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.
We design four kinds of integrated plasmid and succeed in demonstrating function of three, GFP-TGATG-gdh (A), and GFP-TGATG-T7 RNA polymerase-PT7-gdh (C). We cultivated bacterias contains these plasmid and their control group in the same condition and value their integral function by monitoring the fluorescent density and its ratio to the OD value.
In integrated plasmid A, GFP is co-expressed with gdh under start-stop codon” TGATG” or with Hairpin. Their control groups is a GFP-gdh uncoupling expressing group and a single GFP group. The relatively density (fluorenscent / Optical) result shows that both two kinds of integrated plasmid A can help increase the final relative expression level of GFP.