Part:BBa_K2462001
RBS
Description
This part is a RBS used in Bacillus megaterium. We got its sequence in pSPAsp-hp, which is a plasmid used for protein expression in Bacillus megaterium. Then the whole part has been synthesized by AuGCT.
Verification
For the verification of this RBS, we use this RBS to construct a circuit with our dehalogenase RdANP. Due to the limitation of time and experimental equipment, we didn’t do the SDS-PAGE, instead, we did HPLC to test the our enzyme. We insist the idea that if we succeed in the expression of our enzyme, we shall verify the RBS at the same time. We use the HPLC to test the effects of our enzyme, but we didn’t get convincing results because there are proteins interrupting the estimation.
The details related to the experiment are described in the part page of BBa K2462000:
https://parts.igem.org/Part:BBa_K2462000 In order to make the experiment more convincing, we also made another group of experimrnts. We took bacteria with RdhANP induced by xylose as the experimental group, and noninduced bacteria as the control. Dots colored differently indicate result under different conditions: green for transformed and induced group; blue for untransformed but induced group; yellow for transformed but induced group. The results are depicted as following:
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]
Disscusion
We decided to take this RBS as one of our parts not only because we used it in our own project but we do hope that the following teams can have more options when they choose to use Bacillus megaterium as their host for heterogeneous expression . We failed to verify this RBS by ourselves, but we are pleasant to give other teams the information of the original source for this RBS. As we get this RBS from Addgene, information can be found in their web.
More details about pSPAsp-hp:
http://www.addgene.org/48122/
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