BBa_K2020006 1 subE_Y77X mutated expression system for subtilisin E in E. coli (Y77X) 2016-10-07T11:00:00Z 2016-10-08T12:30:28Z 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, tyrosine⁷⁷ in the propeptide of the enzyme was exchanged against the amber stop codon UAG. Therefore, the expression of the enzyme is interrupted. <br> This composite part was created to integrate a non-canonical amino acid into the sequence of the propeptide 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 TAT of tyrosine⁷⁷ was substituted with the stop codon TAG. false Nicola Freyer, Lea Steinbeck, Svenja Meyer, Alexander Deitert annotation2488255 1 subtilisin E range2488255 1 523 1351 annotation2488257 1 BBa_B0010 range2488257 1 1360 1439 annotation2488248 1 LacI range2488248 1 1 200 annotation2488249 1 BBa_B0034 range2488249 1 209 220 annotation2488250 1 PelB range2488250 1 227 292 annotation2488251 1 propeptide range2488251 1 293 522 annotation2488252 1 TGG520TAG range2488252 1 520 522 BBa_K2020006_sequence 1 caatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacatactagagaaagaggagaaatactagatgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcgggcaaaagcagcaccgaaaaaaaatatattgtgggctttaaacagaccatgagcgcgatgagcagcgcgaaaaaaaaagatgtgattagcgaaaaaggcggcaaagtgcagaaacagtttaaatatgtgaacgcggcggcggcgaccctggatgaaaaagcggtgaaagaactgaaaaaagatccgagcgtggcgtatgtggaagaagatcatatcgcgcatgaataggcccagagcgtgccgtatggcattagccagattaaagccccggcgctgcatagccagggctataccggcagcaacgtgaaagtggcggtgattgatagcggcattgatagcagccatccggatctgaacgtgcgcggcggcgcgagctttgtgccgagcgaaaccaacccgtatcaggatggcagcagccatggcacccatgtggcgggcaccattgcggcgctgaacaacagcattggcgtgctgggcgtgagcccgagcgcgagcctgtatgcggtgaaagtgctggatagcaccggcagcggccagtatagctggattattaacggcattgaatgggcgattagcaacaacatggatgtgattaacatgagcctgggcggcccgaccggcagcaccgcgctgaaaaccgtggtggataaagcggtgagcagcggcattgtggtggcggcggcggcgggcaacgaaggcagcagcggcagcaccagcaccgtgggctatccggcgaaatatccgtcaacgattgctgttggcgccgtaaattcaagcaatcagcgtgcgtcattctcatccgcaggtagcgaactggatgtgatggcgccgggcgtgagcattcagagcaccctgccgggcggcacctatggcgcgtataacggcaccagcatggcgaccccgcatgtggcgggcgcggcggcgctgattctgagcaaacatccgacctggaccaacgcgcaggtgcgcgatcgcctggaaagcaccgcgacctatctgggcaacagcttttattatggcaaaggcctgattaacgtgcaggcggcggcgcagtaatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc 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