GENETIC MANIPULATION AND CULTURING OF AZOTOBACTER VINELANDII FOR THE PRODUCTION OF NITROGENASE FOR USE IN PROTEIN-ENGINEERED ELECTROCHEMICAL SYSTEMS by ROYCE D. DUDA Submitted in partial fulfillment of the requirements for the degree of Master of Science Chemical and Biomolecular Engineering CASE WESTERN RESERVE UNIVERSITY August, 2018 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Royce D. Duda candidate for the degree of Master of Science *. Committee Chair Prof. Julie Renner Committee Member Prof. Harihara Baskaran Committee Member Prof. Heidi Martin Date of Defense June 20, 2018 *We also certify that written approval has been obtained for any proprietary material contained therein. 1 Table of Contents List of Tables ......................................................................................................................4 List of Figures .....................................................................................................................5 Acknowledgements ............................................................................................................7 Abstract ...............................................................................................................................9 Chapter 1: Introduction ..................................................................................................10 1-1 Importance of Ammonia Production, The Haber-Bosch Process, and Electrochemical Ammonia Production .........................................................................11 1-2 Review of the Nitrogenase Enzyme .........................................................................14 1-3 Utilization of the Nitrogenase Enzyme in Photo- and Electrochemistry .................17 1-4 Challenges in the Modification of the Nitrogenase Enzyme in A. vinelandii ..........19 Chapter 2: Experimental Methodology .........................................................................24 2-1 Culturing and Derepression of A. vinelandii ............................................................24 2-2 Preparation of Nitrogenase ......................................................................................25 2-3 Nitrogenase Acetylene Reduction Activity Assay ...................................................28 2-4 Competence and Transformation into A. vinelandii ................................................30 2-5 Modification of the pCRISPR vector in E. coli .......................................................33 Chapter 3: Preparation of MoFe Nitrogenase ...............................................................34 3-1 Derepression of the nifHDK operon in A. vinelandii ...............................................34 3-2 MoFe Nitrogenase Purification ................................................................................36 2 Chapter 4: Incorporation of the CRISPR-Cas9 System into A. vinelandii .................41 4-1 Technical Approach .................................................................................................41 4-2 Cloning Designed DNA Spacer into the pCRISPR Plasmid ...................................46 4-3 Transformation and Replication of the pCRISPR and pCas9 Plasmids into A. vinelandii ........................................................................................................................49 4-4 Conclusions and Future Work .................................................................................54 Chapter 5: Summary and Conclusions ..........................................................................55 Appendices ........................................................................................................................57 Appendix A: Widening Opportunities for Women in Science Program ........................57 A-1 Introduction: Women in Stem Fields ......................................................................... 57 A-2 Biomolecular Cloning in Escherichia coli Curriculum for the WOWS Program .................................................................................................................................. 58 A-3 WOWS Survey Results ............................................................................................... 59 Appendix B: WOWS Program Documents ....................................................................64 Appendix C: Azotobacter vinelandii and Nitrogenase Laboratory Manual ...................76 Appendix D: A. vinelandii Genomic DNA Repair Fragment Sequence ......................100 Appendix E: Cloning of CRISPR Spacer into pCRISPR Plasmid Sequencing Results ..........................................................................................................................102 Bibliography ..................................................................................................................111 3 List of Tables Table 1-1 Basic components and functions of the nitrogenase enzyme. (p. 15) Table 3-1 Gas chromatography results from the derepressed A. vinelandii acetylene reduction activity assay. (p. 35) Table A-1 Schedule for the E. coli cloning curriculum for the WOWS program. (p. 59) 4 List of Figures Figure 1-1 Representation of the nitrogenase enzyme and the electron-transfer pathway contained within. (p. 17) Figure 1-2 Flowsheet demonstrating the overall steps toward creating a protein-engineered nitrogenase and the testing of this enzyme on an electrode surface. (p. 23) Figure 2-1 Schematic of the glassware setup utilized for acetylene production. (p. 29) Figure 2-2 (A) Growth of Azotobacter vinelandii cells on modified Burk agar plates containing NH4OAc. (B) Growth of iron and molybdenum-starved competent A. vinelandii. (p. 31) Figure 3-1 SDS-PAGE gel confirming the purity of MoFe nitrogenase. (p. 37) Figure 3-2 Calibration curve of the concentration of bovine serum albumin standards along with the concentration of the MoFe proteins determined by the Biuret method. (p. 39) Figure 4-1 Methods used to immobilize MoFe nitrogenase onto an electrode surface including (A) with an immobilization polymer and (B) through the potential use of protein- engineered nitrogenase. (p. 43) Figure 4-2 Mechanism of genetic modification in A. vinelandii with the pCRISPR-pCas9 system. (p. 45) Figure 4-3 (A) Sequences of the location of interest of the pCRISPR plasmid and the pCRISPR spacer insert. (B) Cloning scheme for the insertion of the pCRISPR spacer into the digested pCRISPR plasmid. (p. 48) 5 Figure 4-4 Results of agarose gel electrophoresis showing (A) the full digestion of the pCRISPR plasmid and (B) incomplete digestion of the pCRISPR plasmid. (p. 49) Figure 4-5 Growth of A. vinelandii cultures in chloramphenicol and kanamycin after transformation of pCRISPR and pCas9 plasmids. (p. 51) Figure 4-6 Growth of A. vinelandii cultures in media containing varied concentrations of chloramphenicol. (p. 52) Figure A-1 Average responses to survey questions by students in the WOWS Program from before and after participation in the program. (p. 60) Figure A-2 Average responses to survey questions about career opportunities for chemical engineering majors by students in the WOWS Program from before and after participation in the program. (p. 61) Figure A-3 Average responses to survey questions about the WOWS program by student participants after participation in the program. (p. 62) 6 Acknowledgements I would like to express sincere gratitude to my advisor, Dr. Julie Renner, for her consistent support throughout the process of creating this document. Her patience, knowledge, and availability were instrumental to my completion of this thesis. I thank her for providing me the opportunity and resources to conduct research that helps to address some of the world’s most pressing challenges. I would also like to thank Dr. Heidi Martin and Dr. Harihara Baskaran for being on my thesis committee and for their suggestions and advisement during my time at Case Western. I also thank Dr. Mohan Sankaran for his assistance on the day of my thesis defense. I would like to thank a large group of fellow graduate students, including Chuck Loney, Zhiqiang Zhong, Zihang Su, Chul-Oong Kim, and Nuttanit Pramounmat who would always bring a smile to my face on even the most frustrating days. Students in other groups were also immensely helpful as well, including Joseph Toth and Ryan Hawtof, who were important in aiding me in collecting important data for this document. I would like to thank some members of the Case Western Reserve University staff. I would not have been able to purchase the necessary equipment for this work without Jennifer Pyles and Nichole Thomas, and I would not have been able to set up the equipment without the help of Laurie Dudik and especially Bill Marx. I would also like to thank Evan Guarr, who was very helpful in collecting data for this document. I would like to thank Dr. Shelley Minteer, Dr. Ross Milton, and Rong Cai for their generosity in inviting me to learn from them. Without their knowledge and willingness to share, I would not have been able to prepare a thesis in this interesting field of research. 7 In addition to those who have helped me while at Case Western Reserve University, I would like to thank the mentors who helped guide me to this point. I would like to thank Professor B. Wayne Bequette and Professor Yuri Gorby for helping to cultivate my interest in research. Diana Prout and Donnamarie Vlieg were also mentors to me who helped nurture my curiosity and love of science