BBa_K1831000 1 BBa_K1831000 NpuDnaE intein for circularization of Type lll AFP+ inker 2015-08-29T11:00:00Z 2015-09-26T08:06:17Z This part was derived from BBa_K1362000, a composite part submitted by Team Heidelberg 2014's NpuDnaE intein RFC[105] circularization construct. We modified the part by adding part BBa_I14018, a constitutive T7 promoter with a medium transcription level prior to the RBS. NpuDnaE intein circularization construct using Type III AFP and a computer-optimized linker. To further characterize the circularization construct developed by Team Heidelberg 2014, a moderately active, Type lll antifreeze protein (AFP) from (PDB: 1AME) was used to replace the RFP initially in the part. While the Type lll AFP normally denatures and loses activity at 37C, the AFP expressed from this part retained 78% of its activity even after heating to 90C. This shows that this construct effectively creates circular proteins which have increased stability over a wide range of temperatures. false false _2257_ 27229 27228 9 false To ensure that the circular AFP would not be strained, and potentially lose function, several linker sequences ranging from 1 to 6 amino acids were modeled using Growmax. The linkers were developed using a modified version of Team Heidelberg 2014's linker generator software, CRAUT. After the potential linkers were compared in Growmax, it was determined that adding one glycine to the extein scar least disturbed the ice-binding residues of the AFP and this linker was used in part BBa_K1831000 This construct was designed to circularize any linear protein, however part BBa_K1362000 from which is was based lacked a T7 promoter. Therefore sub-cloning of the construct would be required by other teams prior to expression of their circular protein. QGEM 2015 was able to add a constitutive T7 promoter, part BBa_I14018, to simplify the use of the NpuDnaE intein for future teams. false Joanna Semrau, Justine Ring, Morgan Litschko, Malak Elbatarny annotation2441265 1 BsaI scar range2441265 1 186 188 annotation2441270 1 BsaI scar range2441270 1 411 413 annotation2441262 1 BBa_I712074 range2441262 1 1 46 annotation2441268 1 Type III AFP range2441268 1 201 401 annotation2441269 1 Extein(N) range2441269 1 402 410 annotation2441271 1 NpuDnaE(N) range2441271 1 414 714 annotation2441264 1 NpuDnaE(C) range2441264 1 123 185 annotation2441263 1 BBa_K1362090 range2441263 1 47 122 annotation2441266 1 Extein(C) range2441266 1 189 197 annotation2441272 1 T7 terminator range2441272 1 715 746 annotation2441267 1 Linker range2441267 1 198 200 BBa_K1831000_sequence 1 taatacgactcactatagggaatacaagctacttgttctttttgcaataattttgtttaactttaagaaggagatactagatgatcaaaatagccacacgtaaatatttaggcaaacaaaatgtctatgacattggagttgagcgcgaccataattttgcactcaaaaatggcttcatagcttccaactgctgggaaggcgcggcgaaccaggcgagcgtggtggcgaaccagctgattccgattaacaccgcgctgaccctggtgatgatgcgcagcgaagtggtgaccccggtgggcattccggcggaagatattccgcgcctggtgagcatgcaggtgaaccgcgcggtgccgctgggcaccaccctgatgccggatatggtgaaaggctatgcggcgcgtggtaaatgcttaagctatgaaacggaaatattgacagtagaatatggattattaccgattggtaaaattgtagaaaagcgcatcgaatgtactgtttatagcgttgataataatggaaatatttatacacaacctgtagcacaatggcacgatcgcggagaacaagaggtgtttgagtattgtttggaagatggttcattgattcgggcaacaaaagaccataagtttatgactgttgatggtcaaatgttgccaattgatgaaatatttgaacgtgaattggatttgatgcgggttgataatttgccgaattaataaatcggtgaaatgcacgactgatag igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 James Alastair McLaughlin Chris J. Myers 2017-03-06T15:00:00.000Z