Part Documentation
We have submitted two parts that encode the enzymes involved in the D-isoascorbic acid biosynthesis pathway. Both parts have had their codon usage optimized for high expression in a Pichia pastoris chassis.
Gene optimization was carried out to overcome codon bias. We chose to use the COOL web tool instead of the service provided by vendors because we wanted to optimize our sequences based on codon context and individual codon usage instead of codon adaptation index as recommended by Pek et al (2015). As per the work of Bai et al (2011) we used a codon table comprised only of highly expressed Pichia pastoris genes and set the sequence's GC content to match that of the table's genes, id est, 45%.COOL outputs a list of possible sequences, which we purged by choosing the sequence that most closely matched our optimization parameters and that did not include any forbidden or common restriction sites (to comply with RFC 10).
Part Table
iGEM015 Example
Glucose oxidase
This part encodes a glucose oxidase originally from Aspergillus niger. This enzyme catalyzes D-glucose's oxidation to D-glucono-1,5-lactone.
ATGCAGACTTTGCTAGTTAGTAGTTTAGTAGTTAGCTTAGCCGCAGCTTTGCCACACTACATTAGGTCTAACGGTATTGAAGCTAGTTTGCTGACTGACCCTAAAGACGTCAGCGGTAGAACTGTGGACTACATCATTGCTGGTGGCGGATTGACTGGTTTGACCACTGCTGCTAGATTGACCGAGAACCCAAACATTAGTGTTCTTGTCATTGAATCCGGTTCTTACGAGTCTGACAGAGGTCCAATCATCGAAGACTTGAATGCTTACGGAGATATCTTTGGTTCTTCCGTTGACCACGCATATGAGACCGTCGAATTGGCTACTAATAACCAAACTGCTCTGATTAGATCCGGTAACGGTTTAGGTGGTAGCACCTTAGTTAACGGCGGTACTTGGACTAGACCACATAAGGCTCAAGTAGATAGTTGGGAGACCGTCTTCGGTAATGAGGGTTGGAACTGGGATAACGTAGCAGCGTATAGCTTGCAAGCTGAAAGAGCTAGAGCACCTAATGCTAAGCAAATAGCTGCTGGACATTACTTTAACGCTAGTTGCCACGGTGTTAATGGTACCGTTCATGCTGGTCCACGTGACACCGGAGATGACTATAGTCCTATAGTTAAGGCCTTGATGAGTGCCGTTGAAGATAGAGGTGTTCCAACTAAGAAGGACTTCGGTTGTGGTGACCCCCACGGCGTTAGTATGTTCCCTAACACACTGCATGAGGATCAAGTTAGAAGTGATGCCGCCCGTGAATGGTTGTTACCTAATTACCAAAGACCTAACCTGCAAGTCTTGACAGGTCAATACGTAGGTAAGGTTCTGTTGAGTCAGAATGGTACCACTCCTAGAGCCGTAGGAGTTGAGTTCGGAACGCATAAGGGTAACACTCATAACGTTTACGCTAAGCATGAAGTTTTACTAGCGGCGGGTAGTGCTGTTAGTCCAACCATTTTAGAGTATAGTGGAATAGGTATGAAGTCTATCCTTGAGCCACTAGGTATTGATACTGTAGTTGACCTGCCAGTAGGGTTGAACTTGCAAGACCAAACAACTGCCACAGTGCGTAGTAGGATAACCTCTGCCGGTGCTGGTCAAGGCCAGGCTGCCTGGTTCGCCACTTTTAATGAAACCTTCGGTGACTATAGTGAGAAGGCTCATGAACTACTGAATACTAAACTAGAGCAATGGGCTGAGGAGGCTGTAGCTAGAGGAGGTTTCCATAACACTACCGCTCTTTTGATTCAATATGAGAACTATAGAGACTGGATAGTTAACCATAACGTAGCATATAGTGAATTGTTCTTAGACACAGCGGGAGTAGCTAGTTTTGACGTTTGGGACTTGCTTCCATTCACTAGGGGTTACGTCCACATCTTAGATAAAGATCCATACTTGCACCACTTTGCCTATGATCCTCAGTACTTCTTGAATGAGCTTGACTTGTTAGGTCAAGCCGCTGCCACCCAATTAGCTAGAAACATTAGTAATAGTGGTGCCATGCAAACCTACTTCGCTGGTGAGACTATTCCAGGTGACAACTTGGCTTACGACGCTGATCTTTCGGCTTGGACTGAGTACATTCCTTACCATTTCAGACCTAACTACCATGGAGTAGGAACTTGTAGCATGATGCCTAAGGAGATGGGTGGAGTAGTAGATAATGCCGCTAGAGTCTACGGTGTCCAAGGTCTGAGAGTTATTGATGGTTCCATTCCACCAACTCAAATGTCTTCTCACGTTATGACTGTTTTCTACGCTATGGCTCTCAAGATTTCTGATGCTATTTTGGAGGACTACGCCTCCATGCAATAA
Gluconolactone oxidase
This enzyme is responsible for the further oxidation of D-glucono-1,5-lactone to D-keto-glucono-1,5-lactone. The original sequence belongs to Penicillium griseoroseum.
ATGTTGTCTCCAAAACCAGCTTTCTTGTTGTTGCTTTTGCACGCTGTTTTCGGTTCTGCCTACAGATGGTTCAACTGGCAATTCGAGGTTACTTGTCAGTCTGATGCTTACATTGCCCCACATAACGAGCACGCCGCTGCTGAGTTCTTGAAAGAGCAATATCCTAAGTCTTCTCACATTAAGGTTGTTGGTAATGGTCACGGTTTCGGCAATCTCACTACCTGTGTTGATAATGCGTTGACTGAGAAGCCAACGTACATTGTTTCTCTTACCAACTTAAAGAAGCTGCATATTGACAAGAAGAACTTGACTGTCACTTTCGGTGCTGGTTGGGATGTTGACGACTTGATCCAAGAGCTTAAGGCCAACGATCTGAGTTTTTCCAACCTGGGAGTCGAGAGAGTGCAAAACTTTGTAGGTGCAGCTTCTACTGGTACCCATGGTTCCGGTTCTGACCTTGGCAACATCGCTACCCAAATCATTGGTTTACGTGTTTTGGACTCTCAAGGTGGTTTGAGAGTTATCAACGAAAAGCACAATGCTGAGGAACTCAAGGCTTTCAGAATCAGTTTGGGTGCTTTGGGTTTAATTACCGAGCTGACTATCAAGGTGCAGCCAACCCAGCTTCTAAAAAAGACTACCAAGGTCCTCAATGCTACCAGCGACTACTCCAAGATGTACAACGAGTTAGCTCAACTTTACAAGGAACACGACAGAATGACTGTCTGGGGTCCTCATTTCGATTGGAATGCGAAATCCCAGTCTTGGGACTTGGAGCCAACTTACTTTCTGTCATATTGGGAACCTACAAACTATACCGGCGTCAGAAACTGCACTCTGAACTATTGTGCCAACGGATGTGGTGACTGTAAAAAGGAGTACATATGTTACGACGAAGTCACCGATGCCGCCTCCTGCTCCCCTCAAGGAGTTTGTTCTCGTGGATTCTATGCTGAAATTGAACATTTCTTGCCTATCGAATACTTCGCTGAGGCTGCTACTAACTACACCATTTTCCAACAAGGACAAACTTCAAGAATGAAGGCTCCATACAACAAGCAAATGGTTATGCAGCACAGATCATTGAAAGGTGATGACACATACTTGTCCCCAGTTAACACCTACAACCTTGGGCCGGATTTGTCTGGTGTTTTCGGTGTCATCGAGATTGATTGGATTCAAGAATACAACAACTTCACCACGTTGTGGCAGAACCAAGAATTGGCCCACGAGTTTTTGCCTCAATTTGGAGAGACTTACAATGCTCGTTCCCACTGGAATAAGATGTCTGCCCCCAACGCCACTTACACTCTCGAAAAGTTCCCAAAGCTGCCAGAGTTTTTGGCTATTCAAAAAAGACAAGATCCAAAGTGTCAGTTCGTCAACGAGTTTTTGGTCGAACAGTTGGGTATTACACGTTGTGCCAACTACATCTCGGTGTAA
