Team:British Columbia/Contribution

UBC iGEM 2021

EXPERIMENTS

Contributions


psicA and the regulatory network of the Salmonella Pathogenicity Islands


The sicA promoter is an important regulatory element in the Salmonella Pathogenicity Island 1 (SPI-1) type III secretion protein system that controls the expression of secreted effector proteins that is involved in the invasion of host cells [1]. SicA and InvF complexes, in particular, are required for effective transcription of sicA operons, which then activates expression of Salmonella enterica serovar Typhimurium virulence genes [2]. As a virulence gene, it is of high interest to researchers who study Salmonella pathogenicity and virulence mechanisms for better understanding human immunology responses and evasion of pathogen invasion.


psicA's response to TNFa


Due to its high research potential, molecules that induce activation of the sicA operon promoter have been previously studied [3]. In this study, it was found that upregulation of sicA occurred in the presence of TNFa, identifying it as a promoter that is allegedly inducible by a molecule that, according to our Integrated Human Practices interviews, could be indicative of immune activity in the tumour. To first test induction of psicA by TNFa as a proof-of-concept, we proposed to construct a plasmid that used psicA to drive expression of the reporter protein RFP. To test the function of this construct, the following protocol was proposed from our Experimental Design page:


Day before the experiment was run:

  • Salmonella will be grown under the following conditions: non-agitated microaerophilic bacterial cultures prepared by inoculation of 10 ml of Luria-Bertani broth with 0.01 ml of a stationary phase culture with varying TNFa concentrations (0ng/mL, 2ng/mL, 4ng/mL, 6ng/mL 8ng/mL, and 10ng/mL), followed by overnight incubation (~18 h) at 37°C.

Day of the experiment:

  • Overnight cultures of bacteria will be concentrated 33-fold in Hank’s balanced salt solution (HBSS) supplemented with 10 mM HEPES, pH 7.4. The overnight cultures from the TNFa pretreated Salmonella strains will be washed thoroughly with HBSS three times to get rid of potential TNFa residue in the media. The bacteria will then be resuspended in fresh HBSS for cell lysis.
  • Next, cell-lysed bacteria will be loaded onto a 96 well plate using a multichannel pipettor, and fluorescence readout will be measured using a plate reader. Even though we used psicA from gBlock synthesis, we have added the above documentation in the literature about psicA function and induction by TNFa to the psicA part on the iGEM registry.

Though we did not reach the final steps of circuit construction and circuit characterization with this plan, we effectively amplified the ordered psicA gBlock to be used in our future circuit construction. Even though IDT’s gBlocks are sent at the concentration appropriate for downstream reactions such as PCR and Golden Gate, we successfully performed PCR on the gBlock itself since we used up the 250ng sent to us in circuit construction attempts. Given that IDT recommends against this action since gBlocks are extremely small and hence PCR has very low efficiency, the fact that our PCR fragment was the correct size was a feat.

For future iGEM teams who would like to do the same for their own purposes, our method of gBlock PCR is described in our Protocols page under “PCR”. For this specific PCR, the number of cycles was limited to 10 due to the small size.

PCR amplification of psicA gblock fragment (143bp)
PCR amplification of psicA gblock fragment (143bp)

Figure 1. PCR amplification of the psicA gblock was successful. In lane 1, the NEB 1 kb plus DNA ladder is observed. Lanes 2 and 3 contain the amplified psicA gblock. Lane 3 is a replicate amplification. As labelled in the figure, the DNA ladder confirms the 143bp size of the psicA gblock. This confirms the presence of the psicA gblock part in preparation for Golden Gate assembly of the construct.


[1] Temme, K., Salis, H., Tullman-Ercek, D., Levskaya, A., Hong, S. H., & Voigt, C. A. (2008). Induction and relaxation dynamics of the regulatory network controlling the type III secretion system encoded within Salmonella pathogenicity island 1. Journal of molecular biology, 377(1), 47-61.

[2] Lou, L., Zhang, P., Piao, R., & Wang, Y. (2019). Salmonella pathogenicity island 1 (SPI-1) and its complex regulatory network. Frontiers in Cellular and Infection Microbiology, 9.

[3] Ma, J., Zhang, Y. G., Xia, Y., & Sun, J. (2010). The inflammatory cytokine tumor necrosis factor modulates the expression of Salmonella typhimurium effector proteins. Journal of Inflammation, 7(1), 1-14.