band present slightly below the 4k DNA ladder marker. The thermal cycling conditions for the PCR are shown in Appendix B, Section B2.  From the gel electrophoresis images, the colony PCR verification step was confirmed to be successfully completed. For the second verification step, Sanger Sequencing, the colony from the fourth lane in Figure 2.6a was picked and grown in LB media with kanamycin antibiotics overnight, in an incubator at 37ºC and 250 rpm. After overnight growth, a plasmid extraction was completed and the DNA was sent to GENEWIZ to develop the nucleotide sequence. The sequence from GENEWIZ was compared with the SnapGene sequence, as shown in Appendix C, Section C2. The two sequences have a greater than 99% match, so the Sanger sequence verification step was successfully completed, and the next gene could be inserted.       31                   a                                    b   Figure 2.6: Colony PCR verification for the OmcA insert; a) linearization of the pET-28a(+)_SO2 plasmid - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO2 plasmid - each lane was from a separate colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO2 plasmid  32  2.3.3 MtrC Insert The third gene to be inserted by Gibson Assembly was the MtrC gene. The vector was now the pET-28a(+)_SO2 plasmid. Again, both the pET-28a(+)_SO2 plasmid and MtrC gene from SO MR-1 were linearized by PCR and the resulting images of the gel electrophoresis are shown in Figure 2.7, while the thermal cycling conditions are shown in Appendix B, Section B3. Using the appropriate primers shown in Appendix B, Section B3, the SnapGene PCR simulations show a DNA length of 8203 bp for the linearized pET-28a(+)_SO2 vector, and 2368 bp for the linearized MtrC gene. Similar to the OmcA gene, the forward primer for linearization of the MtrC gene was designed to bind upstream of the start codon to ensure that a ribosome binding site was present in the assembly. Figure 2.7a shows a bright band slightly above the 8k DNA ladder marker, and Figure 2.7b shows a bright band in between the 2k and 2.5k DNA ladder markers. These results align with the SnapGene simulations. Figure 2.7c shows the SnapGene simulation of the Gibson Assembly of the two linearized fragments. The assembled plasmid was now named pET-28a(+)_SO3 since there were 3 SO MR-1 genes attached to the pET-28a(+) vector.     33  a  b                              c   Figure 2.7: Linearization steps and assembly of MtrC into pET-28a(+)_SO2; a) linearization of the pET-28a(+)_SO2 vector - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO2 vector; b) linearization of MtrC gene insert - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized MtrC gene fragment; c) SnapGene simulation of pET-28a(+)_SO3 assembly 34  Colony PCR was once again used to verify the Gibson Assembly after transformation and overnight growth of the cells. The results of the colony PCR are shown in Figure 2.8. Again, four colonies were analyzed, and each colony had its own PCR reaction. For the colony PCR, one primer that attaches to the f1 origin of replication and another primer that attached upstream of the MtrC gene were used since they entirely cover the attachment sites of the Gibson Assembly reaction, as shown in Figure 2.8b.  The primer that attaches to the LacI gene was not used in this instance because using the other primer resulted in a quicker PCR reaction time. From Figure 2.8b, the DNA length of the colony PCR shows a length of 2730 bp. Figure 2.8a shows the gel electrophoresis results of the colony PCR and the image shows a bright band slightly above the 2.5k DNA ladder marker. The thermal cycling conditions for the PCR are shown in Appendix B, Section B3. After successfully completing the colony PCR, the colony from the fourth lane from Figure 2.8a was picked and grown overnight in LB media with kanamycin antibiotics, in an incubator at 37ºC and 250 rpm. Then, a plasmid extraction was conducted, and the DNA was sent to GENWIZ for Sanger Sequencing. The results of the Sanger Sequencing are shown in Appendix C, Section C3, alongside the nucleotide sequence from the SnapGene simulation. There was more than a 99% match between the two sequences, therefore the Gibson Assembly was deemed to be successful, and the final two genes could then be inserted.         35                 a                         b   Figure 2.8: Colony PCR verification for the MtrC insert; a) linearization of the pET-28a(+)_SO3 plasmid - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO3 plasmid - each lane was from a separate colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO3 plasmid  36  2.3.4 MtrA & MtrB Insert The final two genes to be inserted by Gibson Assembly, MtrA and MtrB, were done as one fragment since, together, they have a moderate DNA length of roughly 3000 bp, and they are side by side on the SO MR-1 genome. The pET-28a(+)_SO3 plasmid now became the vector for the final Gibson Assembly. Again, both the pET-28a(+)_SO3 plasmid and MtrA and MtrB genes from SO MR-1 were linearized by PCR. Figure 2.9 shows the gel electrophoresis results from each of the PCR reactions. The thermal cycling conditions are shown in Appendix B, Section B4. From the SnapGene PCR simulations, and using the primers shown in Appendix B, Section B4, the DNA length of the linearized pET-28a(+)_SO3 vector is 10524 bp, and the DNA length of the linearized MtrA and MtrB genes is 3214 bp. Again, the forward primer used for linearization of the MtrA and MtrB genes was designed to bind upstream on the start codon of MtrA to ensure a ribosome binding site was part of the Gibson Assembly. There is a bright band slightly above the 10k DNA ladder marker in Figure 2.9a, and there is a bright band slightly above the 3k DNA ladder marker in Figure 2.9b. Therefore, the images suggest the PCR reactions amplified the desired region. The SnapGene simulation of the final Gibson Assembly of the two linearized fragments is shown in Figure 2.9c. The final assembled plasmid was named pET-28a(+)_SO5 since all 5 desired genes from SO MR-1 were inserted into the pET-28a(+) vector.   37  a  b                                 c   Figure 2.9: Linearization steps and assembly of MtrA & MtrB into pET-28a(+)_SO3; a) linearization of pET-28a(+)_SO3 vector - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO3 vector; b) linearization of MtrA & MtrB gene insert - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized MtrA and MtrB gene fragment; c) SnapGene simulation of pET-28a(+)_SO5 assembly  38  Again, colony PCR was used to verify the Gibson Assembly after transformation and overnight growth of the cells. The colony PCR results are shown in Figure 2.10. Four colonies were analyzed, and each colony had its own PCR reaction. Primers that attach to the f1 origin of replication and the LacI gene of pET-28a(+) were used as they encompass the entirety of all 5 of the inserted  SO MR-1 genes, as shown in Figure 2.10b. Additionally, from the SnapGene simulation shown in Figure 2.10b, the DNA length of the colony PCR shows a length of 9352 bp. The gel electrophoresis results, shown in Figure 2.10a, show a faint band in between the 8k and 10k DNA ladder markers. The thermal cycling conditions for the PCR are shown in Appendix B, Section B4. There is also a bright band present in the 5k-6k bp region. The band in this region could possibly be there due to pET-28a(+)_SO3 plasmid also being transformed, as it was used as a template for the linearization shown in Figure 2.9a. If the template pET-28a(+)_SO3 plasmid was transformed alongside the assembled pET-28a(+)_SO5 plasmid, the DNA length from a colony PCR using the primers that attached to the f1 origin of replication and the LacI gene would yield 6183 bp. These findings suggest that there is still some pET-28a(+)_SO3 plasmid alongside the pET-28a(+)_SO5 plasmid in the transformed bacteria. The Gibson Assembly and transformation could be repeated, but with a longer DpnI digestion after the pET-28a(+)_SO3 PCR reaction in order to cleave the pET-28a(+)_SO3 plasmid, and decrease the probability of it being transformed alongside pET-28a(+)_SO5.  Additionally, the band in the 5k-6k bp region could be from the primers binding in a different location than expected. However, a nucleotide blast was conducted and there were no other identifiable binding regions. One final possibility could be that the primers could be falling off during the extension phase, which would show a decrease in the DNA length.  39                        a                        b   Figure 2.10: Colony PCR verification for the MtrA & MtrB insert; a) linearization of the pET-28a(+)_SO5 plasmid - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO5 plasmid - each lane was from a separate colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO5 plasmid 40   Although there could potentially be pET-28a(+)_SO3 present in the bacterial colonies, the presence of a band in the anticipated region, 8k to 10k, was adequate verification that the colony PCR was successful. Next, the colony from the fourth lane from Figure 2.10a was picked, grown overnight in LB media and kanamycin antibiotics, in an incubator at 37ºC and 250 rpm. Then, a plasmid extraction was conducted, and the DNA was sent to GENWIZ for Sanger Sequencing. The nucleotide sequence from GENEWIZ and the nucleotide sequence from the SnapGene simulation are shown in Appendix C, Section C4. The sequences have greater than a 99% match, therefore the Gibson Assembly was successfully completed, and all 5 genes were verified to be inserted into the pET-28a(+) plasmid.   41  Chapter 3: Protein Expression  3.1 Introduction To ensure that the genes of the Mtr pathway and the lycopene synthesis pathway were being sufficiently transcribed and translated, protein expressions experiments were conducted to visualize the expression levels. Before the protein expression could be validated, the two separate plasmids, the pET-28a(+)_SO5 plasmid containing the genes of the Mtr pathway and the pAC-Lyc plasmid containing the genes of the lycopene synthesis pathway, needed to be transformed into one bacterial strain. As stated in the previous chapter, all transformations during the molecular cloning steps were done using Trans1-T1 phage chemically competent cells from Civic Bioscience due to their high transformation efficiency. Additionally, the pAC-Lyc plasmid was being studied by another graduate student in my lab, who already had the plasmid in DH5α cells, so the lycopene synthesis cells were gifted for use in my research. For applications in biogenic solar cells, each of these plasmids were to be extracted from their host cells, transformed into the E. coli strain BL21(DE3), and used for solar cell assembly. The BL21(DE3) strain was chosen as the host organism of the two-plasmid system due to its ability of high-level expression of recombinant proteins, and also because it carries the T7 ribonucleic acid (RNA) polymerase gene under control of the lacUV5 promoter (Jeong, 2015). Therefore, IPTG could be used to induce expression of T7 RNA polymerase and the genes of the desired Mtr pathway proteins further downstream. The IPTG only had an effect on the pET-28a(+)_SO5 plasmid, since the genes in the pAC-Lyc are under an endogenous promoter. After transformation of both plasmids into BL21(DE3), the protein expression levels were to be evaluated. The cells were induced and given a sufficient amount of time for growth, and the amount of soluble and insoluble protein was measured using a Bradford 42  Assay. A Bradford Assay is a prevalent assay that allows for fast and simple protein quantification in cell lysates, recombinant protein samples, and cellular fractions for the goal of normalizing biochemical measurements (Ernst, 2010). Once the Bradford Assay was completed, the required amount of protein could be loaded into a protein gel to visualize the expression of each of the desired proteins. Protein gel electrophoresis is very similar to DNA gel electrophoresis, as each are loaded into a well, a current is applied, and the genetic material is separated based on size. The larger the protein or DNA fragment, the less it travels down the gel. Bacterial strains lacking one or both of the plasmids, and bacterial strains with both plasmids were used to verify the presence or absence of protein expression of the desired genes in the Mtr pathway and the lycopene synthesis pathway.            3.2 Materials and Methods 3.2.1 Two Plasmid Co-Transformation  To obtain a sufficient amount of each of the plasmids, pET-28a(+)_SO5and pAC-Lyc, for co-transformation, each plasmid had to first be grown in the host cell before their DNA could be extracted. The pET-28a(+)_SO5 plasmid was grown using the same colony that was picked and used for colony PCR and Sanger Sequencing verification, since that specific colony had been known to have the genes of the Mtr pathway. The colony was picked and transferred into a flask with 50 mL of LB media and 50 μL of 50 mg/mL stock solution of kanamycin antibiotics. The bacterial culture was grown overnight in an incubator at a temperature of 37ºC and shaken at 250 rpm. Additionally, a glycerol stock of DH5α cells containing the pAC-Lyc plasmid was used for the other culture. A small amount of the glycerol stock was scraped and transferred into a flask with 50 mL of LB media and 50 μL of 34 mg/mL stock solution of chloramphenicol antibiotics. 43  Similarly, the bacterial culture was grown overnight in an incubator at a temperature of 37ºC and shaken at 250 rpm. After the bacterial culture was grown overnight, a Thermo Scientific, GeneJET Plasmid Miniprep Kit was used to extract the plasmid DNA. The pET-28a(+)_SO5 plasmid and the pAC-Lyc plasmid were both stored at -20ºC for future use.  After each of the plasmids had been extracted, the co-transformation could be performed. As previously stated, the E. coli strain BL21(DE3) was to be the host cells for the two-plasmid system. BL21(DE3) chemically competent cells purchased from Civic Bioscience were transferred from the -80ºC freezer, and was thawed on ice for 20 minutes. Once the competent cells had thawed, 25 μL of the competent cells were placed in the fresh microcentrifuge tube on ice along with 10 ng of DNA from the pET-28a(+)_SO5and 10 ng of DNA from the pAC-Lyc plasmid. These tubes were setup in triplicate since the transformation efficiency of two plasmids is very low. Each of the tubes were gently flicked to help mix the DNA, and they were then left to incubate on ice for 30 minutes. After incubating on ice, each of the tubes was heat-shocked in a water bath for 30 seconds and immediately placed on ice for 2 minutes. Next, 450 μL of LB media was added to the reaction mixture, and was placed in an incubator at 37ºC and 250 rpm for 1 hour. LB-agar plated containing both kanamycin and chloramphenicol antibiotics were setup and pre-warmed at 37ºC. After 1 hour in the incubator, 100 μL of the competent cells for each tube were spread onto their own plate and incubated overnight at 37ºC.  3.2.2 Culture Conditions To compare the presence or absence of the desired proteins, four different bacterial strains were cultured. Each bacterial culture was grown for a sufficient amount of time to ensure that there was 44  enough protein expressed from each of the pathways. The control culture was E. coli BL21(DE3) bacteria with no added DNA or plasmids, the second culture was BL21(DE3) bacteria transformed with pET-28a(+) plasmid DNA (referred to as BL21(DE3)-pET-28a(+)), the third culture was BL21(DE3) transformed with the pAC-Lyc plasmid (referred to as BL21(DE3)-Lyc), and the final culture was BL21(DE3) bacteria transformed with both the pET-28a(+)_SO5 plasmid and the pAC-Lyc plasmid (referred to as BL21(DE3)-Mtr-Lyc). The control culture was grown overnight in 100 mL of 2X yeast extract-tryptone (YT) media with no antibiotics in an incubator at 37ºC and 250 rpm. The BL21(DE3)-pET-28a(+) was grown overnight under the same conditions, but with 100 μL of 50 mg/mL stock solution of kanamycin antibiotics. The BL21(DE3)-Lyc culture was also grown in 100 mL of 2xYT media for 48 hours in an incubator at 25ºC and 250 rpm with 100 μL of 34 mg/mL stock solution of chloramphenicol antibiotics. Finally, the BL21(DE3)-Mtr-Lyc culture was grown in 2xYT media for 48 hours in an incubator at 25ºC and 250 rpm with 100 μL of 50 mg/mL stock solution of kanamycin antibiotics, 100 μL of 34 mg/mL stock solution of chloramphenicol antibiotics, and was also immediately induce with 100 μL of 1M IPTG. The longer incubation time was implemented to allow for sufficient protein expression of the lycopene plasmid to occur. At 48 hours the cultures could be visibly seen to have a bright orange-red color indicating the lycopene synthesis genes were being expressed. The lower incubation temperature was implemented to decrease the stresses on the cell during protein synthesis.   3.2.3 Protein Standard Curve A standard curve utilizing a protein of known concentration was to be generated and used as a reference for analysis of the subsequence samples of unknown protein concentration. The standard curve was used to determine the protein concentrations of the soluble and insoluble proteins in 45  each of the cultures. Bovine Serum Albumin (BSA) was chosen as the reference protein used in the standard assay. Pierce Bovine Serum Album Standard Ampules, 2 mg/mL, were purchased from Thermo Fischer Scientific. These ampules were then diluted into samples of the following concentrations: 0.5 mg/mL, 0.25 mg/mL, 0.0125 mg/mL, and 0.0625 mg/mL. The dye reagent that was used to react with the protein for the experiment was the Bio-Rad protein assay dye reagent concentrate (5X). As the dye reacts with the protein, the color of the sample changes. A darker sample indicates a greater protein concentration. The dye was taken from the fridge, diluted 5-fold, creating a 1X solution, and left to equilibrate at room temperature. Next, 5 μL from each of the diluted BSA samples, and 5 μL of double distilled water were transferred using a pipette into a well in their own column on a microplate, and 250 μL of the 1X dye reagent was added to each of the wells. This step was repeated two more times creating triplicates of each sample. The samples were then left to incubate at room temperature for a minimum of 5 minutes. Next, the microplate with all of the samples was moved to a spectrophotometer and the absorbance was measured at 595 nm. The absorbance data was then averaged for the triplicate samples. The blank samples were also averaged and were subtracted from the standard values. The standard curve was then created by plotting the standardized absorbance data at 595 nm (y-axis) versus their concentration in mg/mL (x-axis). The plot could then be used for analysis of samples with unknown protein concentrations.   3.2.4 Bradford Assay A Bradford protein assay was conducted to measure the concentration of insoluble (CymA, CrtB, CrtE, IpdI) and soluble proteins (MtrA, MtrB, MtrC, OmcA) being expressed by the BL21(DE3) bacteria. A volume of 10 mL was taken from each of the cultures, labelled, and transferred to 12 46  mL culture tubes. The tubes were then centrifuged at 3000 rpm for 20 minutes. After being centrifuged, the supernatant was discarded from each of the samples, and the resulting pellets were weighed. The cells, or pellets, were then gently resuspended with a pipette and lysed using Bacterial Protein Extraction Reagent purchased from Thermo Fisher Scientific at a ratio of 3 mL/gcells. The samples were then gently mixed on a rotating plate for 30 minutes at room temperature. Next, the lysed cells were then transferred into 2 mL microcentrifuge tubes and centrifuged at 14000 rpm for 30 minutes at 4ºC. After centrifugation, the supernatant from each sample was transferred to fresh 2 mL microcentrifuge tubes. These samples contained the soluble proteins from each of the 4 different bacterial cultures. Each soluble protein sample was diluted 20-fold with double distilled water so that the protein concentration could be within the range of the standard curve. To obtain the insoluble proteins from the samples, 1 mL of 8M urea was added to the pellets from the 2 mL microcentrifuge tubes and the samples were resuspended using a pipette. The samples were left to incubate at room temperature for 30 minutes. Once the incubation had finished, the samples were centrifuged at 14000 rpm for 15 minutes. The supernatant was kept, and transferred to fresh 2 mL microcentrifuge tubes. These samples had the insoluble proteins from each of the 4 different bacterial cultures. Each of the insoluble protein samples were diluted 100-fold with double distilled water so that the protein concentration could be within the range of the standard curve. Next, in triplicate, 5 μL from each of the diluted samples containing soluble or insoluble proteins was transferred to a well inside a microplate, with each sample in its own column. Next, 250 μL of 1X Bio-Rad protein assay dye reagent was added to each of the wells and the samples were left to incubate at room temperature for 5 minutes. Once the incubation had completed, the microplate was transferred to a spectrophotometer and the absorbance at 595 nm was calculated. The absorbance data was logged and the values were averaged for each of the 47  triplicate samples. The unknown sample concentrations could then be determined using the standard curve that was previously generated. The soluble protein samples used for analysis were multiplied by a dilution factor of 20, and the insoluble protein samples used for analysis were multiplied by a dilution factor of 100 to obtain the final concentration of the non-diluted samples. With a known concentration, a protein gel could then be run to verify the expression of the desired proteins.      3.2.5 Protein Gel  To visualize the presence of proteins from the Mtr pathway and lycopene synthesis pathway, a Bolt 10%, Bis-Tris, 1.0mm, 10-well Mini Protein Gel purchased from Thermo Fisher Scientific was used for analysis. First, the gel cassette was taken out of its package and placed inside the electrophoresis tank. The tank was then filled with 1X Bolt Sodium Dodecyl Sulfate running buffer just below the top of the cassette. Next, the soluble and insoluble protein samples were loaded into each well. Using the results from the Bradford assay, 20 μg of protein was transferred to a PCR reaction tube, along with 10 μL of 4X Bolt LDS Sample Buffer purchased from Thermo Fisher Scientific, 10X NuPAGE Sample Reducing Agent purchased from Thermo Fisher Scientific, and double distilled water up to 40 μL. The samples were heated in a PCR thermal cycling machine at 70ºC for 10 minutes before being loaded into the protein gel wells. The well directly in the middle was loaded using 10 μL of PageRuler Prestained Protein Ladder, 10 to 180 kDa, purchased from Thermo Fisher Scientific. The lid was placed and secured on the tank and electrodes were connected to the tank and the power supply. A voltage of 200V was applied for roughly 20 minutes, until the green 10 kDA marker was at the bottom reference line on the tank. After running the protein gel, the cassette was then stained to visualize the bands of the proteins. SimplyBlue 48  SafeStain purchased from Thermo Fisher Scientific was used as the stain for the process. The gel was taken from the tank, placed in a Tupperware container with enough depth to fully submerge the gel, and washed 3 times with 100 mL of double distilled water. After washing, roughly 20 mL of the stain was added to the container and was left to incubate at room temperature for 1 hour with gentle shaking. The stain was then discarded and washed with 100 mL of double distilled water for two hours. A final rinse with 100 mL of double distilled water was done for 1 hour. The protein gel was then able to be analyzed for the presence of the desired proteins.    3.3 Results and Discussion 3.3.1 Mtr Pathway & Lycopene Synthesis Pathway Co-Transformation To verify successful transformation of the pET-28a(+)_SO5 plasmid into BL21(DE3), colony PCR was conducted. The colony PCR results are shown in Figure 3.1. Four colonies were picked from the LB-agar plates after transformation and overnight growth of the cell, and PCR reactions were setup for each of the colonies. Again, primers that attach to the f1 origin of replication and the LacI gene of the pET-28a(+)_SO5 plasmid were used, as shown in Figure 3.1b. From Figure 3.1b, the DNA length of the colony PCR shows a length of 9352 bp. Figure 3.1a shows the gel electrophoresis results of the colony PCR. There is a band present in between the 8kb and 10kb DNA ladder markers, suggesting that the pET-28a(+)_SO5 plasmid was successfully transformed.    There is also a band in the 5k-6k bp region that could be there as a result of pET-28a(+)_SO3 template DNA that was not completely digested. Nevertheless, due to the presence of a band in the predicted region, the DNA from the pET-28a(+)_SO5 plasmid was deemed to be successfully transformed into the BL21(DE3) bacteria. Further information in regard to the thermal cycling conditions for the PCR reaction are specified in Appendix B, Section B5.  49                           a                            b   Figure 3.1: Colony PCR verification for the pET-28a(+)_SO5 plasmid into BL21(DE3); a) linearization of the pET-28a(+)_SO5 plasmid - lane 1 (farthest left) and lane 6 (farthest right): 1kb Plus Opti-DNA Marker, lanes 2, 3, 4, 5: linearized pET-28a(+)_SO5 plasmid - lanes 1 and 2 were from the same colony, lanes 3 and 4 were from the same colony; b) SnapGene simulation of the linearization of the pET-28a(+)_SO5 plasmid 50  After successfully completing the colony PCR, the transformation of pAC-Lyc plasmid into the same BL21(DE3) colonies needed to be verified. To verify the presence of the pAC-Lyc plasmid, one of the colonies was picked and cultured to see if the bacteria had the distinct orange-red color of the lycopene pigment. So, the colony from the second lane in Figure 3.1a was picked, and grown in LB media with kanamycin and chloramphenicol antibiotic for 48 hours at 25ºC and 250 rpm. The culture after 48 hours of growth is shown in Figure 3.2. There is a clear orange-red color in the culture, therefore, the pAC-Lyc plasmid, along with the pET-28a(+)_SO5plasmid, were considered to be successfully transformed into the same BL21(DE3) bacteria. A glycerol stock from the culture was made and stored at -80ºC for future use.    Figure 3.2: Bacterial culture grown overnight from the pET28a(+)-Mtr plasmid and pAC-Lyc plasmid co-transformation  51  3.3.2 Mtr-Pathway & Lycopene Synthesis Pathway Protein Expression The standard curve from the BSA solutions of known concentration is shown in Figure 3.3. With the standard curve, the absorbance of unknown samples could be measured and the protein concentration could be determined. The standard curve was required for loading the appropriate amount of protein into each well for protein gel electrophoresis.    Figure 3.3: BSA standard curve measured with absorbance at 595 nm   52  The protein gel illustrating the expression levels of soluble and insoluble proteins in BL21(DE3) bacteria, BL21(DE3) cells transformed with the pET-28a(+) plasmid, BL21(DE3) cells transformed with the pAC-Lyc plasmid, and BL21(DE3) cells co-transformed with the pET-28a(+)_SO5 plasmid and pAC-Lyc plasmid is shown in Figure 3.4. The insoluble proteins include membrane bound proteins CymA, CrtB, CrtE, and Ipdi, and the soluble proteins include outer membrane proteins MtrA, MtrB, MtrC, and OmcA.   From the protein gel, it can be seen that there are some differences between each of the lanes, however it is difficult to draw conclusions since natively synthesized proteins were also being expressed in addition to the cloned proteins of interest in the Mtr pathway and lycopene synthesis pathway.   Figure 3.4: Protein gel depicting the insoluble and soluble protein expression of BL21(DE3) with and without cloned DNA 53  These natively synthesized protein could be several essential proteins within the cell such as ribosomal proteins, chaperone proteins, or enzymes. Moreover, there are many bands in each of the lanes from the 40 kDA ladder marker to the 130 kDA ladder marker, so it is hard to determine with any certainty if the genes OmcA, MtrC, MtrB, CrtI, and IpdI are being expressed in the BL21(DE3)-Lyc strain and BL21(DE3)-Mtr-Lyc strain. Additional experiments such as real-time PCR could be beneficial in order to get a more thorough review of the desired proteins being expressed in each strain, while being able to decrease the amount of interference from other proteins being expressed in the system. More on additional protein expression methods is outlined in Chapter 5.  54  Chapter 4: Biogenic Solar Cells 4.1 Introduction To evaluate the effectiveness of the implementation of the genes of the Mtr pathway into bacteria for biogenic solar cell production, the photovoltaic characteristics of strains with and without the Mtr genes were compared and analyzed. The strain used as a baseline for comparison for these experiments was the BL21(DE3)-Lyc bacteria since the strain included the lycopene synthesis pathway that was necessary to generate the photosensitive dye required for electron transfer in the system. The experimental group that was compared to the baseline was the BL21(DE3)-Mtr-Lyc bacteria since the strain included the lycopene synthesis pathway and the Mtr pathway that was hypothesized to increase the performance of the system.  The procedures involved in the experiments were simple – construct biogenic solar cells using the BL21(DE3)-Lyc and BL21(DE3)-Mtr-Lyc strains and evaluate the photovoltaic characteristics using a solar simulator. The first step was to develop the solar cells using the BL21(DE3)-Lyc strain to generate a baseline set of data for comparison. The idea was to make several sets of solar cells using the BL21(DE3)-Lyc bacteria and evaluate their performance using the solar simulator. Once consistent results were achieved with each new set of solar cells being made, the results could be averaged and used as a standard for comparison with the BL21(DE3)-Mtr-Lyc strain.  The same procedures were repeated for the BL21(DE3)-Mtr-Lyc strain. Several sets of solar cells were made until consistent photovoltaic characteristics were seen with each new set of solar cells that were being analyzed. The results could then be averaged and compared with the baseline to see if an increase in photovoltaic performance, most notably the conductivity and efficiency, could be achieved.  55  4.2 Materials and Methods 4.2.1 Culture Conditions The culture conditions for the growth of the BL21(DE3)-Lyc bacterial strain and the BL21(DE3)-Mtr-Lyc bacteria strain were the same conditions that were used for to verify the protein expression. The BL21(DE3)-Lyc strain, only expressing the lycopene synthesis pathway, was grown for 48 hours in 50 mL of 2xYT media and 50 μL of 34 mg/mL stock solution of chloramphenicol antibiotics in an incubator at 25ºC and 250 rpm. Similarly, the BL21(DE3)-Mtr-Lyc strain, expressing both the Mtr pathway and lycopene synthesis pathway, was also grown for 48 hours in 100 mL of 2xYT media in an incubator at 25ºC and 250 rpm, but with 50 μL of 50 mg/mL stock solution of kanamycin antibiotics in addition to 50 μL of 34 mg/mL stock solution of chloramphenicol antibiotics. The BL21(DE3)-Mtr-Lyc strain was immediately induced with 50 μL of 1M IPTG to start transcription and translation of the T7 RNA polymerase which is required to stimulate the expression of the genes of the Mtr pathway. The 48-hour culture time was necessary to allow for sufficient expression of each genes from each pathway.    4.2.2 Cell Extraction The cells from the BL21(DE3)-Lyc culture and the BL21(DE3)-Mtr-Lyc culture were each transferred into a 50 mL falcon tube and centrifuged at 3400 rpm for 10 minutes. The supernatant was discarded, and the pellets were resuspended and washed at 3000 rpm for 10 minutes with 50 mL of double distilled water. The wash step was repeated twice for a total of three washes or until the supernatant was devoid of any coloration. The resulting cell pellets were either used immediately for further experiments, or stored in a refrigerator at 4ºC for use later that same day.   56  4.2.3 Titanium Dioxide Coating The pelleted cells from the BL21(DE3)-Lyc and BL21(DE3)-Mtr-Lyc cultures were resuspended using a pipette and 1 mL of double distilled water before being transferred to their own 0.5mM tryptophan stock solution (20 mL) that was prepared using double distilled water. Next, 5 mg of TiO2 nanoparticles was added to each solution. The TiO2 nanoparticles were purchased from Thermo Fisher Scientific. The resulting mixture for each strain was mixed and shaken on a rotating plate for 2 hours to complete the deposition of the TiO2 nanoparticles over the cells expressing the lycopene dye. After mixing for 2 hours, each mixture was centrifuged at 3000 rpm for 10 minutes and the supernatant was discarded. The resulting pellet was then washed three times with double distilled water by centrifuging at 3000 rpm for 15 minutes each time.      4.2.4 Ultraviolet-Visible Spectroscopy The successful deposition of the TiO2 nanoparticle complex around the lycopene producing cells was verified using ultraviolet-visible (UV-vis) spectroscopy. The analysis was replicated from Srivastava and his biogenic solar cells (Srivastava, 2018). The samples were analyzed at different time frames of the titanium dioxide coating process. The UV-vis analysis was conducted just before the mixture was shaken, during the time it was being shaken, and after the shaking process had finished. The spectrophotometer used was a Perkin Elmer Lambda 25 and the wavelength range was set to scan from 300 to 700 nm. Pure TiO2 samples and pure BL21(DE3)-Lyc samples were also analyzed by the spectrophotometer to help draw conclusions on the absorbance data, and verify the coating of the TiO2 nanoparticles.    57  4.2.5 Solar Cell Fabrication The solar cell fabrication methods were repeated from Srivastava and his biogenic solar cells (Srivastava, 2018). To construct the biogenic solar cells, FTO coated glass segments (50 x 50 x 2 mm) purchased from Sigma Aldrich were used as the conductive glass for each of the electrodes. The FTO glass segments had a surface resistivity of 7 Ω/sq and transmittance of 80%. Before assembling the electrodes, the FTO glass segments were cleaned to ensure that any organic and water-soluble contaminants were removed. The steps involved consecutive sonication for 5 minutes in absolute ethanol, 5 minutes in iso-propanol, 5 minutes in di-chloromethane, and 2 minutes in double distilled water. The FTO glass segments were also cleaned in the same fashion after analysis with the solar simulator was completed. To assemble the anode electrode, the pellet from the TiO2 coating procedure was smeared onto the conductive side of the glass using a doctor-blade method. The anode electrode was then heated at 110ºC for roughly 10 minutes, or until the cells appeared to be hardened on the glass segment, to reduce the water content in the lycopene producing cells. The cathode electrode was made by drop casting a 0.5 mg/mL aqueous solution of graphene oxide purchased from Graphenea onto the conductive side of the other FTO glass segment. The cathode electrode was then heated at 450ºC for roughly 20 minutes or until the solution appeared to be hardened onto the glass segment to reduce the water content. An iodide/tri-iodide electrolyte, Iodolyte HI-30, purchased from Solaronix was used for the system. The electrolyte was drop casted onto the anode electrode and the cathode electrode was then placed on top of the anode electrode, thus, entrapping the electrolyte within the cell. The biogenic solar cell was held together using small binder clips. The biogenic solar cell was then covered in order to minimize lycopene degradation before solar simulator analysis.   58  4.2.6 Solar Simulator Setup A TriSOL solar simulator was used to characterize the photovoltaic properties of the biogenic solar cells. The solar simulator included a 300 W xenon light source powered by a model 6911 Newport power supply. The dimensions of the light output were set to 5 cm x 5 cm. A Newport Oriel correction filter was then used to calibrate the light output to an air-to mass ratio of AM 1.5. Proper calibration was key in order to decrease the spectral mismatch in the 350-700 nm region to smaller than 1.5%. A certified silicone reference cell was used to measure the power output of the lamp, which was roughly 1 Sun or 100 mW cm-2. To obtain the photovoltaic characteristics of the biogenic solar cells, an external potential bias was applied to the cells while the system measured the generated photocurrent with a Keithley digital source meter, model 2400.   4.3 Results and Discussion 4.3.1 Ultraviolet-Visible Spectroscopy Analysis The absorbance data from the UV-vis spectroscopy is shown in Figure 4.1. The absorbance was scanned and calculated within the range of 300 to 700 nm. Recall, the purpose of the UV-vis spectroscopy is to verify the deposition of TiO2 around the lycopene producing cells. Therefore, there were 4 different groups measured in the experiment: The first group was the BL21(DE3)-Lyc bacteria without any addition of TiO2. These cells were taken after being cultured for 48 hours. The second group was the BL21(DE3)-Lyc bacteria mixture with the tryptophan and TiO2 nanoparticles after being shaken for 1 hour. Similarly, the third group was also the BL21(DE3)-Lyc bacteria mixture with the tryptophan and TiO2 nanoparticles, but after being shaken for 24 hours. The final group was simply a mixture of dilute TiO2 in double distilled water.   59   Figure 4.1: Ultraviolet-visible spectroscopy analysis of BL21(DE3)-Lyc cells with and without the addition of TiO2  The shift of the BL21(DE3)-Lyc@TiO2 cells upward from the BL21(DE3)-Lyc cells indicates successful complexation of the TiO2 nanoparticles around the lycopene producing cells. The upward shift of the absorbance can be attributed to the interfacial displacement that materializes during the formation of the bilayer, thus confirming the encapsulation of TiO2 nanoparticles around the bacterial cells.  Additionally, the different between the BL21(DE3)-Lyc@TiO2 cells shaken for 1 hours versus shaken at 24 hours is negligible. Therefore, mixing and shaking for 1 hour appears to be sufficient time to allow for the coating of the cells. The experiment could be 60  repeated with measurements at smaller time intervals to have a better idea of the minimal amount of time needed to fully coat the cells.   4.3.2 Photovoltaic Characterization The current-voltage (I-V) curve used to depict some of the electrochemical measurements for the BL21(DE3)-Lyc biogenic solar cell is shown in Figure 4.2. The I-V curve illustrates the relationship between the applied voltage and the current flow through the system. The I-V curve suggests that there were insignificant differences between light and low light, suggesting acceptable usage under ambient light conditions. The same findings were seen by Srivastava and his first-generation biogenic solar cells (Srivastava, 2018).    Figure 4.2: Current-voltage curve of BL21(DE3)-Lyc biogenic solar cell 61  However, the mechanism explaining why there are insignificant differences between the two light conditions is still not completely understood. It is difficult to develop a thorough understanding of the mechanism since it is a heterogeneous system, and no two solar cells have the exact same composition. Therefore, it is hard to establish energy level diagrams of the system, which would allow for a better understanding of how the intensity and energy levels within the light affect the biogenic solar cells. One postulation that could explain why there is no difference in performance under light and low light conditions could be that the bright light condition has enough intensity to degrade the material too rapidly. Having the materials degrade too quickly, thus having a surplus of electrons, could create a rate of electron transfer that is too high, resulting in more losses and a decrease in performance of the system. On the other hand, the low light intensity could allow for electrons to be shuttled more efficiently since the rate of electron generation is lower. Thus, giving the system ample opportunity to adjust, and resulting in less material degradation, lower losses, and less of a hinderance on the performance of the system. To be clear, the biogenic solar cells are a very complicated, heterogeneous system and these mechanisms are only speculations. There have been no experiments developed to test these hypotheses, so there is future work necessary in order to design the methods and analyses that are required to have a better understanding of the system, which is beyond the scope of the current work.  Additional data for the BL21(DE3)-Lyc biogenic solar cell from the solar simulator showed a direct open-circuit potential (VOC-Direct) of 12.2 mV, and a direct short-circuit current (ISC-Direct) of 1.0 μA. Data could not be produced by the solar simulator for other characteristic data including the open-circuit potential (VOC), short-circuit current (ISC) and the corresponding short-circuit current density (JSC), and the efficiency. These findings suggest that there could be short circuiting 62  within the system, and that troubleshooting and additional trials should be conducted to help address the issues at hand. Suggestions to potentially alleviate these issues are outlined in Chapter 5.  It can be noted that the results generated for Figure 4.2 are only from one set of biogenic solar cells. Many additional biogenic solar cells were made, however, the prior cells appeared to be short-circuiting, so no data was generated by the solar simulator. Additional biogenic solar cells under the same conditions should be made and measured with the solar simulator to have a more comprehensive sense of the electrochemical characteristics. Furthermore, as mentioned earlier, the data for Figure 4.2 was generated by biogenic solar cells made using the BL21(DE3)-Lyc strain. The BL21(DE3)-Mtr-Lyc strain was also used to make several biogenic solar cells, but the same problem of short-circuiting was occurring and no characteristic data was produced. The experiments should be repeated until the issues of short-circuiting are resolved, and enough consistent data has been generated in order to compare the baseline group, BL21(DE3)-Lyc, with the experimental group, BL21(DE3)-Mtr-Lyc. Again, more information on resolving the issues of short-circuiting and how to generate more consistent data is outlined in Chapter 5.    63  Chapter 5: Conclusion and Future Work 5.1 Conclusion The main goal of the research was to investigate how the presence of bacterial nanowires affect the conductivity and efficiency of the biogenic solar cells. Although achieving the main goal of the research may require additional experiments, there were fundamental steps taken to help steer future research in the right direction. Hopefully, these scientific findings may be built upon and the desired goals can be reached in the future.  The major research findings are as follows: As discussed in Chapter 2, the primary objective was to clone the genes that are responsible for bacterial nanowires formation and extracellular electron transfer from SO MR-1 into the pET-28a(+) vector plasmid. These genes were CymA, OmcA, MtrC, MtrA, and MtrB – the five-primary component of the Mtr respiratory pathway. The genes were successfully cloned into the pET-28a(+) vector, and their insertion was verified using colony PCR and Sanger Sequencing.   In Chapter 3, the main goals were to co-transformed the Mtr pathway plasmid and lycopene synthesis plasmid into the same bacterial strain that was well-suited for protein expression, and to verify the protein expression of each of the desired genes. The pET-28a(+)-Mtr plasmid and pAC-Lyc plasmid were co-transformed into BL21(DE3) bacteria for amplified expression. Successful transformation of the pET-28a(+)_SO5 plasmid was verified by colony PCR, and successful transformation of the pAC-Lyc plasmid was verified by culturing selected colonies overnight, and observing an orange-red hue present within the culture indicating that lycopene was being synthesized. To verify the protein expression of the genes of the Mtr pathway and lycopene synthesis pathway, protein gel electrophoresis was conducted. The results from the gel 64  electrophoresis were vague in regards to determining the expression of the desired proteins. The uncertainty may have arisen from other proteins of similar size being natively synthesized, thus, impeding any clear indication of the desired proteins being translated. Other verification methods such as real-time PCR could be implemented in order to detect the expression of specific genes without the hindrance from other genes being expressed.   In Chapter 4, the objectives were to verify the coating of TiO2 nanoparticles around the lycopene producing cells, and to construct biogenic solar cells using BL21(DE3)-Lyc cells and Bl21(DE3)-Mtr-Lyc cells and compare their photovoltaic measurements. UV-vis spectroscopy was used to analyze the deposition of TiO2 over the lycopene producing cells. The absorbance data of the UV-vis scan from a wavelength of 300 to 700 nm showed that the BL21(DE3)-Lyc cells mixed in a tryptophan solution with TiO2 had an upward shift in absorbance when compared to the BL21(DE3)-Lyc cells that were not mixed in the tryptophan solution with TiO2. The upward shift in the absorbance spectra indicates successful complexation of the TiO2 nanoparticles about the BL21(DE3)-Lyc cells. A solar simulator was used for photovoltaic characterization of the biogenic solar cells made with BL21(DE3)-Lyc cells and Bl21(DE3)-Mtr-Lyc cells. The BL21(DE3)-Lyc cells were used to develop a baseline set of data for comparison with the Bl21(DE3)-Mtr-Lyc cells. The solar simulator was able to generate I-V curves for the BL21(DE3)-Lyc cells showing the relationship between current and potential. The solar simulator also showed a direct open circuit potential (VOC-Direct) of 12.2 mV, and a direct short circuit current (ISC-Direct) of 1.0 μA. However, several other photovoltaic measurements could not be produced by the solar simulator including the open circuit potential (VOC), short circuit current (ISC) and the corresponding short-circuit current density (JSC), and the efficiency, indicating that there could be some problems with short-65  circuiting. Additionally, the biogenic solar cells made with Bl21(DE3)-Mtr-Lyc cells were unable to produce I-V curves as well as the other photovoltaic data mentioned above. Therefore, troubleshooting and strategies to address the issues of short circuiting should be examined.   Ultimately, the experimental methods outlined offer a detailed protocol for the optimization of lycopene production and bacterial nanowire formation in E. coli. These experiments have never been done in literature, and can help pave the way for future research to be conducted.   5.2 Future Work 5.2.1 Molecular Cloning and Biogenic Solar Cell Fabrication One possibility for the lack of photovoltaic measurements from the solar simulator and short circuiting could be an inadequate amount of lycopene being produced by the cells. The biogenic solar cells developed by Srivastava (Srivastava, 2018) were made using BL21(DE3) bacteria and two transformed plasmids. The first plasmid was the same pAC-Lyc plasmid, and the second was a pTrc-RDE’ plasmid that significantly over-expressed the production of lycopene within the cell. There is roughly a 10-fold difference between the amount of lycopene production, with and without the pTrc-RDE’ plasmid. Originally, the pET-28a(+)_SO5 plasmid was to be co-transformed into the BL21(DE3) bacteria alongside the pAC-Lyc plasmid and the pTrc-RDE’ plasmid. However, a 3-plasmid transformation has a significantly low transformation efficiency, so it is unlikely that all three plasmids would be integrated at the same time. Additionally, cell stress can hinder protein expression in bacterial hosts. Therefore, having 3 separate plasmids each encoding several different genes and requiring 3 different antibiotics can induced a high amount of metabolic stress on the cells. One potential strategy to genetically engineer each of the genes 66  into the host cells while decreasing cell stress would be to integrate the genes of interest into the bacterial chromosomes from each of the plasmids. With each of the 3 plasmids integrated into the chromosomal DNA, the plasmid would have the genetic machinery required to express and overproduce lycopene, while also being able to produce the proteins used for bacterial nanowire production in the Mtr pathway. Thus, a significantly greater amount of lycopene could be produced, and the conductivity of the cell could theoretically be increased as well, with the aid of the electrically conductive nanowires. Biogenic solar cells could then be constructed with the genes being expressed from the chromosomal DNA, and the photovoltaic measurements could be determined with a solar simulator. These results could be compared with the original two plasmid system of biogenic solar cells made using BL21(DE3) bacteria with the pAC-Lyc plasmid and pTrc-RDE’ plasmid, to validate if the addition of the genes of the Mtr pathway have a significant effect on the photovoltaic characteristics of the system.    5.2.2 Real-time PCR and Electron Microscopy Additional protein expression verification techniques could be used to see if the genes of the Mtr pathway and lycopene pathway are being expressed. One example previously mentioned is real-time PCR for quantification of multiple genes in the BL21(DE3)-Mtr-Lyc cells. Real-time PCR is different from traditional PCR reactions since it measures PCR amplification as the reaction takes place. The constant amplification measurements in real-time make it possible to determine the starting concentration of nucleic acid; whereas, in traditional PCR, the initial concentration of nucleic acid is unable to be calculated since the results are collected after completion of the reaction. Real-time PCR quantifies gene expression with the use of a fluorescent reporter molecule. As the real-time PCR reaction proceeds and the replication of the target DNA increases, the 67  fluorescence emitted from the fluorophore increases as well. Therefore, real-time PCR reactions could be setup for each of the genes of interest from the lycopene synthesis pathway and Mtr pathway. The expression levels could be determined for each specific gene and the analysis would be simplified since any disturbance from other genes in the system would be eliminated.  In addition, to help verify the presence of bacterial nanowires being produced by the BL21(DE3)-Mtr-Lyc cells, as well as showing the porous nature of the material, electron microscopy could be used to obtain high resolution images of the cells. For example, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) would be good methods to obtain clear images of the cells. Each of these methods operates under a vacuum, has an electron source and electron apertures, and controls the shape of the path of the electron beam by a series of electromagnetic and electrostatic lenses. TEM allows for 2-dimension projections of the sample, whereas SEM provides a 3-dimension image of the surface of the sample. Both would be sufficient for the analysis since either a 2-dimension or 3-dimension image would allow one to see if appendages were present on the extracellular surface of the cells.   5.2.3 Viability of the Cells and Solid-State Solar Cell Integration Another goal would be to improve the viability of the cells. The cells used in all of the experiments were in fact dead cells. There was still lycopene within the cells which allowed for the photovoltaic measurements to be analyzed with a solar simulator. However, developing methods that would not kill the bacteria, or allow for the bacteria to be cultured and continuously renewed, would allow for indefinite production of lycopene.  68  Furthermore, since these biogenic solar cells have been showed to work as efficiently in dim light conditions as in bright light conditions, they could be integrated with solid-state photovoltaic cells in order to harness more solar energy under varying light conditions (Srivastava, 2018). Solid-state photovoltaic cells, such as silicone, gallium arsenide, and copper indium selenide have been well developed and have efficiencies that are amongst the highest of all photovoltaic cells (National Renewable Energy Laboratory, 2019). These photovoltaic cells are most efficient under direct sunlight and their efficiency can reduce substantially under overcast or cloudy weather (Premalatha, 2017). The implementation of biogenic solar cells with solid-state photovoltaics working in conjunction to one another would allow the solid-state cells to work at maximum efficiency under bright conditions, and the biogenic solar cells to maintain a higher efficiency than normal when conditions are cloudy. In addition, the integration of the two technologies would make the implementation of solar power more feasible in areas such as British Columbia and parts of Northern Europe where overcast skies are common and the brightness levels in the sky can vary throughout the day.   5.2.4 Shewanella Oneidensis MR-1 Genetic Engineering A final interesting set of experiments would be to create biogenic solar cells out of genetically engineered SO MR-1 cells that are capable of synthesizing lycopene. In the current experiments that were outlined, the bacterial nanowires from SO MR-1 were cloned into E. coli cells that were capable of synthesizing lycopene. The next set of proposed experiments would essentially be the opposite, since SO MR-1, which already has the genes required for nanowires formation, would be cloned with the genes responsible for lycopene synthesis. 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RSC Advances 5.57 (2015): 45755-45759.  76  Appendices  Appendix A  Mtr Pathway Protein Sequences A.1 CymA MNWRALFKPSAKYSILALLVVGIVIGVVGYFATQQTLHATSTDAFCMSCHSNHSLKNEVLASAHGGGKAGVTVQCQDCHLPHGPVDYLIKKIIVSKDLYGFLTIDGFNTQAWLDENRKEQADKALAYFRGNDSANCQHCHTRIYENQPETMKPMAVRMHTNNFKKDPETRKTCVDCHKGVAHPYPKG  A.2 OmcA MMKRFNFNTATKAMLGAGLLSLLLTGCGGSDGKDGEDGKPGVVGVNINSTSTLKAKFTNATVDAGKVTVNFTLENANGVAVLGLTKDHDLRFGIAQLTPVKEKVGETEADRGYQWQAYINAKKEPGTVPSGVDNLNPSTQFQANVESANKCDTCLVDHGDGSYSYTYQVNVANVTEPVKVTYSADATQRATMELELPQLAANAHFDWQPSTGKTEGIQTRNVVSIQACYTCHQPESLALHGGRRIDIENCASCHTATSGDPESGNSIEFTYMIHAIHKGGERHTFDATGAQVPAPYKIIGYGGKVIDYGKVHYPQKPAADCAACHVEGAGAPANADLFKADLSNQACIGCHTEKPSAHHSSTDCMACHNATKPYGGTGSAAKRHGDVMKAYNDSLGYKAKFSNIGIKNNALTFDVQILDNKDQPIGKEFISDPSAYTKSSIYFSWGIDKDYPAYTAGSRYSDRGFALSNSKVSTYNEATKTFTIDSTNSNLKLPADLTGMNVELYAGVATCFNKGGYGVEDVVATPCSTDTRYAYIQDQPFRFKWNGTDTNSAAEKRRAIIDTAKCSGCHNKEIVHYDNGVNCQACHTPDKGLKTDNTYPGTKVPTSFAWKAHESEGHYLKYAGVQSGTVLKTDCATCHTADKSNVVTGIALGRSPERAWLYGDIKNNGAVIWVSSDAGACLSCHQKYLSDAAKSHIETNGGILNGTSAADVQTRASESCATCHTPSQLMEAHGN 77   A.3 MtrC MMNAQKSKIALLLAASAVTMALTGCGGSDGNNGNDGSDGGEPAGSIQTLNLDITKVSYENGAPMVTVFATNEADMPVIGLANLEIKKALQLIPEGATGPGNSANWQGLGSSKSYVDNKNGSYTFKFDAFDSNKVFNAQLTQRFNVVSAAGKLADGTTVPVAEMVEDFDGQGNAPQYTKNIVSHEVCASCHVEGEKIYHQATEVETCISCHTQEFADGRGKPHVAFSHLIHNVHNANKAWGKDNKIPTVAQNIVQDNCQVCHVESDMLTEAKNWSRIPTMEVCSSCHVDIDFAAGKGHSQQLDNSNCIACHNSDWTAELHTAKTTATKNLINQYGIETTSTINTETKAATISVQVVDANGTAVDLKTILPKVQRLEIITNVGPNNATLGYSGKDSIFAIKNGALDPKATINDAGKLVYTTTKDLKLGQNGADSDTAFSFVGWSMCSSEGKFVDCADPAFDGVDVTKYTGMKADLAFATLSGKAPSTRHVDSVNMTACANCHTAEFEIHKGKQHAGFVMTEQLSHTQDANGKAIVGLDACVTCHTPDGTYSFANRGALELKLHKKHVEDAYGLIGGNCASCHSDFNLESFKKKGALNTAAAADKTGLYSTPITATCTTCHTVGSQYMVHTKETLESFGAVVDGTKDDATSAAQSETCFYCHTPTVADHTKVKM  A.4 MtrA MKNCLKMKNLLPALTITMAMSAVMALVVTPNAYASKWDEKMTPEQVEATLDKKFAEGNYSPKGADSCLMCHKKSEKVMDLFKGVHGAIDSSKSPMAGLQCEACHGPLGQHNKGGNEPMITFGKQSTLSADKQNSVCMSCHQDDKRMSWNGGHHDNADVACASCHQVHVAKDPVLSKNTEMEVCTSCHTKQKADMNKRSSHPLKWAQMTCSDCHNPHGSMTDSDLNKPSVNDTCYSCHAEKRGPKLWEHAPVTENCVTCHNPHGSVNDGMLKTRAPQLCQQCHASDGHASNAYLGNTGLGSNVGDNAFTGGRSCLNCHSQVHGSNHPSGKLLQR  78  A.5 MtrB MKFKLNLITLALLANTGLAVAADGYGLANANTEKVKLSAWSCKGCVVETGTSGTVGVGVGYNSEEDIRSANAFGTSNEVAGKFDADLNFKGEKGYRASVDAYQLGMDGGRLDVNAGKQGQYNVNVNYRQIATYDSNSALSPYAGIGGNNLTLPDNWITAGSSNQMPLLMDSLNALELSLKRERTGLGFEYQGESLWSTYVNYMREEKTGLKQASGSFFNQSMMLAEPVDYTTDTIEAGVKLKGDRWFTALSYNGSIFKNEYNQLDFENAFNPTFGAQTQGTMALDPDNQSHTVSLMGQYNDGSNALSGRILTGQMSQDQALVTDNYRYANQLNTDAVDAKVDLLGMNLKVVSKVSNDLRLTGSYDYYDRDNNTQVEEWTQISINNVNGKVAYNTPYDNRTQRFKVAADYRITRDIKLDGGYDFKRDQRDYQDRETTDENTVWARLRVNSFDTWDMWVKGSYGNRDGSQYQASEWTSSETNSLLRKYNLADRDRTQVEARITHSPLESLTIDVGARYALDDYTDTVIGLTESKDTSYDANISYMITADLLATAFYNYQTIESEQAGSSNYSTPTWTGFIEDQVDVVGAGISYNNLLENKLRLGLDYTYSNSDSNTQVRQGITGDYGDYFAKVHNINLYAQYQATEKLALRFDYKIENYKDNDAANDIAVDGIWNVVGFGSNSHDYTAQMLMLSMSYKL 79  Appendix B  PCR Reaction Setup B.1 CymA Insert Thermal Cycling Conditions Table B.1: PCR primers and thermal cycling conditions for the linearization of the pET-28a(+) vector DNA Primer Sequence 5’-3’[1] Tm[2] (ºC) DNA Template pET-28a(+) + cymA F ACGCCAGTTCATGGTATATCTCCTTCTTAAAGTTAAACAAAATTAT 56 pET-28a(+) pET-28a(+) + cymA R TCCAAAAGGATAACTAACAAAGCCCGAAAGGAAG 55-56 pET-28a(+) PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 1:00 1 Denaturation 95 0:20 30 Annealing 51 0:20 Extension 68 4:00 Final Extension 68 10:00 1 [1] Nucleotides in italics are the overhanging sequence of the primer, and the rest is the binding sequence that attaches to the DNA template [2] The melting temperature (Tm) is given for the binding sequence only, excluding the overhanging sequence.   80  Table B.2: PCR primers and thermal cycling conditions for the linearization of the CymA gene insert DNA Primer Sequence 5’-3’[1] Tm[2] (ºC) DNA Template cymA + pET-28a(+) F AAGGAGATATACCATGAACTGGCGTGCACTATTTAAACC 60 SO MR-1 cymA + pET-28a(+) R TCGGGCTTTGTTAGTTATCCTTTTGGATAGGGGTGAGCG 59-60 SO MR-1 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 2:00 1 Denaturation 95 0:20 30 Annealing 55 0:20 Extension 68 0:50 Final Extension 68 5:00 1 [1] Nucleotides in italics are the overhanging sequence of the primer, and the rest is the binding sequence that attaches to the DNA template [2] The melting temperature (Tm) is given for the binding sequence only, excluding the overhanging sequence.       81   Table B.3: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO1 Gibson Assembly DNA Primer Sequence 5’-3’ Tm (ºC) DNA Template f1 origin F GGCCCACTACGTGAACC 57 pET-28a(+)_SO1 LacI R CGACATCGTATAACGTTACTGGT 57 pET-28a(+)_SO1 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 5:00 1 Denaturation 95 0:20 30 Annealing 52 0:20 Extension 68 1:20 Final Extension 68 10:00 1      82  B.2 OmcA Insert Thermal Cycling Conditions Table B.4: PCR primers and thermal cycling conditions for the linearization of the pET-28a(+)_SO1 vector DNA Primer Sequence 5’-3’[1] Tm[2] (ºC) DNA Template pET-28a(+)_SO1 + OmcA F GGTAGATAACAGCATCATCTTTATCCTTTTGGATAGGGGTGAGCGAC 61 pET-28a(+)_SO1 pET-28a_SO1 + OmcA R CACACGGTAACTAAGCCCGAAAGGAAGCTGAGTTG 62 pET-28a(+)_SO1 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 1:00 1 Denaturation 95 0:20 30 Annealing 57 0:20 Extension 68 4:20 Final Extension 68 10:00 1 [1] Nucleotides in italics are the overhanging sequence of the primer, and the rest is the binding sequence that attaches to the DNA template [2] The melting temperature (Tm) is given for the binding sequence only, excluding the overhanging sequence.  83  Table B.5: PCR primers and thermal cycling conditions for the linearization of the OmcA gene insert DNA Primer Sequence 5’-3’[1] Tm[2] (ºC) DNA Template OmcA +  pET-28a(+)_SO1  F CCTATCCAAAAGGATAAAGATGATGCTGTTATCTACCTCAAAAGAAATAGTC 60 SO MR-1 OmcA +  pET-28a(+)_SO1  R CCTTTCGGGCTTAGTTACCGTGTGCTTCCATCAATTG 60 SO MR-1 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 2:00 1 Denaturation 95 0:20 30 Annealing 55 0:20 Extension 68 1:45 Final Extension 68 10:00 1 [1] Nucleotides in italics are the overhanging sequence of the primer, and the rest is the binding sequence that attaches to the DNA template [2] The melting temperature (Tm) is given for the binding sequence only, excluding the overhanging sequence.    84  Table B.6: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO2 Gibson Assembly DNA Primer Sequence 5’-3’ Tm (ºC) DNA Template f1 origin F GGCCCACTACGTGAACC 57 pET-28a(+)_SO2 LacI R CGACATCGTATAACGTTACTGGT 57 pET-28a(+)_SO2 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 5:00 1 Denaturation 95 0:20 30 Annealing 52 0:20 Extension 68 3:00 Final Extension 68 10:00 1        85  B.3 MtrC Insert Thermal Cycling Conditions Table B.7: PCR primers and thermal cycling conditions for the linearization of the pET-28a(+)_SO2 vector DNA Primer Sequence 5’-3’[1] Tm[2] (ºC) DNA Template pET-28a(+)_SO2 + MtrC F CGTCTCTCGATTCAGATAATTATATCGACTTAGTTACCGTGTGCTTCCATCAATTGC 61 pET-28a(+)_SO2 pET-28a_SO2 + MtrC R CACTAAAGTGAAAATGTAAGCCCGAAAGGAAGCTGAGTTG 60-61 pET-28a(+)_SO2 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 1:00 1 Denaturation 95 0:20 30 Annealing 56 0:20 Extension 68 6:00 Final Extension 68 10:00 1 [1] Nucleotides in italics are the overhanging sequence of the primer, and the rest is the binding sequence that attaches to the DNA template [2] The melting temperature (Tm) is given for the binding sequence only, excluding the overhanging sequence.  86  Table B.8: PCR primers and thermal cycling conditions for the linearization of the MtrC gene insert DNA Primer Sequence 5’-3’[1] Tm[2] (ºC) DNA Template MtrC +  pET-28a(+)_SO2  F CGGTAACTAAGTCGATATAATTATCTGAATCGAGAGACGAAAC 62-63 SO MR-1 MtrC +  pET-28a(+)_SO2  R CTTCCTTTCGGGCTTACATTTTCACTTTAGTGTGATCTGCAACTGTTGG 62 SO MR-1 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 2:00 1 Denaturation 95 0:20 30 Annealing 57 0:20 Extension 68 1:45 Final Extension 68 10:00 1 [1] Nucleotides in italics are the overhanging sequence of the primer, and the rest is the binding sequence that attaches to the DNA template [2] The melting temperature (Tm) is given for the binding sequence only, excluding the overhanging sequence.    87  Table B.9: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO3 Gibson Assembly DNA Primer Sequence 5’-3’ Tm (ºC) DNA Template f1 origin F GGCCCACTACGTGAACC 57 pET-28a(+)_SO3 MtrC R GCACACGGTAACTAAGTCGATATAATTATCTG 59 pET-28a(+)_SO3 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 5:00 1 Denaturation 95 0:20 30 Annealing 52 0:20 Extension 68 2:00 Final Extension 68 10:00 1       88  B.4 MtrA & MtrB Insert Thermal Cycling Conditions Table B.10: PCR primers and thermal cycling conditions for the linearization of the pET-28a(+)_SO3 vector DNA Primer Sequence 5’-3’[1] Tm[2] (ºC) DNA Template pET-28a(+)_SO3 + MtrA F GCTTGGGCAAATTACATTTTCACTTTAGTGTGATCTGCAACTG 59 pET-28a(+)_SO3 pET-28a_SO3 + MtrB R GCATGAGTTACAAACTCTAAAGGAAGCTGAGTTGGCTGC 60 pET-28a(+)_SO3 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 1:00 1 Denaturation 95 0:20 30 Annealing 55 0:20 Extension 68 8:00 Final Extension 68 10:00 1 [1] Nucleotides in italics are the overhanging sequence of the primer, and the rest is the binding sequence that attaches to the DNA template [2] The melting temperature (Tm) is given for the binding sequence only, excluding the overhanging sequence.  89  Table B.11: PCR primers and thermal cycling conditions for the linearization of the MtrA & MtrB gene insert DNA Primer Sequence 5’-3’[1] Tm[2] (ºC) DNA Template MtrA +  pET-28a(+)_SO3  F GATCACACTAAAGTGAAAATGTAATTTGCCCAAGC 62 SO MR-1 MtrB +  pET-28a(+)_SO3  R CAACTCAGCTTCCTTTAGAGTTTGTAACTCATGCTCAGCATCAGC 61-62 SO MR-1 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 2:00 1 Denaturation 95 0:20 30 Annealing 57 0:20 Extension 68 2:30 Final Extension 68 10:00 1 [1] Nucleotides in italics are the overhanging sequence of the primer, and the rest is the binding sequence that attaches to the DNA template [2] The melting temperature (Tm) is given for the binding sequence only, excluding the overhanging sequence.   90  Table B.12: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO5 Gibson Assembly DNA Primer Sequence 5’-3’ Tm (ºC) DNA Template f1 origin F GGCCCACTACGTGAACC 57 pET-28a(+)_SO5 LacI R CGACATCGTATAACGTTACTGGT 57 pET-28a(+)_SO5 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 5:00 1 Denaturation 95 0:20 30 Annealing 52 0:20 Extension 68 7:00 Final Extension 68 10:00 1     91  B.5 Mtr Pathway Co-Transformation Thermal Cycling Conditions  Table B.13: PCR primers and thermal cycling conditions for the Colony PCR verification of the pET-28a(+)_SO5 plasmid co-transformation DNA Primer Sequence 5’-3’ Tm (ºC) DNA Template f1 origin F GGCCCACTACGTGAACC 57 pET-28a(+)_SO5 LacI R CGACATCGTATAACGTTACTGGT 57 pET-28a(+)_SO5 PCR Step Temperature  (ºC) Time  (MM:SS) Number of Cycles Initial Denaturation 95 5:00 1 Denaturation 95 0:20 30 Annealing 52 0:20 Extension 68 7:00 Final Extension 68 10:00 1  92  Appendix C  Sanger Sequencing C.1 Verification for CymA Insert Table C.1: Nucleotide BLAST of GENEWIZ Sanger Sequencing vs SnapGene simulation for CymA Insert   [1] Query represents the pET-28a(+)_SO1 plasmid nucleotide sequence from SnapGene simulation [2] Subject represents the GENEWIZ Sanger Sequencing nucleotide sequence from the pET-                    28a(+)_SO1 plasmid using the GENEWIZ T7 TERM universal primer  93  C.2 Verification for OmcA Insert Table C.2: Nucleotide BLAST of GENEWIZ Sanger Sequencing vs SnapGene simulation for OmcA Insert  [1] Query represents the pET-28a(+)_SO2 plasmid nucleotide sequence from SnapGene simulation [2] Subject represents the GENEWIZ Sanger Sequencing nucleotide sequence from the pET-                    28a(+)_SO2 plasmid using the GENEWIZ T7 TERM universal primer 94  C.3 Verification for MtrC Insert Table C.3: Nucleotide BLAST of GENEWIZ Sanger Sequencing vs SnapGene simulation for MtrC Insert  [1] Query represents the pET-28a(+)_SO3 plasmid nucleotide sequence from SnapGene simulation [2] Subject represents the GENEWIZ Sanger Sequencing nucleotide sequence from the pET-                    28a(+)_SO3 plasmid using the GENEWIZ T7 TERM universal primer 95  C.4 Verification for MtrA & MtrB Insert Table C.4: Nucleotide BLAST of GENEWIZ Sanger Sequencing vs SnapGene simulation for MtrA & MtrB Insert  [1] Query represents the pET-28a(+)_SO5 plasmid nucleotide sequence from SnapGene simulation [2] Subject represents the GENEWIZ Sanger Sequencing nucleotide sequence from the pET-                    28a(+)_SO5 plasmid using the GENEWIZ T7 TERM universal primer 

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