During wet lab work, we tested several different constructs for our Synporter protein in the Vitamin B12 producing organisms R. planticola and S. blattae. The organisms were either grown under aerobic or anaerobic conditions.
The TorA-BtuF Synporter protein (MW = 37.22 kDa) and the TorA-GlmS Synporter protein (MW = 22.6 kDa) contain both a polyhistidine-tag. The expression could therefore be shown with Western Blot analysis.
For both Synporter proteins the expected protein bands were detected (Fig. 1).
Figure 1: Western blot analysis of Synporter proteins. For BtuF, the samples were taken from different cultures, which were induced with different concentrations of arabinose (C0-C4). The samples of GlmS were taken before and after induction with arabinose.
Additionally, to test the function of our Synporter proteins, we also performed a photometric B12 assay.
Significantly higher amounts of Vitamin B12 in the cytoplasm and in the periplasm could be detected, when the Synporter proteins were expressed (Fig. 2).
Figure 2: Figure 6: Photometric assay. The relative Vitamin B12 amount in the cytoplasm (left) and periplasm (right) of aerobic and anaerobic S. blattae and R. planticola cultures with BtuF and GlmS plasmids was compared to a control culture with an empty plasmid. The OD was measured at 367 nm.