Part:BBa_K1381001
yenbox_J23113
A customized version of BBa_K138000 (yenbox_WT) were we have tried to change the wildtype promoter, orginated from Yersinia enterocolitica, to a standardized promoter from the Anderson promoter library. yenbox_J23113 consists of the promoter BBa_J23113 (J23113) fused together, upstreams of a homolog to the luxbox, called the yenbox. The yenbox is a recognition site from Y. enterocoliticas quorum sensing system, the Yen system. The yenbox is recognised by an activator, BBa_K138004 (YenR) and when interaction between these two occur, an induction of the promoters expression occur.
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.
Characterisation
For characterisation we created the constructs BBa_K138009 (yenbox_J23113-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_K138005 (J23110-B0034-YenR), BBa_K138006 (J23102-B0034-YenR) or BBa_K138007 (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.
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 1 above, 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.
- <a id="reference1">[1]</a> 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
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 41
Illegal NheI site found at 52
Illegal NheI site found at 75 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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