BBa_K2020004 1 subE_S221X mutated expression system for subtilisin E in E. coli (S221X) 2016-10-07T11:00:00Z 2016-10-08T11:53:10Z The sequences of the BioBrick parts were obtained through the Registry. {| class="wikitable" style="text-align:center" |- ! part !! colspan="2"| BioBrick No. |- | LacI promoter || colspan="2"| [[Part:BBa_R0010|BBa_R0010]] |- | RBS || colspan="2"| [[Part:BBa_B0034|BBa_B0034]] |- | rowspan="2" | CDS || colspan="2"| [[Part:BBa_K2020001|BBa_K2020001]] |- | [[Part:BBa_J32015|BBa_J32015]] || [[Part:BBa_K2020000|BBa_K2020000]] |- | terminator || colspan="2"| [[Part:BBa_B0010|BBa_B0010]] |- |} The construct was ordered at IDT. This composite part consists of the promoter [[Part:BBa_R0010|BBa_R0010]], the ribosome binding site [[Part:BBa_B0034|BBa_B0034]], the newly created BioBrick part [[Part:BBa_K2020001|BBa_K2020001]] and the terminator [[Part:BBa_B0010|BBa_B0010]]. BioBrick BBa_K2020001 is a composite part itself and includes the secretion tag pelB ([[Part:BBa_J32015|BBa_J32015]]) and a subtilisin E gene optimized for ''Escherichia coli'' codon usage ([[Part:BBa_K2020000|BBa_K2020000]]). Once introduced into ''E. coli'', this BioBrick is able to produce an inactive version of subtilisin E and simultaneously secret the enzyme into the periplasm of the cell. Subtilisin E is an alkaline serine protease which non-specifically digests proteins. By performing a site-directed mutagenesis, serine in the catalytic triade of the enzyme was exchanged against the amber stop codon UAG. Therefore, the expression of the enzyme is interrupted. This composite part was created to integrate a non-canonical amino acid into the sequence of subtilisin E by adding an orthogonal tRNA/aminoacyl-synthetase pair that is capable of incorporating the non-canonical amino acid of choice at the UAG codon. false false _2487_ 30763 30763 9 false The sequence of the subtilisin E gene from ''Bacillus subtilis'' was codon optimized for ''E. coli'' with the DNA and protein sequence analysis software "Geneious" and additionally with the "Codon Optimization Tool" from IDT. The sequence was partly ordered from IDT ([[Part:BBa_K2020001|BBa_K2020001]] + [[Part:BBa_B0010|BBa_B0010]]) and then cloned into [[Part:BBa_J04500|BBa_J04500]], a protein expression backbone which already carries the LacI promoter [[Part:BBa_R0010|BBa_R0010]] and the ribosome binding site [[Part:BBa_B0034|BBa_B0034]]. Afterwards, a mutation in the active site of the enzyme was introduced by performing a site-directed mutagenesis. The codon AGC of serine²²¹ was substituted with the stop codon TAG. false Nicola Freyer, Lea Steinbeck, Svenja Meyer, Alexander Deitert annotation2488230 1 BBa_B0034 range2488230 1 209 220 annotation2488234 1 AGC1182TAG range2488234 1 1182 1184 annotation2488231 1 PelB range2488231 1 227 291 annotation2488232 1 propeptide range2488232 1 293 522 annotation2488235 1 BBa_B0010 range2488235 1 1360 1439 annotation2488233 1 subtilisin E range2488233 1 523 1351 annotation2488229 1 LacI range2488229 1 1 200 BBa_K2020004_sequence 1 caatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacatactagagaaagaggagaaatactagatgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcgggcaaaagcagcaccgaaaaaaaatatattgtgggctttaaacagaccatgagcgcgatgagcagcgcgaaaaaaaaagatgtgattagcgaaaaaggcggcaaagtgcagaaacagtttaaatatgtgaacgcggcggcggcgaccctggatgaaaaagcggtgaaagaactgaaaaaagatccgagcgtggcgtatgtggaagaagatcatatcgcgcatgaatatgcccagagcgtgccgtatggcattagccagattaaagccccggcgctgcatagccagggctataccggcagcaacgtgaaagtggcggtgattgatagcggcattgatagcagccatccggatctgaacgtgcgcggcggcgcgagctttgtgccgagcgaaaccaacccgtatcaggatggcagcagccatggcacccatgtggcgggcaccattgcggcgctgaacaacagcattggcgtgctgggcgtgagcccgagcgcgagcctgtatgcggtgaaagtgctggatagcaccggcagcggccagtatagctggattattaacggcattgaatgggcgattagcaacaacatggatgtgattaacatgagcctgggcggcccgaccggcagcaccgcgctgaaaaccgtggtggataaagcggtgagcagcggcattgtggtggcggcggcggcgggcaacgaaggcagcagcggcagcaccagcaccgtgggctatccggcgaaatatccgtcaacgattgctgttggcgccgtaaattcaagcaatcagcgtgcgtcattctcatccgcaggtagcgaactggatgtgatggcgccgggcgtgagcattcagagcaccctgccgggcggcacctatggcgcgtataacggcacctagatggcgaccccgcatgtggcgggcgcggcggcgctgattctgagcaaacatccgacctggaccaacgcgcaggtgcgcgatcgcctggaaagcaccgcgacctatctgggcaacagcttttattatggcaaaggcctgattaacgtgcaggcggcggcgcagtaatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc 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