Part:BBa_K4604015

piG_01b (tetR_bluB)

piG_01b is 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). Our 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, which is an essential nutrient required 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 1: BluB enzyme expression for different inducer concentrations. Detection of recombinantly expressed, his-tagged BluB enzyme with SDS-PAGE followed by Western Blot. Detection of the BluB protein was performed with an anti-his antibody. Loading control: RNA polymerase β-subunit.

We examined the expression of BluB using Western Blot. Cells containing this BioBrick were compared to ones with BBa_K4604020 (piG_07).

LC-MS is a method used to determine the concentration of molecules based on their mass. With this, it was possible to detect how much Hydroxocobalamin (OHCbl) is produced (relative to dry cell mass). When AdoCbl is exposed to light, it is converted to another derivative of B12, namely OHCbl. To make the preparation of the samples and measurement easier, we worked in sunlight, thereby converting AdoCbl into OHCbl and measured the amount of this compound. We cultivated bacteria containing either a functional or a mutated version of the blub gene and afterwards sent them to Hannibal Lab at the University Medical Center Freiburg, to be measured via mass spectrometry.

As seen in the graph above, induced cultures show clear bands that prove BluB expression. There are slight bands indicating BluB expression for non-induced cells containing this BioBrick. There was no bluB expression in cells carrying piG_07 (BBa_K4604020) or pGGAselect. Next a LC-MS measurement of OHCbl was performed using constructs with either a functional (piG_01b) or non-functional (piG_07/BBa_K4604020) version of bluB.

Figure 2: OHCbl content measurement with LC-MS in dry cell pellet. E. coli MG1655 cultures induced with 100 ng/mL DOX were supplemented with 500nM cobinamide. Samples taken immediately before induction and after cultivation for 24 h. LC-MS performed at Hannibal Lab, University Medical Center Freiburg.

LC-MS measurement shows that the induced cultures of E. coli containing this BioBrick produce AdoCbl as opposed to the ones containing piG_07 (BBa_K4604020). Thereby we have both proven the expression of BluB as well as AdoCbl synthesis.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 710
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1618