BBa_K2020003 1 subE_S221Y mutated expression system for subtilisin E in E. coli (S221Y) 2016-10-04T11:00:00Z 2016-10-05T12:05:25Z 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 tyrosine. Therefore, the enzyme looses its proteolytic activity. 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 TAC which codes for tyrosine. false Nicola Freyer, Lea Steinbeck, Svenja Meyer, Alexander Deitert annotation2486160 1 BBa_B0010 range2486160 1 1360 1439 annotation2486161 1 AGC1182TAC range2486161 1 1182 1184 annotation2486156 1 BBa_B0034 range2486156 1 209 220 annotation2486159 1 subtilisin E range2486159 1 523 1351 annotation2486157 1 PelB range2486157 1 227 292 annotation2486155 1 LacI range2486155 1 1 200 annotation2486158 1 propeptide range2486158 1 293 522 BBa_K2020003_sequence 1 caatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacatactagagaaagaggagaaatactagatgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcgggcaaaagcagcaccgaaaaaaaatatattgtgggctttaaacagaccatgagcgcgatgagcagcgcgaaaaaaaaagatgtgattagcgaaaaaggcggcaaagtgcagaaacagtttaaatatgtgaacgcggcggcggcgaccctggatgaaaaagcggtgaaagaactgaaaaaagatccgagcgtggcgtatgtggaagaagatcatatcgcgcatgaatatgcccagagcgtgccgtatggcattagccagattaaagccccggcgctgcatagccagggctataccggcagcaacgtgaaagtggcggtgattgatagcggcattgatagcagccatccggatctgaacgtgcgcggcggcgcgagctttgtgccgagcgaaaccaacccgtatcaggatggcagcagccatggcacccatgtggcgggcaccattgcggcgctgaacaacagcattggcgtgctgggcgtgagcccgagcgcgagcctgtatgcggtgaaagtgctggatagcaccggcagcggccagtatagctggattattaacggcattgaatgggcgattagcaacaacatggatgtgattaacatgagcctgggcggcccgaccggcagcaccgcgctgaaaaccgtggtggataaagcggtgagcagcggcattgtggtggcggcggcggcgggcaacgaaggcagcagcggcagcaccagcaccgtgggctatccggcgaaatatccgtcaacgattgctgttggcgccgtaaattcaagcaatcagcgtgcgtcattctcatccgcaggtagcgaactggatgtgatggcgccgggcgtgagcattcagagcaccctgccgggcggcacctatggcgcgtataacggcacctacatggcgaccccgcatgtggcgggcgcggcggcgctgattctgagcaaacatccgacctggaccaacgcgcaggtgcgcgatcgcctggaaagcaccgcgacctatctgggcaacagcttttattatggcaaaggcctgattaacgtgcaggcggcggcgcagtaatactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc 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