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This year LINKS_China employs the previous part RiboJ, BBa_K2615996 into our new constructed ptac promoter. In our experiment, we added three types of ribozymes, RiboJ, SccJ and Sar J as insulators into the circuit of the new ptac promoter.
We first carried out an experiment in the culture medium with glucose as the source of carbon for the cultivation of bacteria in M9 medium. In this experiment, we tested four ptac promoter, BBa_K180000, the new one, the one with RiboJ and with SccJ. We convinced that all for ptac promoters are able to be activated when induced by IPTG, and as the concentration of IPTG increases, the expression of GFP genes promotes.
Furthermore, the addition of RiboJ or SccJ shows prominent improvement of sensitivity towards the concentration of IPTG. At zero concentration of IPTG, all four promoter starts at a similar level, as the concentration of IPTG increases, the gradient of the graph increased dramatically.
Then we substituted the carbon source to glycerol which was applied in our own experiments. We also added another ribozyme, SarJ to investigate their function. These results confirmed the higher sensitivity to IPTG concentration and better adaptation to all carbon source when using the new ptac promoter with ribozymes, RiboJ, SccJ and SarJ.
Then we inserted the ptac-RiboJ into our own plasmid instead of the BBa_K180000. The promoter is placed before pilA and we carried out the extraction experiment of pili to verified the function of the ptac-RiboJ promoter.
We transformed the plasmid with ptac-RiboJ promoter into E.coli BL21. Then, cultivated the pili and expressed in vivo. We extracted them finally to 150mM ethanol-amine and filtrate to obtain the required supernatant. We took a SEM photography of the pili which confirm the growth of pilA as there are many hole-like structure in the photography.