Part:BBa_K5222012:Design
PETase-MHETase B3-B5
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 424
Illegal PstI site found at 1045
Illegal PstI site found at 2281 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 2178
Illegal PstI site found at 424
Illegal PstI site found at 1045
Illegal PstI site found at 2281 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 424
Illegal PstI site found at 1045
Illegal PstI site found at 2281 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 424
Illegal PstI site found at 1045
Illegal PstI site found at 2281 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Recently, Japanese researchers identified the bacterium Ideonella sakaiensis, which is capable of producing the enzymes necessary to break down PET plastic into its two main components: TPA and EG. This transformation is made possible by the expression of two specific enzymes, named PETase and MHETase. Here we created a fusion protein PETase-MHETase optimized for Chlamydomonas reinhardtii. Also this CDS is adapted for B3-B5 positions in MoClo To introduce this CDS into Chlamydomonas, we will employ the MoClo technique to construct the necessary plasmid for transformation. Our CDS needs to be adapted for MoClo assembly, so we plan to insert our gene into positions B3-B5 of the MoClo system. To do this, we added appropriate fusion sites at the ends of the sequence. At the 5' end, we incorporated a B3 scar (AATG) along with the BbsI restriction site, and at the 3' end, a B5 scar with a BbsI restriction site (see Chlamy Guide made by 2019 iGEM Sorbonne University team for more details). Additionally, to prevent any internal cleavage of our gene during the digestion/ligation process, we removed internal type IIS restriction sites (BsaI, BbsI, and SapI
Source
pCJ190 as initial code