Team:UI Indonesia/Contribution

Contribution

Universitas Indonesia 2021 iGEM team created a project focused on H pylori eradication treatment using an engineered E. coli Nissle 1917. We use several parts for our project, including Proteinase K and copA promotor. We chose Proteinase K because it has the activity to degrade biofilms of microorganisms, including H pylori which is the target of our project. CopA promoter is used to induce the downstream gene expression for the kill switch system using ammonia. Due to the COVID-19 restriction, we can't do experiments in the lab, so we contributed to those parts documentation through literature studies and got the following results.

Proteinase K (BBa_K1149035) has the catalytic triad (active site) at Asp 39, His 69, and Sr224. The optimum pH range for maximal activity of Proteinase K is 7.5–12.0¹. In addition, Ca²⁺ ions have a role in providing protection against autolysis and increasing the thermal stability of proteinase K². As we mentioned before, Proteinase K can inhibit biofilm formation and degrade preformed biofilm of H. Pylori³. CopA promoter (BBa_K1555000) usually can sense copper, but our literature study reported that copA promoter also could sense gold and ammonia for promoter activation^4-5. For further information and characterization, you can check it at this link BBa_K1149035 and BBa_K1555000. Hopefully, the information we provide can be used as a reference parameter for the use of parts so that experiments carried out in the future can increase the chances of success.

References

1. Barret A, Rawlings N, Woessner J. Handbook of Proteolytic Enzymes. 3rd ed. Academic Press. 2012
2. Bajorath J, Hinrichs W, Saenger W. The enzymatic activity of proteinase K is controlled by calcium. Eur J Biochem. 1988;176(2):441–7.
3. Windham I, Servetas S, Whitmire J, Pletzer D, Hancock R, Merrell D. Helicobacter pylori Biofilm Formation Is Differentially Affected by Common Culture Conditions, and Proteins Play a Central Role in the Biofilm Matrix. Applied and Environmental Microbiology. 2018;84(14)
4. Stoyanov J, Brown N. The Escherichia coli Copper-responsivecopA Promoter Is Activated by Gold. Journal of Biological Chemistry. 2003;278(3):1407-1410.
5. 5. Shen P, Niu D, Permaul K, Tian K, Singh S, Wang Z. Exploitation of ammonia-inducible promoters for enzyme overexpression in Bacillus licheniformis. Journal of Industrial Microbiology and Biotechnology. 2021;48(5-6):1-8.