Difference between revisions of "Part:BBa K1381004"

Revision as of 14:40, 14 October 2014

B0034-YenR

YenR is an activator originated from Yersinia enterocolitica. When YenR binds to the recognition region, the yenbox, the expression level of the fused promoter is induced. Both YenR and the yenbox are parts in the Yen system, which is a homolougous system to the Lux system.

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, tho the two interesting ones are the recognition region the yenbox (BBa_K1381000) and the activator YenR, that recognise and interacts with the yenbox. When YenR binds to the yenbox it induces the expression level of the promoter fused to the yenbox. Later, in the presence of Y. enterocolitica, its signaling molecules, 3-oxo-hexanoyl homoserine lactone (OHHL), will start flowing into our system, interacting with YenR. When binding occurs between the OHHL and YenR, YenR will lose its active shape and thereby its ability to interact with the yenbox. The induction will then be lost and the expression level will return to its base level. [1]

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 it overnight to grow. Cells containing only the constructs 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-B0032-yenbox_WT-GFP + pSB1K3-J23101-B0034-YenR
3. pSB1C3-B0032-yenbox_WT-GFP + pSB1K3-J23110-B0034-YenR
4. pSB1C3-B0032-yenbox_WT-GFP + pSB1K3-J23102-B0034-YenR
B) 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

Graph 1 above, 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 in what amount the two respective plasmids are present. 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.

Futher characterisation
We wanted to construct a customized version of BBa_K1381000 (yenbox_WT) were we tried to change the wildtype promoter, orginated from Y. enterocolitica, to a standardized promoter from the Anderson promoter library. yenbox_J23113 consists of the promoter BBa_J23113 (J23113) fused together, upstreams of the yenbox.

While designing this part, we tried to mimic the wildtype version. Hence, the wildtype version has an overlap of two bases between its promoter and the yenbox. Since the two last bases of the yenbox and the two first bases of J23113 did not match, we were faced with the choice of either changing two bases in the promoters sequence or in yenbox's sequence. In the article by Ching-Sung Tsai and Stephen C. Winanas [1] they discovered that the binding between the activator YenR and the recognition region of the yenbox is not dependent on the entire sequence of the yenbox. Depending on which part of the yenbox that is changed or replaced, YenR binds to the yenbox with different strengths. However, it still interacts with the yenbox and induces the strength of the promoter. Based on this fact, together with the knowledge that the Anderson promoters are very sequence dependent, we chose to change two bases in the sequence of the yenbox. The part BBa_K1381001 (yenbox_J23113) was created.

For the measurements we constructed, as above, the construct BBa_K1381009 (yenbox_J23113-B0032-GFP), where the yenbox fused with the wildtype promoter was coupled to the green fluorescent protein (GFP). The same procedures as for the characterisation experiments with BBa_K1381008 (yenbox_WT-B0032-GFP) was done. The result can be seen below.

Graph 2. The production of the green fluorescence protein GFP in cells containing the following constructs:
1. pSB3C17-B0032-yenbox_J23113-GFP
2. pSB3C17-B0032-yenbox_J23113-GFP + pSB1K3-J23101-B0034-YenR
3. pSB3C17-B0032-yenbox_J23113-GFP + pSB1K3-J23110-B0034-YenR
4. pSB3C17-B0032-yenbox_J23113-GFP + pSB1K3-J23102-B0034-YenR

In graph 2, we did not observe any induction This implies that our customized version of the yenbox fused with the promoter J23113 did not work as intended. It seems like the activator YenR could not recognise nor interact with the yenbox due to the changes made in the last two bases of the yenbox. It should be tested to redesign this part so that the yenbox is not fused to the promoter but just simply put beside it without any overlap. If the induction is not extremely distance dependent, that might work better. Otherwise, you could try to find other promoters, were the first two bases match with the two last bases of the yenbox, so that we don’t have to modify the yenbox.

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