BBa_M50056 1 BBa_M50056 PETase double mutant I208V and R90A full plasmid information 2016-12-11T12:00:00Z 2016-12-11T11:26:28Z omp-T is a secretion tag native to E.coli. Strong Ribosome binding site (RBS), ampicillin-resistance, rhamnose-inducible promoter, and origin of replication are sequences designed from DNA2.0. Wild type PETase sequence is from Ideonella sakaiensis and is identified by Yoshida et al. Two mutation sites are originally designed by 2016 iGEM Tianjin team: the 208th amino acid (changed from isoleucine(I) to valine(V)) and the 90th amino acid (changed from arginine (R) to alanine (A)), and each individual mutant has been showed by the Tianjin team to We use Escherichia coli as the chassis organism to express the PETase gene and optimize our codon accordingly. This composite part is made of a rhamnose-inducible promoter, an ampicillin resistance marker, an origin of replication, a secretion tag, a strong RBS, and a PETase gene that contains two mutations. All basic parts except mutated PETase come from DNA 2.0. Wild type PETase shows the ability to hydrolyze Polyethylene terephthalate (PET), commonly used for plastics. The two mutations of PETase are identified by the 2016 iGEM Tianjin team to improve the catalytic activity of PET independently. The PETase gene here also incorporates 6 his tag at the end. The sequence is optimized for Escherichia coli. false false _848_ 34425 34425 9 false We would like to test PETase catalytic activties from unlysed cells, so we choose to use a secretion tag. Between the two secretion tags omp-T and YebF, YebF has been documented to interfere with PETase activity by the 2016 iGEM Harvard team. Thus, we choose to use omp-T tag that is also naturally encoded by E.coli. Based on the choice for secretion tag and antibiotic resistance (amp), the only available plasmid from DNA 2.0 synthesis contains rhamnose-inducable promotor and strong RBS. For the PETase gene, our design thinking is based on the hypothesis that the combination of two mutations will further enhance the PETase catalytic activity. 6 His tag is also appended at the end of the PETase sequence for western blotting test. The entire sequence is optimized for Escherichia coli by IDT???s codon optimization tool. false Lucy Maynard, Doris Mai and Amy Weissenbach component2532600 1 BBa_M50054 annotation2532600 1 BBa_M50054 range2532600 1 1 894 BBa_M50054 1 BBa_M50054 PETase double mutant I208V and R90A 2016-12-11T12:00:00Z 2016-12-12T12:59:47Z Wild type PETase sequence is originally from Ideonella sakaiensis. This is a mutant of PETase that combines two mutations designed by the 2016 iGEM Tianjin team. Wild type PETase shows emzyme ability to hydrolyze Polyethylene terephthalate (PET), commonly used for plastics. The two mutations are the 208th amino acid (changed from isoleucine(I) to valine(V)) and the 90th amino acid (changed from arginine (R) to alanine (A)), and each individual mutant has been showed by the Tianjin team to improve the catalytic activity of PET. This sequence combines both mutations, and is optimized for Escherichia coli. false false _848_ 34425 34425 9 false The design is based on the hypothesis that the combination of the two mutations will further enhance the PETase catalytic activity. false Lucy Maynard, Doris Mai and Amy Weissenbach annotation2532581 1 R90A range2532581 1 268 270 annotation2532579 1 stop codon range2532579 1 892 894 annotation2532580 1 I208V range2532580 1 622 624 annotation2532578 1 6 hig tag range2532578 1 874 891 annotation2532577 1 PETase coding region range2532577 1 4 873 annotation2532576 1 start codon range2532576 1 1 3 BBa_M50056_sequence 1 atgaactttccccgcgcttcccgtcttatgcaggcggctgtgttgggcggattaatggcggtcagcgcggcggcaacggctcagacaaatccttatgcacgcggccctaatcccactgccgcttcccttgaagctagtgcaggccctttcaccgttcgctcgtttaccgtgtcccgcccgtccgggtatggcgcggggacggtatattatccgacaaatgccggcggaaccgtcggtgctatcgctatcgtgcctggctacactgctgcccagtcctcaattaaatggtggggaccgcgtttagcaagccatggttttgtcgtaattactatcgatactaattcgacccttgaccaaccgtcctctcgcagcagtcagcagatggcggctcttcgtcaagttgcaagcttgaatgggacatcaagctcgccgatttatggtaaggtggataccgcgcgtatgggcgttatgggctggtcgatgggcggaggtgggtcgttgatttctgcggccaacaaccccagtcttaaagctgctgccccccaagcgccctgggactctagtacgaacttcagttccgtcacagtgcctacattaatctttgcgtgcgagaatgacagcgtagccccggtgaattcaagcgccttaccgatctacgatagtatgtcccgtaatgctaaacagtttttagagattaacggggggtcccactcgtgtgctaattctggcaactcgaatcaggctctgattggcaaaaaaggtgttgcctggatgaaacgctttatggacaatgacactcgctactcgacgttcgcttgtgagaaccccaatagtacgcgtgtcagtgatttccgcaccgcgaactgcagtcaccatcatcaccatcattagtag BBa_M50054_sequence 1 atgaactttccccgcgcttcccgtcttatgcaggcggctgtgttgggcggattaatggcggtcagcgcggcggcaacggctcagacaaatccttatgcacgcggccctaatcccactgccgcttcccttgaagctagtgcaggccctttcaccgttcgctcgtttaccgtgtcccgcccgtccgggtatggcgcggggacggtatattatccgacaaatgccggcggaaccgtcggtgctatcgctatcgtgcctggctacactgctgcccagtcctcaattaaatggtggggaccgcgtttagcaagccatggttttgtcgtaattactatcgatactaattcgacccttgaccaaccgtcctctcgcagcagtcagcagatggcggctcttcgtcaagttgcaagcttgaatgggacatcaagctcgccgatttatggtaaggtggataccgcgcgtatgggcgttatgggctggtcgatgggcggaggtgggtcgttgatttctgcggccaacaaccccagtcttaaagctgctgccccccaagcgccctgggactctagtacgaacttcagttccgtcacagtgcctacattaatctttgcgtgcgagaatgacagcgtagccccggtgaattcaagcgccttaccgatctacgatagtatgtcccgtaatgctaaacagtttttagagattaacggggggtcccactcgtgtgctaattctggcaactcgaatcaggctctgattggcaaaaaaggtgttgcctggatgaaacgctttatggacaatgacactcgctactcgacgttcgcttgtgagaaccccaatagtacgcgtgtcagtgatttccgcaccgcgaactgcagtcaccatcatcaccatcattagtag igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z