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Team:University of Sheffield /Project
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Project Overview
Contents |
Introduction
Fusion Receptor
The Native E.coli pathway
A Green Fluorescent Reporter
Introduction
The initial plan was to introduce a Green Fluorescent reporter molecule under one of the genes controlled by small regulatory RNA called CsrA. So as not to reduce the viability of the cell as well as to avoid any possible “noise” in the engineered system, the gene choice required several attempts.
Re-usable testing kit
The plan to have a micro – chip for sensing biological water contamination ideally would include a possibility of reusing it. Thus, the aim of our project was to use GFP with an LVA tag (AANDENYALAA). This would mean that the GFP would have an amino acid tag targeted by housekeeping proteases and would be degraded within 45 minutes once there is no more stimulation for gene expression. This in turn would mean that the E.coli system sensing V.cholerae presence in water would stop fluorescing once the concentration of CAI-1 molecules decreased up to tolerable level.[2]
The idea to include degradable GFP in our project was taken from the Andersen, J.B et al. (1998) New Unstable Variants of Green Fluorescent Protein for Studies of Transient Gene Expression in Bacteria. Applied and Environmental Microbiology.
pgaABCD operon control
This particular four - gene locus in E.coli is responsible for synthesis of the biofilm adhesion protein called PGA. As this is a virulence factor, i.e. non-essential for growth in supplemented media, a possible insertion of GFP and possible disruption the operon would not cause lethal effects to our cells.
As far as it is known, the pgaABCD genes are subject to post-translational inhibition by the small regulatory RNA CsrA as well as activation by the cation-responsive regulatory protein NhaR. The first fact is to our advantage. In theory, once the binding of the CAI-1 to fusion kinase occurred, the CsrA inhibition would be releaved and the GFP would be expressed . Also, according to research it has been proven that the csrA deletion mutant had an impressive 3000 fold increase compared to the wild type.
Possible “noise” in the system
As mentioned before, one of the factors to be taken into account prior choosing the gene within which the GFP would be inserted is the interference or “noise” in the system. This possibility might have caused background fluorescence or GFP expression independent from the sensing event of our interest.
The reasons underlying this “noise” are listed below:
- High salt concentrations (NhaR dependent)
- Alkaline pH (NhaR dependent)
- Ethanol ( NhaR dependent)
- Glucose presence in the media (Independent from both CsrA and NhaR)
The noise in the system appears only if the cells are grown at extreme conditions. However, if the growth media contains only 1% glucose, 1 % ethanol, 1 % NaCl the levels of the pgaABCD transcript should not be elevated by any other factors except CsrA. On the other hand, if additional NaCl or glucose are added to the medium it might cause significant increase in the pgaABCD mRNA appearance [2]
Target of insertion
As the pgaABCD is a four-gene locus another question that arose was the actual place within the operon where the insertion could take place. Another obstacle was that genes b, c and d overlap with each other.
One would theoretically suggest insertion under the promoter sequence, before the actual gene a . However, the very high inhibition with CsrA is actually due to 6 binding sites, one of which overlaps with gene a formyl-methionine codon.
Thus, the only possible way of inserting GFP, with minimal disruption of the whole operon, is at the end of gene a sequence just before the termination codon. This would give a high level of control by CsrA, as it is just after the CsrA regulatory binding sites, and would hopefully prevent extensive damage to genes b,c and d expression.
