Team:St Andrews






University of St. Andrews

Project description

Significance:

One of the growing threats to modern medicine is the resistance of bacteria to antibiotic substances. The increasing doses of antibiotics needed to clear infections have given way to entire strains of bacteria which are largely immune to the standard therapies. Our project centers around one way to combat this issue: that of developing new antibiotics altogether. Many bacteria engage in a kind of ‘germ warfare,’ releasing metabolites which are toxic to their competitors. Our aim is to create a screening system to determine which endogenous molecules within a bacterial sample perform this function, if any. In essence, we sought a way to assess the antimicrobial properties of any novel metabolites found in the course of metabolomic research.

Moreover, there are two ways in which a potential new lead compound for development into an antibiotic might be useful. First, it might be antibiotic in the classical sense--that it kills prokaryotes. Secondly, an antimicrobial substance might disrupt the formation of biofilms or the stability of extant ones. Biofilms are essentially the scaffolding around which a colony of bacteria live. They allow the members of the group to anchor to a surface, protect the bacteria from antibiotics, and trap food. Biofilm dependent infections are notoriously hard to clear, and consistently affect patients with conditions such as Cystic Fibrosis. If a metabolite were to disrupt a biofilm, then it too would have valuable properties not to overlook. As such, our project has two components: one to detect cell death, and another to detect the presence of biofilms.

Cell lysis:

The first part of our project focuses on the detection of cell lysis in E.Coli. To do this, we employ a split mNeonGreen(mNG) Fluorescent protein. mNG has 11 strands which make up a barrel structure. The last of these strands can be removed to halt the fluorescence and then reattached to regenerate the fluorescence. We have constructed two plasmids which will be inserted into two populations of E.Coli. The first E.Coli population will make a fragment of mNeonGreen called mNG 1-10 and retain it within the bacteria. The Second E.Coli will make the other fragment of the mNeonGreen (mNG 11) and will export it into the medium. The smaller of the two halves has been chosen to be exported in order to minimise protein unfolding during export from a bacteria. If this is successful we will have a population of E.Coli full of mNG 1-10 and a media full of mNG 11. At this point antibiotics can be added to lyse the bacteria and in doing so release the mNG 1-10 halves. The split mNG parts should then recombine and form full length mNG which will glow and therefore, give us a signal that the first population of E.Coli has been lysed.

We have also ordered plasmids which contain a viral protein tag attached to each mNG half. These are a ChickV nsp3 tag (attached to mNG 11) and a SH3 tag (attached to mNG 1-10). These two viral protein tags have high affinity for each other and can therefore, provide another site of binding if the affinity between the mNG halves is not high enough. The viral protein tags are connected to their respective mNG halves with a 24 residue linker in between. This linker is constructed from glycine, serine and prolines to ensure maximum flexibility. As there is a potential for the viral protein tags to affect the binding of the mNG halves, we have also ordered plasmids without them.

Biofilms:

The second part of our project revolves around the presence or absence of biofilms--a sticky material that some populations of bacteria generate to both adhere to the surface that they are colonizing and to evade threats like cells of the immune system or antibiotics. Moreover, antibiotic resistance is frequently passed between cells within the depths of these biofilms. We aim to detect said biofilm formation from the bacteria Pseudomonas aeruginosa.

We have identified that PSL polysaccharide is a molecule present in the biofilm of Pseudomonas aeruginosa and that it may be required for the biofilm formation. Staphylococcus aureus Protein A (SpA) was then identified as a protein which could bind to PSL. With this in place, the experimental procedure was developed as follows- A Plasmid map is constructed for the exportation of SpA linked to a full length Fluorescent protein (potentially mOrange2). This plasmid will be inserted into the bacteria Bacillus subtilis which will act as our chassis. The reason Bacillus subtilis has been chosen is that it is possible it can export the large protein we will produce without unfolding it and destroying its functionality. After the SpA-mOrange protein has been exported into the media the SpA can bind with PSL present in the biofilm of Pseudomonas aeruginosa. After binding a wash can be done to remove anything which is not bound, if at this point the glow from mOrange is still visible in the area the biofilm is expected to be we know that the presence of a biofilm is confirmed and the experient is a success.

There remain however problems with this experiment. The plan is to combine both Cell lysis and biofilm experiments into one single pot. If this were done it would mean the co-culture of 3 different bacteria which may be an issue. In an attempt to step up to this goal we have also set up 2 intermediate steps in the biofilm experiment. Firstly we are going to order a sequence for the production and retention of the SpA-mOrange within E.Coli. This plasmid backbone will have already been used in the cell lysis experiment so we can be sure that it will work at the time of use in the biofilm experiment. In doing this we would have to lyse the E.Coli and protein purify the SpA-mOrange and then add this manually to the biofilm produced by Pseudomonas aeruginosa. Another intermediate step is using the flexibility of our Bacillus subtilis backbone to our advantage. The backbone we will order also contains his tags which if we chose different restriction site would allow us to retain the SpA-mOrange within Bacillus subtilis, lyse it, protein purify and then add it manually to the biofilm. With these 2 intermediate steps we removed the black box element of this experiment making it easier to diagnose problems in the wet lab.

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