# Project design & description

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Project description

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 each be cloned into two distinct populations of E.Coli. The first E.Coli population will express the fragments of mNeonGreen, $$mNG_{1-10}$$, and retain it within the cell membrane of each bacterium. The second population of E.Coli will continuously express the remaining fragment of mNeonGreen ($$mNG_{11}$$) and will export it into the surrounding growth medium. In order to minimise protein misfolding during export, the smaller of the two $$mNG$$ parts (strand 11) was fused to a signal peptide, whilst the larger $$mNG$$ part was chosen to be the retained part. This means that $$mNG_{1-10}$$ will build up in one population of E.Coli and $$mNG_{11}$$ will be present in the media. At this point, novel metabolites can be added to test whether they lyse the bacteria and in doing so, release $$mNG_{1-10}$$ into the media containing $$mNG_{11}$$. The split $$mNG$$ parts then associate and form full length $$mNG$$ to generate a fluorescent signal, indicating that the metabolite successfully lysed the bacterial cells.

During the experimental planning of the cell lysis system, caution was taken in order to ensure that the sensitivity of the system was high enough. As we were unsure whether the affinity of the two $$mNG$$ parts was high enough, we also ordered plasmids containing viral tags. These are a ChickV nsp3 tag (attached to ($$mNG_{11}$$) and a SH3 tag (attached to $$mNG_{1-10}$$), which bind extremely tightly once in contact. The viral protein tags are connected to their respective $$mNG$$ halves with a 8 residue linker in between, with the smaller, nsp3 tag, fused to $$mNG_{11}$$ for export. This linker was put in place in order to minimise interactions of the protein domains and is constructed from glycine, serine and prolines to ensure maximum flexibility. After completing the experiment, the plasmids containing these viral tags resulted in higher fluorescence than those without. However, both variants did provide fluorescence higher than the background level and we will therefore, for simplicity, submit the constructs without the tags.

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

These diagrams demonstrate the theoretical working of our cell lysis system. The mNeonGreen strand 11 fragment exits the cell to exist in the media, and then upon lysis of the second class of cell, the mNeonGreen strands 1-10 fragment joins it to produce a fluorescent signal.

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

This set of diagrams depict largely the same system as the ones above; however the difference lies in the small viral tag (represented as a black dot) which increases the affinity of the two fragments for each other.

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 in populations of E.coli and P. aeruginosa.

We have identified four major components of these biofilms: Double stranded RNA, cellulose, the polysaccharide PSL, and Alginate, the latter two of which are specific only to P.aeruginosa. In order to detect the presence of these molecules, specific binding proteins (BPs) were identified and fused to the fluorescent protein, mcherry; Staphylococcus aureus Protein A (SpA) for PSL, Alginate binding protein (Algp7) for Alginate, cellulose binding protein (Cel71 CBM) for Cellulose and dsRNA binding protein (FHV B2) for double stranded RNA . With this in place, the experimental procedure was developed as follows-

Four separate plasmid maps were constructed for the exportation of each binding protein linked to a full length fluorescent protein (mCherry). These plasmids were transformed into our chassis, E.Coli. The exportation of the BP-mCherry protein into the media allowed the BP to interact with its respective molecule present in the biofilm under investigation. After binding, a wash was applied to remove anything unbound, meaning that if a biofilm was present, a red signal persisted after the removal of the wash.

Figure 3.1

Figure 3.2

Figure 3.3

The above diagrams depict a binding protein being synthesised, exiting the cell, and binding a polysaccharide such as PSA or alginate.