Effect of Ph on fluorescence output for cells with and without colanic acid

Background

RcsB (biobrick BBa_K200000 is the gene responsible for generating a colanic acid capsule around our bacteria. Having a colanic acid capsule around them will protect them from being degraded by the high acidic nature of the stomach environment.

Aim

Here, as proof of concept, we have designed a simple experiment that will show that colanic acid has a protective effect on our growing bacteria in acidic environments (simulating gastric conditions). The method is described below.

Experimental Method

In a multiple well plate, 2 different types of cell cultures were grown, both containing the BBa_I13522 construct, which is a constitutive promoter expressing GFP. The differences were:

• Some cultures contained colanic acid (chemically induced by p-Fluoro-L-phenylalanine (FPA))
• Some cultures did not contain colanic acid.

Growth rate of the cell cultures and levels of GFP expression were monitored over time, at different Ph values in order to decide if induction using colanic acid provides extra protection to the construct.

Result analysis: GFP expression

Figure1: Illustration. Our bacteria contain a constitutive promoter expressing GFP and we are testing the levels of fluorescence

Colanic acid cells

Neutral Ph

We can see that in the presence of water, cells with colanic acid give out a fluorescence output around values of 350000 to 400000. Cells can survive without difficulty at this Ph so the output remains fairly constant.

Figure 2: Effect of neutral Ph on cells with colanic acid for 3 different wells<b>