The first step in our experiments was to perform a csgA knockout (KO) using the lambda red method outlined by Datsenko and Wanner [1]. The purpose of the csgA KO is to replace the native csgA gene with our modified csgA-SpyTag plasmid construct.
csgA KO Protocol: https://drive.google.com/file/d/1P-U4E-h5uwOKAzR4gf0AbFunBmjLYv-z/view?usp=sharing
Revised csgA knockout Protocol: https://drive.google.com/file/d/10Zagbu3OgQ0deAOk_LXAEj10ORWVhXyk/view?usp=sharing
We planned to co-transform csgA-SpyTag-GFP and SpyCatcher-nrtA-His into ΔcsgA mutants. We created 5 different variations of csgA-SpyTag and SpyCatcher-nrtA plasmids to test. Plasmid components are based off of existing iGEM biobrick parts designed by Marburg 2015.
Plasmids 1, 3, and 4 act as controls to verify the correct expression of csgA-SpyTag, SpyCatcher, and SpyCatcher-nrtA. CsgA production in plasmids 1 and 2 would have been verified with Congo Red (CR) dye, which stains amyloid fibers. Successful transformations of plasmids 1, 2, and 3 would have been verified through detecting GFP fluorescence in transformed colonies. The His tag in plasmids 3, 4, and 5 would have been used in a Western blot to verify that the correct construct was produced. A Western blot would also have been used to verify the binding of SpyTag with SpyCatcher, since we know the size of the entire csgA-SpyTag-GFP-SpyCatcher-nrtA-His construct.
The narG CC(-40.5) promoter was characterized by transforming Plasmid 2 into E. Coli DH5-A using the protocol outlined by New England Biolabs [2], then growing overnight cultures and using a microplate reader as outlined in the protocol we devised. Our goal was to determine the impact of varying concentrations on protein production, which was directly proportional to the amount of fluorescence we measured.
References
[1] K. A. Datsenko and B. L. Wanner, “One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products,” Proceedings of the National Academy of Sciences, vol. 97, no. 12, pp. 6640–6645, Jun. 2000, doi: 10.1073/pnas.120163297.
[2]“High Efficiency Transformation Protocol (C2987H/C2987I).” NEB, https://www.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987.
[3] Hothersall, J., Lai, S., Zhang, N., Godfrey, R. E., Ruanto, P., Bischoff, S., Robinson, C., Overton, T. W., Busby, S. J. W., & Browning, D. F. (2022). Inexpensive protein overexpression driven by the NarL transcription activator protein. Biotechnology and Bioengineering, 119, 1614– 1623. https://doi.org/10.1002/bit.28071
Thanks for the collaborator teams and the sponsor of our university
