Part:BBa_K2588001
hybB Cold Shock Promoter (shortened)
In our project, oHybB is used in the construct oHybB-ompA-AOX1 to activate AOX1 and thereby generate heat through uncoupling the electron transport chain from ATP synthesis, when the surrounding temperatures are low. This is a very important aspect, as mosquitoes are attracted by heat.
Usage and Biology
oHyBb is a cold-shock induced promotor. It is active at temperatures below 17 °C.
Improvement
When we started planning our project, we were looking for a cold shock inducible promotor, but learned that the existing one did not work properly (see for example: Team Groningen 2011). Therefore, we figured out that the original HybB construct was too long. Besides containing a 5´UTR, it also included a part of a coding sequence from the original downstream gene. Because we were sure that this would lead to an interference with any gene of interest which would be cloned downstream of this promotor, we shortened the original sequence and named it oHybB (optimized HybB). For further information see our Improve page.
Characterization
To characterize the function of oHybB, we generated a composite part consisting of pSB1C3-oHybB-E0840. A direct comparison between our new part oHybB (BBa_K2588001) and the original HybB (BBa_J45503) was made. The activity of our promotor was characterized via measurement of the fluorescence at different temperatures (16 °C, RT and 37 °C). As a negative control, untransformed DH5α were chosen and as a positive control DH5α with pSB1C3-BBa_I20270.
Fig. 1: Improvement of BBa_K2588001 over BBa_J45503. GFP fluorescence of E. coli cultures expressing both HybB-Promoter/GFP combinations at different temperatures was measured. Values are mean + SD from three technical replicates. Significances: * corresponds to p<0.05, ** corresponds to p<0.01, *** corresponds to p<0.001.
Discussion
A successful improvement of the original promotor BBa_J45503 could have been showen. In comparison, oHybB showed an increased activity after a cold shock for 13 h. The highest activity was observed at 16 °C, the lowest at 37 °C. In contrast to the original oHybB promotor our data was significant. For further information see our Improve page.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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