Difference between revisions of "Part:BBa J64001"

Revision as of 22:45, 21 October 2021

psicA from Salmonella

The sicA promoter is a regulatory element in the Salmonella SPI-1 secretion system. It controls the expression of secreted effector proteins in the natural system and turns on late. Please reference Temme et al, 2008, JMB for exact dynamics of this promoter and associated feedback mechanisms. This promoter only functions in Salmonella strains containing SPI-1.

Sequence and Features

British_Columbia team's documentation

Biology of psicA

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 (Temme et al, 2008). 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 (Lou et al., 2019). As a virulence gene, it is of high interest of 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 (Ma et al. 2010). 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.

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 <a href="https://www.idtdna.com/pages/support/faqs/should-i-amplify-my-gblocks-gene-fragments-when-i-receive-them-">IDT recommends against this action</a> 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.

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 143 bp size of the psicA gblock. This confirms the presence of the psicA gblock part in preparation for Goldengate assembly of the construct.

References

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. 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.