Demonstration
1.
We have designed the basic functional parts including gdh, cI protein, emrR protein and emrR binding promoter. Gdh and cI protein is well character by using SDS-Page gel electrophoresis and western blot. These parts can worked well separately in E.coli when cultivated in LB medium containing appropriate amount of sAmp antibiotics and IPTG inducer.
In our project, gdh works as a growth factor that successfully accelerated the growth of bacteria, which is clearly showed in figure1A. Gdh is a 89kDa protein and in our design, we added V5-tag to the C-terminus of the coding sequence, so we can process the SDS-Page gel electrophoresis and western blot (figure1B) to make sure it is gdh that cause the growth acceleration.
Besides, we created the loop described in Figure 3a to test the function of emrR protein and its binding promoter. Bacteria is induced by SA. By monitoring the fluorescent density and its ratio to the OD value in the bacteria (Figure3), we confirm that emrR protein and its binding promoter can correctly work and have the potential to be used in integrated plasmids.