BBa_K1954004 1 BBa_K1954004 Mutacin III biosynthetic device 2016-10-16T11:00:00Z 2016-10-19T02:13:09Z Our device was based on the sequence of AF154675.1 The biosynthetic locus was engineered by our team in a form allowing for high-yield and fine-tuned expression of mutacin III (Fig. 1). We placed a strong T7 promoter upstream of mutA to obtain high levels of the propeptide, a repressible pTet promoter upstream of the mutBCDP co-transcription unit and an inducible araBAD promoter for the mutT gene, coding for the ATP-binding-cassette-like transporter of mutacin III. All illegal restriction sites were removed from the endogenous sequences by silent mutagenesis. Fig. 1. Simplified diagram of the mutacin III biosynthetic locus designed by UCL iGEM 2016. Mutacin III is a ribosomally synthesized 22 amino acid screw-shaped lanthionine-containing peptide. The biosynthesis of mutacin III involves the expression of the structural gene mutA to make a prepropeptide, comprising a C-terminal propeptide and an N-terminal leader peptide from which the former undergoes processing and the latter is cleaved off before export into the medium (4). The specific post-translational modifications make mutacin III distinct from other bacteriocins and are introduced by enzymes coded for by other genes in the locus (mutBCDP). Basing on sequence homologies of genes in the locus with those of other lantibiotic biosynthetic loci it can be inferred that these enzymes catalyze the dehydration (mutB) and cyclization (mutC) of the propeptide serine and threonine residues which can condense with a neighboring cysteine residue (6) leading to the formation of lanthionine or methyllanthionine (thioether) bridges, respectively. The enzyme coded by mutD catalyzes the oxidative decarboxylation of the C-terminal cysteine residue (7) while the product of mutP is a serine protease which cleaves the leader peptide and is likely the last step in the biosynthesis (8). The mature mutacin III is composed of rings connected by flexible linkers (Fig. 2) which may be important in the mechanism of bacteriocidal activity (9). Following export, the peptide is believed to form transmembrane pores as monomer aggregates leading to membrane disruption and efflux of cellular components (10). The content of anionic phospholipids in the membrane has been suggested to be an important factor influencing initial binding ??? mutacin III has a net positive charge whereas Gram-positive bacteria have a high relative amount of anionic lipids (11). Fig. 2. The structure of mature mutacin III (10). In one investigation of the activity of mutacin-related lantibiotic gallidermin it became clear that lantibiotics are more effective in preventing biofilm formation rather than in exterminating microorganisms already embedded in biofilms (12). To reflect this, our device could be used to transform E. coli cells and employed as an anti-cariogenic strategy in replacement therapy (Fig. 3). Such a novel bacterial strain would demonstrate features of a successful effector strain as it would not cause disease by itself and because it could displace the host pathogenic bacteria. Importantly, there are very few existing examples of lantibiotic resistance compared with antibiotics and only one mechanism of resistance to mutacin III, known as CprRK in Clostridium difficile, has been established (13). Moreover, the fact that a closely related lantibiotic nisin has been shown to exhibit low in vivo toxicity levels (14) and has been widely used as food preservative from as early as mid 1940s (15) further encourages the prospect of considering the employment of mutacin III as an anti-cariogenic agent. false false _2421_ 24377 32880 9 false All restriction sites were removed by silent mutagenesis. false Kamil Żmijewski, Luba Prout annotation2515079 1 T7 promoter range2515079 1 1179 1197 annotation2515085 1 muB range2515085 1 1656 4626 annotation2515084 1 BBa_B0034 range2515084 1 1638 1649 annotation2515093 1 BBa_B1002 range2515093 1 9864 9897 annotation2515076 1 AraC range2515076 1 129 1207 annotation2515090 1 BBa_I0500 range2515090 1 8022 8187 annotation2515081 1 BBa_B0034 range2515081 1 1220 1231 annotation2515073 1 TetR 2 range2515073 1 92 110 annotation2515089 1 rrnB T1 range2515089 1 7872 7943 annotation2515075 1 BBa_I0500 range2515075 1 129 1338 annotation2515067 1 -35 range2515067 1 4 9 annotation2515080 1 Pbad range2515080 1 1208 1338 annotation2515066 1 BBa_I14033 range2515066 1 1 38 annotation2515070 1 mutR range2515070 1 59 913 annotation2515074 1 -10 range2515074 1 109 114 annotation2515077 1 rrnB T1 range2515077 1 958 1029 annotation2515068 1 -10 range2515068 1 27 32 annotation2515086 1 mutC range2515086 1 4615 5889 annotation2515072 1 -35 range2515072 1 86 91 annotation2515078 1 rrnB T2 range2515078 1 1121 1148 annotation2515091 1 BBa_B0034 range2515091 1 8205 8216 annotation2515069 1 BBa_B0034 range2515069 1 47 58 annotation2515087 1 mutD range2515087 1 5913 6479 annotation2515071 1 TetR 1 range2515071 1 67 85 annotation2515083 1 T7 terminator range2515083 1 1507 1554 annotation2515092 1 mutT range2515092 1 8223 9848 annotation2515082 1 mutA range2515082 1 1238 1429 annotation2515088 1 mutP range2515088 1 6484 7827 BBa_K1954004_sequence 1 ggcacgtaagaggttccaactttcaccataatgaaacaagaaagaggagaaaatacaaatgaaggtaaaccaatcaatggagctgggtgaactgtaccgtgaactgcgtatcgctcgtggtttgaagatcaaggatatcgcttgtaagaatctgtccaagtcacaactctctcgttttgaaaatggacaaaccatgttggcagctgataaattgctattagctatttcgggcattcatatgagcttttcggaatttggatatgctttgagccattatgaggagagtgatttcttcaagcgcggtcataagttatcagaattatatgcccagaaggatatcaagggattaaagaagctgctggagttcaacgacaaccatgaggtatttgatgtctacaatcgtctgaacaagctggttattcaagttactattcatttgctagatactgattacataatatcagatgatgataagaatttcctgacaacttacctgtacaatatcgaagagtggactgagtatgaactgtacatcttcgggaatactatgtctatattgtcatctgatgatctgatcttcttaggcaaggcttttgtagaacgtgataagttgtatatatctcttcctagtcataagaagaatgcagagttaaccttcctgaacttaatcctgattctgcttgaacgtaagaagctgtatcaggcaatctacttcgtagagaatctggagaagctgctgaattaccaggatatgttcgcaataacattcctgaagttcctgaagaagatcatcacttacttccatgataagtcagtagatatgtctgaactggagcattacatcaacatcgttgaggagatcaaccctacgatcgcttcaatcctgaagtctaatctgaaccagctgctgtaataacactgatagtgctagtgtaaatcactactagagccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtattgggcgctcttctttataactgataatacgactcactataggggatgtcaaagaatacaagcataaagaggagaaaatacaaatgtcaaacacacaattattagaagtccttggtactgaaacttttgatgttcaagaagatctctttgcttttgatacaacagatactactattgtggcaagcaacgacgatccagatactcgtttcaaaagttggagcctttgtacgcctggttgtgcaaggacaggtagtttcaatagttactgttgttaatgaaagctaagcttgatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttgctgacaggagcaactatatccggatctggcgtaatatccctatcagtgatagagattgacatccctatcagtgatagagatacaaagaggagaaaatactaatgaacgatttccaattccaagattacttcatgtaccgcaagccactgggcaacttctctaacttcttcagtatcactgatacgatggatcccattgagttactacatagtgatccgatatttgctgaaggagtatatttggcctcttcatctcttagagcagccataaataaacttaagaatcatactgcgagtactaaggataaaaagaatgcaagagagactatttttcaatactatgcccgttataacacgagatcaactccgtttggcttgttttcgtccatcggagtaggtgctttctcggcttacctgaagaaggaaaagtctcgttacgaaaagtctatcaacatcgatctgttctgggcttacaaggtagcagataagctggaaagcatgcctgaaatcctgaacactctgaaggtagttgctaacaatgctctgcaaaagtcagataacttctggctgttggatacgcgcagtcatttcggtctgatgaactcacgttctgatatccatgaggacatcacagttaagtctaaccagctgatcgattacgttatcaattgtgcagaacagccaatctgttacccaacactggttgataatatcgccgagaagttctctcagtctagtgatggtgtaaagaaatacttacagaagctgatcaaggaagagttcttgatcactgagctgaagttcagtctgatcgaagacaaccctctggattggttcatcaacatcctggaacgcactcagaacaacctggaactgcttgaaaagctgactgagataaaggcaatgattcaggactatactgactgtgacattggtgagggcaacaatttgattttagctttagaaaataagatgagccaaatagcagaaactaatgcatatttgcgggttgatctttatgatcatgcagagttggagttatctcatcatatcaaggattctcttcagaacatcctgaaggtactgagcagcttctcaccaggtattaatagtcggaaagaaattagaaattaccatgagaaatttatttccagatatggatatgaacagttagtacctcttcaattacttttgaactctactagcggacttggctttccaaaagggtatagtcagacaaaggatttcaagcagaataatggagatagtcggaaccagaagatcacagagttcctccagagaaagttcgagaagtctctgagagatggtaaggaaatcatcctgaatgatgatgatttaaaagatctggattttgataaggaacagcagatatcaggggaactatactgtttctacgatttcaagaaccgcaagctggaggtgagcggcctgggcgtgtcaccgatgatcggagataccttcggacgtttccatgctaaattgccgaacacgatcatcacagagaacgtaaacaagacgaaggagatcttcgctaaggcctaccctaacactatcatcactcagctgaacgaagtgccatacttcgggcgcagcggtaacgttatggtcagcaacagcctgaagagccatcagctggagctgcgcaactatactagcaagaaggatatgagcatcaatgatatctacgtaggtgcaaccagcgaggaactgtacttctacagcaagaagtacggaaagcgcgttatcttcatcatgaacaacatgttcaactacatcaacggtagcaaactgctgcgtttcctgctggaagttagcaatagcgactttcaaaatattactccgattatgtttgataatctggattcttacaaccatgtgcctactatcatctataaggacatcatcatcaagccagaaacatggaatatccgcaagagcgaagttaagactctggacagcctcaagaactggctgagcgataacgatgtgccgagcctggtgcgcatgaagtacaccgatcagatcatctacctggacctgagccgcaccatcgacctgaccatgctgttccgcagcatcaagaagcacagcttcatccagctgctggctacccacagcgaatttgtgaacgacacgaagatcctggagctggtggtgcctttcaccaggagcgatgtcaacgctcgccagatctaccactacgcccagaacatctacaccctggaggatagcgatagcaaggagaagtacttctacgtgaagatctacgtgaacaagcagcgccagaccagcttcctgcaaaaggagtacccgctgctgctgaactacctgaagctgccggaaaacctgcaatggttctacatcagatacgaagatgatgagaaggacagcatccgcctgcgcatccgctacgtagaagataagcaactggttcaactgtacagccgcttcatcgagtgggcgacagaagcacgcaagaacatacagatcctgggttacgaaatcagcgaatacatcccggaaagcgcacgctacggtggcaagaagtacagcacaatcatccacagcttcttctactacgatagcatcctgagcctgctgctgcaaaagaagacagatcaggctatcgaagtgcgcactagcctgagcatcatccgcatgttcctgatgatgaagctgaacctgcaagatcaacaggaactgatgaagaacctgttcgatggagaacacaagctgaagtacgaaaaggaataccataacagcatcagcctgttactggatagcctgtgtacccgcaaccagatcgatgaagctgataccttctgtgtaatgaacatgaagatcatcactgaagaagtgagcagcgtgctgaagcaggaggacttaacaacagattggcagagaatcctgggaagtctgatccacatgcgatgtaatcgagtatacggcatcaacagcgagctggagcgcaagaccatgttcatcgttgacaaggttgttaacagcaagcgctacacagatatgttcctggaggtggataatgagacaaagtaaacgtgtcgagaagatcaagaacatcctgaccgagcagacctacctgttcgactaccaggaggtgctgaagaagatcagtcaagtaaagcaaacagatttctggaatctgctgagcctgagctcgggaatcaccagcctgttaatcttctatcaggagtatgagaatctggaaggagtgaacctgaagccgcaaaagcagtcattgatcggacttataagttactacatcaaccagatagcagataagtcctctctgttcgatggtctggctggggtaggtttcgctattaactacgtcagcaataacggtaagtactatcagaagcttcttaaccagattgacagcaagatctgtcagaacgttaagcggaa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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