IGEM:IMPERIAL/2009/Encapsulation/Phase2/Colanic acid

Background

Colanic acid is an exopolysaccharide produced by many bacteria including E.coli. Since it is naturally produced by E.coli we can manipulate the pathway as opposed to building in a new one (e.g. alginate biosynthesis).

Colanic acid has been shown to offer production against acidic conditions and dessication, it is also non-pathogenic.

• James Chappell 09:51, 23 July 2009 (EDT):Any evidence about how bacteria with this coat would survive through the GI track? Be great if there were some examples, obviously E.coli can survive to some extend as of course we can become infected by some strains but there maybe some more direct evidence out there.

Regulation

Upregulation of RcsB has been shown to induce colanic acid production.[1] Colanic acid production is also upregulated by blue light.[2]

We can decouple this pathway to control the thickness of our cellular capsule.

To efficiently link an input of blue light to an output of colanic acid, the circuit can be simplified by placing RcsB under the control of YcgE repressible promoter. This biobrick has been designed by the 2009 Leuven team (BBa_K238000). There is currently no BioBrick for RcsB although the sequence is known.

There are also RcsB knockout strains of E.coli which could be used to avoid cross-talk between pathways.

http://cgsc2.biology.yale.edu/Mutation.php?ID=77155

• James Chappell 10:01, 23 July 2009 (EDT):Cool but the paper you have cited above shows that by contorlling the RscB you will effect your cell division so defiantly we need to consider this, is it okay to write quick summary of the paper and what unintended affects we might expect. Also it seemed from the diagram that RscB is really at a node to many different pathways, look for recent paper to see if they have filled out any of the downstream pathways more.

It might also be interesting to put an amber stop codon in the RcsB gene and co-express SupD with our gene of interest. This way, capsule formation would be influenced by both a light input and the levels of the gene of interest. Having said this, you only have to look at mucoid colonies to see the excessive colanic acid production, this indicates that all cells within a high density population will become encapsulated (i.e. it will be difficult to efficiently relate the presence of a capsule to a discrete amount of protein).

Based on this, it might be better to add an inducer which initiates the production of a protein of interest at a known rate. After a certain period of time, a blue light is switched on which does two things:

1) Initiates encapsulation (& possibly trehalose synthesis).

2) Turns off the production of the protein of interest. This could be achieved by using blue light to trigger the expression of a repressor which binds to the promoter controlling the protein of interest. Of course this level of dosage control is dependent on our final application. For instance, if the xylanase/cellulase application were chosen, higher levels of protein would be advantageous and therefore it would not be necessary to repress synthesis upon encapsulation.

Useful Papers

1. Gervais FG, Phoenix P, and Drapeau GR. The rcsB gene, a positive regulator of colanic acid biosynthesis in Escherichia coli, is also an activator of ftsZ expression. J Bacteriol. 1992 Jun;174(12):3964-71. DOI:10.1128/jb.174.12.3964-3971.1992 |

3. Mao Y, Doyle MP, and Chen J. Role of colanic acid exopolysaccharide in the survival of enterohaemorrhagic Escherichia coli O157:H7 in simulated gastrointestinal fluids. Lett Appl Microbiol. 2006 Jun;42(6):642-7. DOI:10.1111/j.1472-765X.2006.01875.x |

4. Tschowri N, Busse S, and Hengge R. The BLUF-EAL protein YcgF acts as a direct anti-repressor in a blue-light response of Escherichia coli. Genes Dev. 2009 Feb 15;23(4):522-34. DOI:10.1101/gad.499409 |