Team:SUSTech Shenzhen/Proof Of Concept
Part1: Why developing proof of concept of using colanic acid as our materials to store water?
Colonic acid is a polysaccharide expressed through the Rcs system after receiving a drought signal by E. coli and other Enterobacteriaceae1. Its unique conditions of producing colonic acid make a possible function of preserving drought environment have aroused our interest. We have guessed that colonic acid has a certain effect on inhibiting bacterial dehydration. Thus, a possible biological water retention method may be found. By analogy, we can also believe that colonic acid can to some extent produce less water loss in the human intestine when diarrhea happens, that is, a large amount of expression in the case of dehydration can also be It can alleviate dehydration of cells around the human intestinal tract. But a proof of concept on the ability of colanic acid to retent water should be done.
Part2:Why do we use the Rcs system as our regulating system?
As we all know, the capsule exists on the surface of many bacteria, and its water content is about 90%~98%. After consulting the relevant literature2,3,4,5, we understand that the Rcs system plays an important role in regulating the expression of colonic acid on the capsule. In the Rcs system, there are multiple mutually regulated phosphorylation systems such as RcsA, RcsB, and RcsC. The synthesis of colanic acid was later shown to be under the control of two positive protein regulators encoded by the RcsA and RcsB genes, RcsA cooperates with RcsB to stimulate the transcription of capsular polysaccharide genes, so with the specific plasmid constructed, we were able to enhance this process to overexpress the required colonic acid.
Part3:Why do we need a RcsB overproduction plasmid in our bacterias6?
RcsA protein is unstable and rapidly degraded by the Lon protease. For the expression of RcsB in normal environment is also low. The interaction of two proteins will be difficult. Therefore, usually under laboratory conditions, the expression of colonic acid is usually at a relatively low level. For RcsA is very unstable and has a high rate of loss. We choose to add a plasmid capable of overexpressing RcsB into our bacteria, Under the induction of a specific medium, we can enhance the expression of colonic acid.
Part4:Can the overproduction of colanic acid promotes the efficiency of engineering bacteria colonization ?
The expression and regulation of polysaccharides on the surface of Escherichia coli can simultaneously have an important effect on the adhesion of bacterial species to small intestinal epithelial cells. The study on the expression of polysaccharides on the capsular surface of bacteria Bifidobacterium7 can see that the regulation of capsular polysaccharides can Enhance the adhesion of bifidobacteria to small intestinal epithelial cells so that bifidobacteria can better colonize the small intestine environment. Therefore, we also hope to obtain an E.coli in the intestine by regulating the amount of colonic acid expressed on the capsule. The relationship between colonization ability in the tract and the expression of colonic acid can also solve the problem of engineering bacteria colonization. Finally with the determination of colanic acid expression by HPLC and the following animal experiment we may also figure out the relation between the production of colanic acid and E.coli colonization.
Part5:Further works
v For the limitation of time, we only test a few parts of our pathway, the ability of water retention is still under testing. Here is the plan we design already. For the colonization of bacterias, we also have a plan of animal (fruit fly).
Reference
[1]Grant, W. D.; Sutherland, I. W.; Wilkinson, J. F., Exopolysaccharide colanic acid and its occurrence in the Enterobacteriaceae. J Bacteriol 1969, 100 (3), 1187-93.
[2]Lee, S. H.; Kim, I. C.; Lee, W. S.; Byun, S. M., RcsC-mediated induction of colanic acid by secretion of streptokinase in Escherichia coli K-12. FEMS Microbiol Lett 1996, 139 (2-3), 189-93.
[3]Davalos-Garcia, M.; Conter, A.; Toesca, I.; Gutierrez, C.; Cam, K., Regulation of osmC gene expression by the two-component system rcsB-rcsC in Escherichia coli. J Bacteriol 2001, 183 (20), 5870-6.
[4]Stout, V.; Gottesman, S., RcsB and RcsC: a two-component regulator of capsule synthesis in Escherichia coli. J Bacteriol 1990, 172 (2), 659-69.
[5]Carballes, F.; Bertrand, C.; Bouche, J. P.; Cam, K., Regulation of Escherichia coli cell division genes ftsA and ftsZ by the two-component system rcsC-rcsB. Mol Microbiol 1999, 34 (3), 442-50.
[6]Kamerling, J. P.; Boons, G.-J., Comprehensive glycoscience : from chemistry to systems biology. 1st ed.; Elsevier: Amsterdam ; Boston, 2007.
[7]Crittenden, R.; Laitila, A.; Forssell, P.; Matto, J.; Saarela, M.; Mattila-Sandholm, T.; Myllarinen, P., Adhesion of bifidobacteria to granular starch and its implications in probiotic technologies. Appl Environ Microbiol 2001, 67 (8), 3469-75.