Difference between revisions of "Part:BBa K1381000"

Revision as of 22:31, 17 October 2014

yenbox_WT

yenbox_WT is a recognition region (homologous to the luxbox), fused with a wildtype promoter from Yersinia enterocolitica. When interaction between the activator YenR (BBa_K1381004) and the yenbox occur, the expression level of the fused promoter is induced.

Characterization

The Yen system
Y. enterocolitica has a homologous quorum sensing system to the famous Lux system, the Yen system. This complex system consist of many different parts, though the two interesting ones are the recognition region the yenbox (BBa_K1381000) and the activator YenR, that recognises and interacts with the yenbox. When YenR interacts with the yenbox, it induces the expression level of the promoter fused to the yenbox. To remove this induction, Y. enterocoliticas signaling molecules, 3-oxo-hexanoyl homoserine lactone (OHHL) can be introduced. These unique signaling molecules will bind to YenR, making YenR lose its active shape and thereby its ability to interact with the yenbox. The induction will then, as a consequence, be lost and the expression level will return to its base level. [1] Though characterisation of this feature still remains to be done.

Characterisation
For characterisation we created the constructs BBa_K1381008 (yenbox_WT-B0032-GFP), where the yenbox fused with the wildtype promoter was coupled to the green fluorescent protein (GFP). These constructs were then cloned into the backbones pSB1C3 and pSB3C17 and transformed into competent E. coli cells already containing one of the YenR constructs BBa_K1381005 (J23110-B0034-YenR), BBa_K1381006 (J23102-B0034-YenR) or BBa_K1381007 (J23101-B0034-YenR) on the backbone pSB1K3. The double transformed cells were then streaked on plates containing both the antibiotic Kanamycin and Chloramphenicol and left overnight to grow. Cells containing only the construct yenbox_promoter-B0032-GFP were also streaked and left to grow.

The following day, overnight cultures were prepared and left for 16 h to grow into stationary phase. After that, 10 µL of the overnight culture was put into 500 µL of PBS solution and left for one hour for stabilization. The green fluorescence production was then measured using a flow cytometer. The results of the test is shown below.


Graph 1. The production of the green fluorescence protein GFP in cells containing the following constructs:
A) 1. pSB1C3-B0032-yenbox_WT-GFP
2. pSB1C3-yenbox_WT-B0032-GFP + pSB1K3-J23101-B0034-YenR
3. pSB1C3-yenbox_WT-B0032-GFP + pSB1K3-J23110-B0034-YenR
4. pSB1C3-yenbox_WT-B0032-GFP + pSB1K3-J23102-B0034-YenR
B) 1. pSB3C17-yenbox_WT-B0032-GFP
2. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23101-B0034-YenR
3. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23110-B0034-YenR
4. pSB3C17-yenbox_WT-B0032-GFP + pSB1K3-J23102-B0034-YenR

Graph 1 shows the induction relative to the base level of expression. In graph 1.A. we can observe the induction but cannot predict to what extent. This is because these cells are transformed with two high copy plasmids containing the same ori(origin of replication). The consequence will be that the cells will confuse the two plasmids with each other and have no control of what amount the two respective plasmids are present in. It will only ensure that it is 100-300 plasmids present in total, regardless of which plasmid it is. This is why we also cloned the yenbox-containing constructs into a low copy plasmid with a different ori than the high copy ones.

In graph 1.B., it can be seen that the amount of YenR that is produced is correlated, as expected, to the rate of the induction. When coupled to the strongest of the three promoters, J23102, the production is increased up to five folds.

These kind of constructs were also constructed with a blue fluorescence protein (BFP) that were also transformed into cells already containing one of the YenR constructs BBa_K1381005 (J23110-B0034-YenR), BBa_K1381006 (J23102-B0034-YenR) or BBa_K1381007 (J23101-B0034-YenR) on the backbone pSB1K3. They were then plated and restreaked together with a non-fluorescence containing reference and a base level reference without any YenR. These plates were used to see if the induction could be seen in UV light, with the naked eye. Unfortunately, as you can see in fig. 1 below, this was not the case. The color could be seen, but there was barely any difference in the intensity of the different cells.

Figure 1. The yenbox-constructs with BFP, streaked together with a non-fluorescent reference and the double transformed cells (containing one of the three YenR-producing plasmids and a plasmid with yenbox_WT-B0034-BFP).

It should also be mentioned that we did an attempt to create bigger constructs containing the characterisation constructs with the yenbox coupled to GFP and the YenR constructs (yenbox_WT-B0034-GFP-terminator-promoter-B0034-YenR). But for some reason these assemblies did not work. After some troubleshooting we came to the conclusion that it was too stressful for our bacteria to produce both BFP and YenR on the same high copy plasmid or that these constructs were not stable in E. coli and got rejected or mutated by the cell.

References
[1] Ching-Sung Tsai and Stephen C. Winanas, The quorum-hindered transcription factor YenR of Yersinia enterocolitica inhibits pheromone production and promotes motility via a small non-coding RNA, 2011, Molecular Microbiology 80[2], 556-571

Sequence and Features