We conducted experiments to knockout the csgA gene in curli-producing E. coli strains in order to transform our own, modified csgA with the attached SpyTag peptide sequence. We verified a successful knockout through selection on chloramphenicol plates (Figure 1A). The knockout was further verified with a colony PCR using colonies from successful transformants and Lambda Red Knockout check primers (Figures 1B and Table 1).
Figure 1. Successful knockout of the csgA and ackA genes in Escherichia coli. A) Plates 2-4 are ∆csgA of strains of MC4100, MG1655, and MC4100Z1 respectively and plate 1 is ∆ackA for E. coli MC4100, used as a positive control. All plates are LB + cm10. B) Gel electrophoresis on colony PCR products
After multiple rounds of troubleshooting, the following protocol detailed in the was determined to be the most effective:
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
The knockout protocol was modified slightly from Datsenko and Wanner’s original protocol by growing cells to OD600 = 0.4 rather than OD600 = 0.6 and selecting for transformants on LB + Cm10 plates. We also modified the size of electroporation cuvettes to 2mm and experimented with different glycerol wash steps for creating electrocompetent cells. Selecting for transformants on plates with a lower antibiotic concentration and for over 24 hours ultimately resulted in a successful knockout.
We verified a successful transformation of our SpyCatcher_GFP-His plasmid (Table 2). Verification was done through transforming E. coli DH5α and measuring fluorescence of the transformants.
We designed 5 different plasmids containing variations of csgA-SpyTag and nrtA-SpyCatcher with and without a nitrate-sensitive promoter (narG CC). These designs were modeled using existing iGEM biobrick parts mentioned by iGEM Marburg 2015, who also used the SpyTag/SpyCatcher system, and modified through cross-referencing PDB, UniProt, and Microbesonline. We successfully ordered and had these plasmids synthesized, but due to time constraints, we were unable to verify successful transformations of all plasmids. Given time, we would like to have transformed and characterized all five of our designed plasmids:
Table 2. Plasmid variants of csgA-SpyTag and nrtA-SpyCatcher with and without the narG CC (nitrate sensitive) promoter. Individual plasmid components were based off of existing iGEM parts.
Nitrate sensitivity was characterized by transforming E. Coli strain DH5-Alpha with plasmid 3. The resulting plate was then grown in an overnight culture twice, with sodium nitrate concentrations ranging from 0-20 (and also 0-200) mM added to them to observe the impact on protein production. Those cultures were then placed into wells, where a plate reader measured fluorescence.
However, astonishingly, we noticed that GFP fluorescence decreased with the increased addition of sodium nitrate. The rate of decrease tends to increase from 5-20 mM, but then decreases from 50-200 mM.
While our results were contrary to what was expected, the promoter was still nitrate sensitive and aerobic, and our efforts showed the importance of the choice of vector in the promoter’s effectiveness.
Table 3 and 4 Results from adding 50-20 mM and 5-10 mM sodium nitrate to the culture, respectively. Left is 10x dilution, right side is 20x dilution.
Figure 2. Changes in GFP fluorescence from adding increasing amounts of Sodium Nitrate (50-200 mM). Yellow is 10x dilution, Gray is 20x dilution.
Figure 3. GFP Fluorescence as a function of increasing concentrations of Sodium Nitrate (0-20 mM). Blue is 20x dilution, Red is 10x dilution.
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.
Thanks for the collaborator teams and the sponsor of our university
