BBa_K2020005 1 subE_Y77W mutated expression system for subtilisin E in E. coli (Y77W) 2016-10-07T11:00:00Z 2016-10-08T12:13:18Z 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 tryptophan. Therefore, the enzyme loses its proteolytic activity. <br> With this composite part, iGEM Aachen 2016 was able to prove that tyrosine⁷⁷ in the propeptide of subtilisin E is essential for the activity of the enzyme. 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 TGG which codes for tryptophan. false Nicola Freyer, Lea Steinbeck, Svenja Meyer, Alexander Deitert annotation2488242 1 BBa_B0010 range2488242 1 1360 1439 annotation2488240 1 TAT520TGG range2488240 1 520 522 annotation2488241 1 subtilisin E range2488241 1 523 1351 annotation2488238 1 PelB range2488238 1 227 292 annotation2488236 1 LacI range2488236 1 1 200 annotation2488237 1 BBa_B0034 range2488237 1 209 220 annotation2488239 1 propeptide range2488239 1 293 522 BBa_K2020005_sequence 1 caatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacatactagagaaagaggagaaatactagatgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcgggcaaaagcagcaccgaaaaaaaatatattgtgggctttaaacagaccatgagcgcgatgagcagcgcgaaaaaaaaagatgtgattagcgaaaaaggcggcaaagtgcagaaacagtttaaatatgtgaacgcggcggcggcgaccctggatgaaaaagcggtgaaagaactgaaaaaagatccgagcgtggcgtatgtggaagaagatcatatcgcgcatgaatgggcccagagcgtgccgtatggcattagccagattaaagccccggcgctgcatagccagggctataccggcagcaacgtgaaagtggcggtgattgatagcggcattgatagcagccatccggatctgaacgtgcgcggcggcgcgagctttgtgccgagcgaaaccaacccgtatcaggatggcagcagccatggcacccatgtggcgggcaccattgcggcgctgaacaacagcattggcgtgctgggcgtgagcccgagcgcgagcctgtatgcggtgaaagtgctggatagcaccggcagcggccagtatagctggattattaacggcattgaatgggcgattagcaacaacatggatgtgattaacatgagcctgggcggcccgaccggcagcaccgcgctgaaaaccgtggtggataaagcggtgagcagcggcattgtggtggcggcggcggcgggcaacgaaggcagcagcggcagcaccagcaccgtgggctatccggcgaaatatccgtcaacgattgctgttggcgccgtaaattcaagcaatcagcgtgcgtcattctcatccgcaggtagcgaactggatgtgatggcgccgggcgtgagcattcagagcaccctgccgggcggcacctatggcgcgtataacggcaccagcatggcgaccccgcatgtggcgggcgcggcggcgctgattctgagcaaacatccgacctggaccaacgcgcaggtgcgcgatcgcctggaaagcaccgcgacctatctgggcaacagcttttattatggcaaaggcctgattaacgtgcaggcggcggcgcagtaatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc 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