Team:SIAT-SCIE/Results

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Results

1. Testing torA protein

The signal peptide torA increases the amount of cjCas9 protein in the cytoplasm that can be transferred to the periplasm, thereby increasing the likelihood that cjCas9 is wrapped by OMV.
In order to test the expression and function of torA, GFP-mut3 was used instead of cjCas9 since we can directly observe the effect of torA by gauging the fluorescence level in cytoplasm and periplasm respectively to determine the amount of GFP-mut3 protein successfully transferred to periplasm, which is indicative of the efficiency of torA. The coding sequence of torA signal peptide precedes the coding sequence of cjCas9.
We performed Osmoshock in order to separate the cytoplasm and periplasm of GFP-mut3- sequence-carrying bacteria. The result of the fluorescence test in Figure 1.1 and 1.2 shows that the torA signal peptide significantly increases the efficiency of the transportation of GFP-mut3 into the periplasm in both strains of bacteria.
This indicates that torA signal peptide is capable of helping transport proteins such as cjCas-9 into the periplasm, which is the step that just precedes cjCas-9’s encapsulation by OMVs.

2. Increased production of OMV by using ompA-knockout bacteria

In order to increase the amount of OMV produced in the periplasm to optimize our design, the outer membrane protein A precursor (ompA) is knocked out to induce hyper vesiculation. The result from Figure 2 indicates that in both experiments, the ΔPF/TF value of BW25113, which is a common strain of E.coli, is larger than that of ompA-knockout BW25113. This reflects that the ompA-knockout bacteria produced more OMVs, which may wrap the GFP-mut3 protein in the periplasm, thereby causing a decrease in a measurement of fluorescent value in the periplasm.

To further prove that the GFP can be successfully wrapped by OMV, we isolate the OMV in periplasm after osmoshock and measure the fluorescent value in it. The positive result in the graph reflects that our hypothesis is correct, and indicates that OmpA knock-out bacteria will produce more OMVs than normal bacteria since the fluorescent value is higher in it.

Reference [1]: Yamada, M., Watanabe, Y., Gootenberg, J. S., Hirano, H., Ran, F. A., Nakane, T., . . . Nureki, O. (2017). Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems. Molecular Cell, 65(6).
[2]: Jmol: an open-source Java viewer for chemical structures in 3D. http://www.jmol.org/
[3]: Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: Biogenesis and functions. Nature Reviews Microbiology, 13(10), 605-619.
[4]: Huai, C., Li, G., Yao, R., Zhang, Y., Cao, M., Kong, L., . . . Huang, Q. (2017). Structural insights into DNA cleavage activation of CRISPR-Cas9 system. Nature Communications, 8(1).