Part:BBa_K5094002

The wild type of I. Sakaiensis bacteria( IsPETase) gene. Before creating our team’s enhanced mutants of the IsPETase, the wild type of PETase gene needed to clone downstream of either the T7 promoter or the pGal1,10 promoter first via traditional enzyme digestion cloning technique, to generate two composite parts ( BBa_K5094006 and BBa_K5094010), those two composite parts were also the controls for functional assays. Those two composite parts were used as DNA templates for the site-directed mutagenesis to make several mutations of IsPETase.

Fig1: After the team amplified IsPETase with two enzyme cut sites, BamHI1 and HindIII, flanked on the two sides via primers designed through PCR. After PCR, the team ran a sample through 1% agarose gel, IsPETase PCR product showed a single distinct DNA band with a length of 873 base pairs+20 bp of enzyme cut sites designed on primers and showed a nice single band data after PCR amplification. The DNA fragment corresponds with DNA ladders according to the gel electrophoresis. Fig3: After the team amplified IsPETase with two enzyme cut sites, XmaI and SpeI, flanked on the two sides via primers designed through PCR. After PCR, the team ran a sample through 1% agarose gel, IsPETase PCR product showed a single distinct DNA band with a length of 873 base pairs+20 bp of enzyme cut sites designed on primers and showed a nice single band data after PCR amplification. The DNA fragment corresponds with DNA ladders according to the gel electrophoresis.