LB agar petri dish containing the carbenicillin, X-GAL and IPTG and cultured overnight. Blue-white screen was used to screen out transformant colonies and identify recombinants. Recombination frequency (RF) and transformation efficiency (TE) were calculated using the following formulas:

Due to time limit, we were provided with E.coli (pTaq2), a strain containing the taq gene where it can be overexpressed from the T7 promoter. E.coli (pControl) containing a control plasmid without the taq gene was also provided. Overnight cultures of both strains were diluted with 50mls L-broth containing the carbenicillin (100μg/ml) and grown to OD 0.6, before protein expression was induced with IPTG. After an overnight production of Taq polymerase we purified the enzyme at 37°C for 30 minutes to denature cellular E.coli proteins. The expression of Taq DNA polymerase enzyme was demonstrated by comparing the SDS-PAGE of the proteins present in respective samples. 5μl and 20μl amounts of each sample were loaded. Two groups shared one SDS-PAGE gel. Unstained protein molecular weight marker (NEB P 7704S) was used. Protein bands were visualized by 1 hour staining in Coomassie Blue Protein Stain, followed by overnight destaining in distilled water.

For testing the enzyme activity of Taq polymerase, 35-cycle PCR amplifications were performed with Taq polymerase purified from E.coli (pTaq2) or commercial polymerase. Negative control PCR were set up with E.coli (pControl) cell lysate with template DNA or commercial Taq polymerase without template DNA. Besides the different component, all PCR reaction contained 0.2μM forward primer, 0.2μM reverse primer, 1×PCR buff, 200μM dNTPs and a certain amount of distill water to set up a total volume of 30μl. PCR products were revealed by electrophoresis in 1% agarose gels.