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From 2011.igem.org

Fixation Results

Aims

-To find a suitable material for encapsulation

-To build a construct that will appropriately express this material within a biofilm

-To transform this construct into the chassis and express it

-To characterise the expression and its effects towards fixation and encapsulation of the biofilm

Methods

We found our encapsulating material in Capsular Colanic Acid (Chao & Zhang, 2011). For simplicity it was decided to use an existing colanic acid biobrick, from the Imperial College iGEM 2009 Team.

This biobrick system had been constructed to form a capsule of colanic acid that is anchored to the cell membrane by a ligase> E. coli naturally uses colanic acid to encapsulate itself (Stout & Gottesman, 1990), as shown in the image below.

The parts of the colanic acid system that were required to induce overexpression (Gervais, Phoenix & Drapeau, 1992) and export were selected. Anchoring to the membrane was not required. Top10 cells were transformed with the biobricks and the DNA was extracted to begin building a construct. We began attaching Ribosome Binding Sites and terminators to the biobricks we required, using the AlwNI-Method as detailed in our protocols. Due to time constraints we did not finish the entire construct.

References

Chao, Y. & Zhang, T., 2011. Probing Roles of Lipopolysaccharide, Type 1 Fimbria, and Colanic Acid in the Attachment of Escherichia coli Strains on Inert Surfaces.Langmuir. 2011 Sep 20;27(18):11545-53

Gervais FG, Phoenix P, Drapeau GR, 1992. The rcsB gene, a positive regulator of colanic acid biosynthesis in Escherichia coli, is also an activator of ftsZ expression. Journal of Bacterioogy. 1992 Jun ; 174(12): 3964-71

Stout V, Gottesman S, 1990. RcsB and RcsC: a two-component regulator of capsule synthesis in Escherichia coli. Journal of Bacteriology. 1990 Feb ; 172(2): 659-69