Team:SUSTech Shenzhen/Design

  • SUSTech_Shenzhen

    • Design

    Why do we want to do the program of engineering bacteria ?

    About Design

    Circuit Design

    Our basic engineered bacteria design consists of 2 components, E. coli and lactobacillus. The overall response of this system mainly including 3 parts.

    Circuit

    In part 1, lactobacillus would constitutively reduce AHL, which is a kind of quorum sensing molecule of E. coli through LuxI (AHL synthase)1. And a chloride-induced expression system is employed to trigger the lysis of lactobacillus and release AHL2.

    In part 2, engineered E. coli would sense the released AHL through EsaR protein. EsaR protein is constitutively expressed and it could bind to Esa box in the absence of AHL molecules, and inhibit downstream expression3-5. In the presence of AHL, the inhibitory effect of AHL is destroyed and downstream water storage polysaccharides expression is activated. Therefore, we can realize the specific expression of polysaccharides to achieve the purpose of storing water and preventing dehydration.

    In part 3, upon the presence of AHL, engineered E. coli would secrete antibacterial peptides with an additional trigger – quorum sensing molecules of the specific pathogens. For instance, here we target the V. cholera and introduce the V. cholera quorum sensing system in E. coli based on the work of Maciej B et al6,7. Under the existence of CAI-1 (quorum sensing molecule of V. cholera), our designed system would promote the downstream antibacterial peptides expression with a help of PhlF inverter system8. Finally, we can achieve the purpose of secreting antibacterial substances to kill pathogenic bacteria.

    For more details of our design, please refer to the contents in our engineering part. It is worth noting that the division of part here is slightly different from that of the modeling and experimental parts.

    Reference

    [1]Bassler, B. L.; Miller, M. B., Quorum Sensing. In The Prokaryotes: Prokaryotic Communities and Ecophysiology, Rosenberg, E.; DeLong, E. F.; Lory, S.; Stackebrandt, E.; Thompson, F., Eds. Springer Berlin Heidelberg: Berlin, Heidelberg, 2013; pp 495-509.

    [2]Sanders, J. W.; Venema, G.; Kok, J., A chloride-inducible gene expression cassette and its use in induced lysis of Lactococcus lactis. Applied and environmental microbiology 1997, 63 (12), 4877-4882.

    [3]Shong, J.; Huang, Y.-M.; Bystroff, C.; Collins, C. H., Directed Evolution of the Quorum-Sensing Regulator EsaR for Increased Signal Sensitivity. ACS Chemical Biology 2013, 8 (4), 789-795.

    [4]Shong, J.; Collins, C. H., Quorum Sensing-Modulated AND-Gate Promoters Control Gene Expression in Response to a Combination of Endogenous and Exogenous Signals. ACS Synthetic Biology 2014, 3 (4), 238-246.

    [5]Minogue, T. D.; Trebra, M. W.-v.; Bernhard, F.; Bodman, S. B. v., The autoregulatory role of EsaR, a quorum-sensing regulator in Pantoea stewartii ssp. stewartii: evidence for a repressor function. 2002, 44 (6), 1625-1635.

    [6]Holowko, M. B.; Wang, H.; Jayaraman, P.; Poh, C. L., Biosensing Vibrio cholerae with Genetically Engineered Escherichia coli. ACS Synthetic Biology 2016, 5 (11), 1275-1283.

    [7]Jayaraman, P.; Holowko, M. B.; Yeoh, J. W.; Lim, S.; Poh, C. L., Repurposing a Two-Component System-Based Biosensor for the Killing of Vibrio cholerae. ACS Synthetic Biology 2017, 6 (7), 1403-1415.

    [8]Moon, T. S.; Lou, C.; Tamsir, A.; Stanton, B. C.; Voigt, C. A., Genetic programs constructed from layered logic gates in single cells. Nature 2012, 491 (7423), 249-253.