Part:BBa_K5094010

pGal1, 10-IsPETase (BBa_K5094001-BBa_K5094002)

After amplifying the wild type of IsPETase gene via PCR and using an enzyme digestion strategy to clone it to the downstream of the pGal1,10 promoter to make a composite part, BBa_K5094010 was used to make several mutations on the IsPETase gene via site-directed mutagenesis, and was transformed into a yeast strain, Saccharomyces, for functional assays. The team used BBa_K5094010 as a DNA template to make mutations but failed due to the large size of the pGal1,10 promoter in the ~7kb plasmid. The team will further optimize several primer sets of annealing conditions for the site-directed mutagenesis.

Fig4: After bacterial transformation for pGal-IsPETase cloning, bacteria colonies were picked up directly to do IsPETase PCR. Lane 2 was IsPETase PCR product control. 2 colonies on lanes 5 and 6 had BBa_K5094010 composite part (full-length IsPETase PCR product)

Fig6: After sequencing, the team only created pGal1,10-IsPETase plasmid (lanes2,3,4), and lane1 is a 1kb DNA ladder.

Fig 7b: The mRNA induction of IsPETase didn’t exhibit significant induction at different time courses in the presence of galactose compared to the 0’ sample with glucose only.

Fig 9b: The co-cultured experiment data didn’t show a clear pattern of the TPA product increasing in the BY4741 yeast strain containing pGal1,10-IsPETase in the presence of galactose during the time course experiment. The experimental control, BY4741 yeast strain containing pGal1,10-eGFP, either in glucose or galactose, should not show any TPA product increase due to lack of the IsPETase. However, the team did detect a small amount of TPA products in both co-cultured mediums. A similar unclear pattern of TPA products was observed in BY4741 containing pGal1,10-IsPETase either in glucose or galactose.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 891
  Illegal AgeI site found at 286
  
• 1000
  COMPATIBLE WITH RFC[1000]