Part:BBa_K4604015
piG_01b (tetR_bluB)
piG_01b 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 for experiments is pGGAselect. The tet operator is a BioBrick from iGEM Freiburg 2022 (BBa_K4229059) This BioBrick can be used to produce Adenosylcobalamin (AdoCbl) in E. coli when supplemented with cobinamide. The tetA/B promoter used in this version of the plasmid is a modified version of piG_01a/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
figure 14 von production results
References
1] Hallberg ZF, Su Y, Kitto RZ, Hammond MC. Engineering and In Vivo Applications of Riboswitches. Annual Review of Biochemistry [Internet]. 2017 Jun 20;86(1):515–39. Available from: https://doi.org/10.1146/annurev-biochem-060815-014628
[2] Fowler CC, Brown ED, Li Y. Using a Riboswitch Sensor to Examine Coenzyme B12 Metabolism and Transport in E. coli. Chemistry & Biology [Internet]. 2010 Jul 1;17(7):756–65. Available from: https://doi.org/10.1016/j.chembiol.2010.05.025
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 710
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1618
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