Team:XH-China/Engineering
Engineering
During our implementation of our project, we are using synthetic biology and error analysis methods to make our experiment procedure concise, efficient, achieving engineering success, the processes of which are illustrated below.
Experiment design
A. Our group projected to auxotrophic bacteria, which requires extra organic growth factors and supplements on the culture medium. The auxotroph is unable to synthesize itself for its mutation. Therefore, using mutant bacteria enables us to regulate their enzyme synthesis by manipulating the availability of supplements.
B. We turned to an alternative plan. Yeast auxotroph was readily available in this company.
C. It is better to use E.coli to achieve optimal results. We may add lactose operons or kill switches to attain the same effects.
During the experiment
1) At the beginning of the experiment：
A. We extracted the plasmid sent from the outsourcing company, and ran agar-gel electrophoresis (Nucleic acid electrophoresis) after the double enzyme digestion. In order to test whether the plasmid that had been introduced into E. coli contained the target gene, the experimental result was that it contained the target gene, and ran protein electrophoresis after induced by IPTG for three hours, but it was found that no protein was expressed.
B. Then double enzyme digestion was performed and separated to transfer the target gene to a new plasmid containing his-tag and ampicillin resistance genes.
2) When we tried to insert the target genes to the plasmid vector：
A. We extracted plasmids from cultured Escherichia coli without the target gene insertion, after digest with two restriction endonuclease BamHI/KpnI,the reaction sample was run electrophoresis with low melting point agar-gel to separate and purified the linear DNA fragment plasmid. Meantime, the plasmids containing the target gene also digest by above 2 endonuclease, then the reaction run electrophoresis to separate and purified target gene linear DNA fragment. After linear plasmid DNA and target gene DNA fragment harvest, the ligation of above two fragments had been performed.
Then we collect more target gene DNA band from electrophoresis gel to increase the quantity of target gene DNA to meet the ligation need.
3)When we are using E.coli to produce keratinase protein：
A. We use E.coli DH5α to amplify plasmid and use E.coli Rossetta as protein expression host cell. Above E.coli could grow in culture medium with ampicillin because carrying plasmid with ampicillin resistance.
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B. Then a single colony picked on LB culture disk, inoculated in culture medium. When the bacteria grow to log growing phase (106CFU/ml), add inducer IPTG to one tube, and leave the other tube untreated. After induction 5-7 hours, harvest the bacteria and run electrophoresis on the SDS-PAGE gel. It was found that the target protein band showed on the gel with expected size comparing nothing show on SDS-PAGE gel loaded by sample without induction.
4)When we found that Rosetta with the inducer did not express keratin
A.Rosetta was induced in a large culture flask and run electrophoresis to confirm the protein produced in E.coli Rosetta by western blot .The band from western blot show not significant strong, which maybe caused by lower protein produce.
B. We confirm the protein produced in E.coli with recombinant plasmid. We inoculate host bacteria in 1L large culture flask, IPTG induce 3-5 hours, harvest and purify target protein keratinase and tested.