Part:BBa_K4604020

piG_07 (tetR_mut_bluB)

piG_07 is consisting of the tet promoter/repressor, a mutated bluB gene and the rrnB terminator. The backbone we used for experiments is pGGAselect. The tet promoter is a BioBrick from iGEM Freiburg 2022 (BBa_K4229059). This can be used as a negative control to BBa_K4604015.

Characterization

To characterize the functionality of this part as a negative control to BBa_K4604015 (referred to as piG_01) we used an ethanolamine medium and mass spectrometry to prove its lack of AdoCbl production in E. coli MG1655.

Ethanolamine medium is a minimal medium devoid of any nitrogen source despite ethanolamine. Since nitrogen is crucial for amino acid synthesis and ultimately cell survival, E. coli is unable to grow in such a medium. We were able to produce said minimal medium in order to demonstrate the lack production of AdoCbl by BBa_K4604020. In this assay we compared the growth of piG_01b (BBa_K4604015) to the mutated non-functional bluB expression construct (piG_07) after induced production in M9 medium. Cells were cultivated in M9 medium, induced with 100 ng/ml DOX and supplemented with the essential substrate for AdoCbl synthesis. After 24 hours the cultures were washed and then cultivated in the ethanolamine medium to observe growth.

Figure 1: E. coli MG1655 growth curve comparison with maximum after 72 hours in 1975 ethanolamine medium. OD 600-measurement of culture samples containing plasmids pGGAselect, piG_01b or piG_07 using SpectraMax ID5 plate reader. The data present in these graphs is the result of at least two independent biological replicates.

The growth curves clearly show that only the induced cells that contain the functional bluB gene (piG_01b) are capable of metabolizing the ethanolamine by producing AdoCbl. This proves that the characterized BioBrick is unable to produce AdoCbl in sufficient amounts to metabolize ethanolamine.

Liquid Chromatography Mass Spectrometry (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.

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.

These results clearly show that the AdoCbl yield in cultures containing a non-functional bluB gene is minimal compared to a functional one. Sequence and Features BBa_K4604020 SequenceAndFeatures