DNA amplification product was observed. Successful amplification of DNA template via PCR validated the feasibility of producing Taq polymerase in recombinant E.coli.
Conclusion
In conclusion, the technologies involved in this lab practical include mini-prep DNA isolation,EcoRI restriction digest, gel electrophoresis, gel purification, plasmid recombination, plasmid transformation, protein overexpression, protein extraction, SDS-PAGE analysis and PCR. Following the standard procedures of cloning and overexpressing taq gene in E.coli, we successfully produced a considerable amount of Taq DNA polymerase with high enzyme activity. Firstly, we extracted plasmids containing the gene encoding Taq polymerase fromE.coli (pTaq1) cells via mini-prep method. Purified plasmids were digested by restriction enzyme EcoRI to produce DNA fragments containing the taq gene, which was then harvested by gel extraction after electrophoresis. Subsequently, taq DNA was ligated to pETBlue-1 vector and transformed into E.coli cells, where the gene was overexpressed. Despite the poor insert gene purity and low transformation efficiency, Taq polymerase was successfully produced in and purified from E.coli, along with high enzyme activity in PCR performance.
MethodsBacterial cell culture
E.coli (pTaq1) is an Escherichia coli strain that harbours plasmid containing the gene encoding Taq polymerase. E.coli (pTaq1) cells were cultured in L-broth with the antibody carbenicillin at 37°C overnight. Carbenicillin screened out bacteria harbouring the plasmid pTaq1 since this plasmid contains resistance gene against carbenicillin. In our experiment plasmid DNA was extracted from E.coli cultures prepared by the students (culture S) or the demonstrator (culture D). Culture S came from only one single bacterial colony while culture D had a significantly higher cell concentration.
Plasmid DNA extraction
Plasmid DNA was extracted from bacteria cells through Min-prep procedures. Mini-prep isolation protocol is ideal for small plasmid DNA purification and it limits contamination of proteins and genomic DNA [3]. Bacteria cells were harvested from 5 ml of cell suspension by centrifugation. QIA spin prep kits (Qiagen 27104) were used to purified plasmid pTaq1