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Part:BBa_K4604016
piG_01a (leaky_tetR_bluB)
piG_01a is a construct consisting of the tet promoter/repressor, the modified t7 RBS, a functional bluB gene and the rrnB terminator. The backbone we used in the experiments is pGGAselect. This version on the plasmid is leaky, a modified version of it is uploaded as BioBrick BBa_K4604016.
Usage and Biology
AdoCbl is a bioavailable form of B12. Vitamin B12 is an essential nutrient, humans are dependent on for the production of red blood cells, the synthesis of the DNA and the function of nerves. To form the complete AdoCbl synthesis pathway in E. coli, it would require 28 additional genes. Since this is not realistic nor practical for an iGEM project, we decided on an alternative method. When supplemented with cobinamide, a precursor for AdoCbl, E. coli is capable of producing AdoCbl on their own in small amounts. With the overexpression of a naturally occurring gene of the synthesis pathway of sinorhizobium meliloti 2011, called bluB BBa_K4604005, a greater yield can be achieved [2].
Characterization
Western Blot analysis using an anti-His-Tag antibody confirmed the induced expression of BluB. Different concentrations of the inducer doxycycline were tested to identify the optimal yield of the BluB protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 727
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1647
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