Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs: 1. pSB3C17-B0032-yenbox_WT-GFP 2. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23101-B0034-YenR 3. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23110-B0034-YenR 4. pSB3C17-B0032-yenbox_WT-GFP + pSB1K3-J23102-B0034-YenR

By constructing the measurement construct BBa_K1381008 (yenbox_WT-B0032-GFP) and performing double transformation together with one of the constructs producting the activator YenR BBa_K1381005 (J23110-B0034-YenR), BBa_K1381006 (J23102-B0034-YenR) and BBa_K1381007 (J23101-B0034-YenR). We managed to show that the activator YenR works perfectly fine in E. coli and that it recognise the recognition region, the yenbox and induces the strength of the promoter fused with it. By measuring the production of the green fluorescence protein GFP using a flow cytometer, we could see that we got a five-fold induction when YenR with the strongest promoter out of the three used were present.

Figure 1. A,B and C shows triplicates of swarmplate assays incubated at 2 days. 1: Motile strain RP437 (positive control), 2: cheZ mutant (negative control), 3: J23100-B0034-cheZ, 4: J23113-B0034-cheZ, 5: J23114-B0034-cheZ

We managed to restore chemotaxis in a non-motilite mutant strain, by reintroducing the cheZ gene on plasmids into cheZ knock-out E. coli. The motility assay used to measure the chemotaxis was also improved by optimizing incubation conditions for cells inoculated in swarm plates. Three promoters of different strengths were tested in combination with our construct and were shown to induce different levels of motility, and hence different levels of cheZ.

Figure 2. The picture show the result from an experiment to test the killing efficiency of our bacteriocin colicin Fy. On the left Y. enterocolitica had been grown in liquid culture together with colicin Fy. The right plate is a negative control, where Y. enterocolitica had grown in the same conditions in liquid culture without any colicin Fy added.

We manage to produce the bacteriocin colicin Fy and by fusing it with a His-tag we could peform a SDS-page and prove its presence. To analyse the colicin Fy’s killing efficiency we let Y. enterocolita grow with and without colicin Fy in liquid cultures and compared the result. Fig. 2 shows these two cultures plated. On the left plate, the Y. enterocolitica had grown with the colicin Fy added, while the right plate is the negativ control, where Y. enterocolitica had grown without any colicin Fy. If we compare the two plates, we can see that there is a clear difference in the amount of growing Y. enterocolitica colonies.