Plasmid Design & Construction

We designed these plasmids to engineer our target bacteria. Including 3 plasmids for knockout vfr, exoS, and exoT to ensure the safety, pBAD33-VgrG3 plasmids for engineering V. cholerae, and pPSV37-PA0085(Hcp1), pPSV37-PA0093(Tse6) plasmids for engineering P. aeruginasa.

PCR and generation homologous arms of vfr, exoS, exoT

After getting the primers from a company, we used the DNA of PAO1 as a template and did PCR to get the homologous arms we needed. Then, we used agarose gel electrophoresis to test the size of the product, which should be ~1kb, to ensure that the product is correct. After that, we use the Nucleic Acid Extraction and Purification kit to purify the DNA and test the concentration of the DNA.

Knockout plasmid construction and verification

We generated the plasmid we needed through the method of Gibson Assembly and transformed the plasmids into T-Fast. Then we used PCR and agarose gel electrophoresis to verify that the plasmid was successfully constructed (if successful, the size of the product should be ~2kb). The plasmids were then transformed into WM6026. And later we tested whether the plasmids were successfully transformed using antibiotic plates.


Figure 7 vfr knock out test

Figure 8 exoT knock out test

Figure 9 exoS knock out test

After the above process, we used homologous recombination to knock out the exoS, exoT, vfr genes in the PAO1. Here we used agarose gel electrophoresis to test the size of the DNA and checked whether the toxic genes (exoS, exoT, vfr) between the homologous arms were knocked out. If they are knocked out, the size should be 2 kb.

Test of T6SS assembly of the toxic genes knocked out strains

Figure 10 toxic genes knocked out strains.

Then we fused Fluorescence molecules sfGFP to the tssB1 and used the microscope to test the T6SS activity of the toxic genes knocked out strains.

Expression plasmids construction

Even though we intended to construct several expression plasmids, the small DNA fragments were difficult to fuse with the vectors. Etelcalcetide seemed to have toxicity to the PAO1 since cell death was observed after being mated with WM6026. At last, KLA, Somatotropin, Dulaglutide, and Dulaglutide genes could be fused with tse6; Minihepcidin genes could be fused with hcp1. For vgrG3, Dulaglutide, KLA, Parkin and Tenecteplase genes can successfully fuse (we only showed the results of Parkin and Tenecteplase since we directly sequenced others’ sequences).

Western blot analysis of the expression and secretion of the fused proteins

Figure 15 cycle 0 Western blot analysis

Figure 16 cycle 0 Western blot analysis

The expression and secretion of the fusion protein were detected by Western blot. In the first analysis, Only Hcp1-Minihepcidin (~19 kDa) was successfully expressed and detected in the engineered strain PAO1 but was not successfully secreted. While for VgrG3 fused proteins (VgrG3-Dulaglutide 106 kDa, VgrG3-KLA 78 kDa, VgrG3-Parkin 128 kDa, VgrG3-Tenecteplase 135 kDa), all of them could be expressed. However, we detected signals in the supernatant from T6SS negative strains, which suggested the inducing condition might be inappropriate.

Figure 17 cycle 1 Western blot analysis

The result indicates the RpoB antibody might be insufficient since the signal inside cells was low. The detection of the signal in supernatant still suggests that the inducing condition was inappropriate.

Figure 18 cycle 2 Western blot analysis

Compared with last cycle, the RpoB signal is indeed better. However, the Hcp fused proteins can only be detected in cells. Even though the Pseudomonas aeruginosa lysed (since we detected the RpoB signal in the supernatant), we still couldn't detect the signal of FLAG. This may suggest that P. aeruginosa may be difficult to secrete fused protein. As for Vibrio cholerae, we found that our plasmids can be expressed stably since we can detect the signals. However, the induce conditions were not good enough to prevent all from cell lysis in the supernatant.