Exploring the TAT protein transport pathway using TatC CRISPR-mediated mutants in Cyanobacteria.

Cyanobacteria are model organisms to study protein sorting and transport because of the presence of both the thylakoid and plasma membranes. This experiment investigates if the tatC gene is vital to the functioning of the TAT pathway as a whole in Cyanobacteria by using knockout mutations. The tatC gene knockout mutants are made using CRISPR-mediated plasmids ligated via a Gibson Assembly, cloned in E. coli, and transformed into Synechocystis PCC 6803. The cyanobacteria cells are grown in BG-11 media with chloramphenicol over multiple generations. The genomic DNA is extracted and used in conformation PCRs to ensure the correct transformation of the mutant cyanobacteria. The mutant cells with the knockout present as albino, whereas the native cells are the typical green. The PCR results and phenotypic changes in the mutant cells indicate that the TAT pathway is not functional without the TatC gene. Repression studies should be conducted as a next step to identify what levels of TatC gene expression are required for the pathway to function. Additionally, similar methodology should be used to investigate the other TAT components present in Synechocystis PCC 6803. 

How does the TAT pathway allow certain proteins end up in different membranes in Cyanobacteria and what role does the tatC gene play in the TAT pathway? 

1. Create CRISPR primers that knockout the tatC gene in pSEVA-Cfp1 plasmid

2. Ligate edited plasmid via Gibson Assembly

3. Transform plasmids into E. coli for cloning

4. Isolate plasmids with Promega purification kits

5. Transform into cyanobacteria via electroporation

6. Grow in culture over generations

7. Isolate genomic DNA

8. Run PCR and gel electrophoresis experiments

9. Analyze results to determine future directions

The combination of the absence of bands, which indicate the tatC gene, in the albino mutants and the presence of bands in the green mutants and controls indicates that the tatC deletion was successful. Futher experimentation is needed to confirm. 

The combination of PCRs that confirm the absence of the TatC gene and the phenotypic change in the mutant cells allow us to infer that the absence of the TatC gene disrupts the function of the TAT pathway. This is significant because the TAT pathway is vital to the cell's function, but it's mechanisms have not been thoroughly elucidated. These results provide a basis for further research into the mechanisms of the TAT pathway, which could have important insights on overall protein transport and protein translocation and implications for future biotechnical production using Synechocystis PCC 6803. 

To conclude this study, an alternate constitutive control needs to be identified and tested so that we can positively identify the mutants as Synechocystis PCC 6803. Whole genome sequencing can be done to positively confirm the gene knockout in the mutants. After confirmation of the knockout, functional assays should be done to determine if the photosynthetic activity is altered in the mutants. These experiments should confirm that the link between the tatC gene absence and inactivation of the TAT pathway results in loss of thylakoid function. 

Moving forward, other types of genetic editing techniques could be useful for exploration of TAT genes, including repression studies to determine at what levels the tatC gene must be expressed for the TAT pathway to function. Additionally, this type of work should be continued with knockout mutants of TatA1 and TatA2 genes to elucidate protein sorting and transport mechanisms within the TAT pathway.

We'd like to thank the Dabney-Smith Research Lab and Miami University for their support. 

1. Baldanta S, Guevara G, Navarro-Llorens JM. SEVA-Cpf1, a CRISPR-Cas12a vector for genome editing in cyanobacteria. Microb Cell Fact. 2022;21(1):103.

2. Ungerer J, Pakrasi HB. Cpf1 Is A Versatile Tool for CRISPR Genome Editing Across Diverse Species of Cyanobacteria. Sci Rep. 2016;6(1):1-9.

3. Frain KM, van Dijl JM, Robinson C. The twin-arginine pathway for protein secretion. EcoSal Plus. 2019;8(2). doi:10.1128/ecosalplus.ESP-0040-2018

4. Frain KM, Gangl D, Jones A, Zedler JAZ, Robinson C. Protein translocation and thylakoid biogenesis in cyanobacteria. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2016;1857(3):266-273.

5. Niu TC, Lin GM, Xie LR, et al. Expanding the Potential of CRISPR-Cpf1-Based Genome Editing Technology in the Cyanobacterium Anabaena PCC 7120. ACS Synth Biol. 2019;8(1):170-180.

6. Harrison DJA, Thompson EP. A rapid and low-cost method for genomic DNA extraction from the cyanobacterium Synechocystis. Biol Methods Protoc. 2020;5(1):bpaa011. 

Anya - During my time in the CDS lab, I have learned many valuable skills that will be useful beyond the classroom. One of the most important has been critical thinking, which has helped me analyze problems and find solutions more effectively. I have also developed strong teamwork skills by working together and supporting each other. In addition, I gained experience using technology, which is an essential skill in today’s growing job market.

Maya - The NACE career ready competencies I have gained while working in the CDS lab are Career + Self-Development, Critical Thinking and Communication. I have developed Career + Self-Development skills as my experience in the lab will be useful in my future career. I have exercised Critical Thinking by troubleshooting experimental issues and analyzing and interpreting data. Additionally, I have gained experience in Communucation through lab presentation on research updates, as well as communicating with my groupmates about experimental procedures and research goals.   

Mike - I have learned the career readiness skills of Teamwork, Technology, and Communication through my research this semester. I have learned teamwork through working with my graduate assistant Victory and Maya, a senior, in my lab. I have to do what they ask and I help them with the procedures that we do in the lab. I have also learned communication with them because I have to communicate with them on what times I need to come into the lab. I have learned the most about technology because I work with PCR machines and gel electrophoresis every time I go into the lab.