Part:BBa_K4604016

piG_01a (leaky_tetR_bluB)

piG_01a is 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 of the plasmid shows leakiness, so a modified version of it is uploaded as BioBrick 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 (Cbi), 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.

Figure 1: BluB enzyme production 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. E. coli BL21 DE3, [piG_01a,BBa_K4604016] overnight culture in LB medium, uninduced.

While we observed an increase in BluB protein yield with increasing inducer concentration, we also noted a leaky expression without induction. This is most likely due to a silent mutation that we introduced in TetR to conform to iGEM standards for this part. This lead to the creation of BBa_K4604016.

After characterizing BluB expression of this BioBrick, Liquid Chromatography Mass Spectrometry (LCMS) measurement was performed to confirm its hypothesized ability to produce AdoCbl. To estimate the g/g dry cell weight (DCW) value for the samples, we measured the cell weight of a 1 mL sample of an independent E. coli BL21(DE3) culture with similar optical density.

Figure 2: OHCbl contents in E. coli BL21(DE3)[piG_01a] cells from production cultures, measured by LC-MS. Colors of bars correspond to respective setups shown in the figure above. Orange bar respresents measured OHCbl in supernatant of samples. (A) 1mL samples (B) 50 mL samples (C) re-measurement of 50 mL cell pellet and supernatant samples. LC-MS performed at Hannibal Lab, University Medical Center Freiburg. n=1

We successfully confirmed that BluB expression with this BioBrick provides sufficient amounts of DMB for AdoCbl production in E. coli BL21(DE3) from Cbi.

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 727
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1647