BBa_K322922 1 BBa_K322922 cat + sacB for BRIDGE: A biobrick compatible markerless recombineering method 2010-10-06T11:00:00Z 2015-05-08T01:12:00Z sacB BBa-K322921 Source: Bacillus subtilis 168 genomic DNA. Sequence cat BBa-K322210 Source: plasmid pTG262 (broad host range vector for Gram positive and Gram negative bacteria). BRIDGE stands for BioBrick Recombineering In Direct Genomic Editing. It is an alternative method for inserting BioBricks into the genome by using homologous recombination instead of restriction digestion, with the added bonus of not leaving a marker behind in the product. The first step of BRIDGE requires the deletion of existing DNA (probably a non-coding piece or a non-essential gene) to introduce a construct of two genes; one an antibiotic resistance gene cat BBa-K322210, the other sacB BBa-K322921, which prevents the host from growing on sucrose. After the first step we can select for cells which have taken up the construct by growing them on the relevant antibiotic. false false _441_ 0 6225 9 It's complicated true BBa-K322210 Notes: The PCR product, P1601, was initially cloned in pSB1A2, which made it easy to confirm that the gene was active, since the transformants became chloramphenicol resistant. A considerable amount of upstream DNA was included in the hope that this would include the promoter region. In the course of the iGEM project, the same PCR product was cloned in pSB1C3 to comply with new submission rules. Since this BioBrick was originally prepared for internal use, it uses abbreviated forms of the prefix and suffix lacking the NotI sites, but is in all ways compatible with RFC10 assembly. BBa-K322921 Note: MABEL was used to mutate an internal EcoRI site to GAGTTC (underlined). Features: coding sequence runs from 13 to 1434, RBS 1 to 3, EcoRI site mutated 211 to 216. false Chris French, Maria Kowal and Geraldine Avila component2083576 1 BBa_K322210 component2083577 1 BBa_K322921 annotation2083576 1 BBa_K322210 range2083576 1 1 1081 annotation2083577 1 BBa_K322921 range2083577 1 1090 2526 BBa_K322210 1 BBa_K322210 Chloramphenicol acetyltransferase (cat) 2010-09-24T11:00:00Z 2015-05-08T01:11:59Z Source: plasmid pTG262 (broad host range vector for Gram positive and Gram negative bacteria). cat is an antibiotic resistance gene, used in the first step of BRIDGE which requires the deletion of existing DNA (probably a non-coding piece or a non-essential gene) to select for cells which have taken up the construct by growing them on the relevant antibiotic. In the BRIDGE, With this system, the markers are removed every time you insert a new gene, so they can be used again and again indefinitely. You could essentially replace the entire genome with new genes. Notes: has complete suffix but prefix lacks NotI site. Features: coding sequence runs from base 294 to 944, rbs 280 to 285. false false _441_ 0 6225 9 It's complicated true Notes: The PCR product, P1601, was initially cloned in pSB1A2, which made it easy to confirm that the gene was active, since the transformants became chloramphenicol resistant. A considerable amount of upstream DNA was included in the hope that this would include the promoter region. In the course of the iGEM project, the same PCR product was cloned in pSB1C3 to comply with new submission rules. Since this BioBrick was originally prepared for internal use, it uses abbreviated forms of the prefix and suffix lacking the NotI sites, but is in all ways compatible with RFC10 assembly. false Geraldine Avila annotation2083599 1 cat range2083599 1 294 944 annotation2083598 1 RBS range2083598 1 280 285 BBa_K322921 1 sacB B. subtilis levansucrase. Lethal to E. coli in presence of sucrose. 2010-09-23T11:00:00Z 2015-05-08T01:12:00Z Source: Bacillus subtilis 168 genomic DNA. Sequence (from SacBPlan11June10.docx) sacB encodes the Bacillus subtilis levansucrase,which is the enzyme catalyzing hydrolysis of sucrose and synthesis of levans(high-molecular-weight fructose polymers). It is lethal to gram-negative bacteria E-coli. It works with cat as an alternative method for inserting BioBricks into the genome by using homologous recombination instead of restriction digestion, with the added bonus of not leaving a marker behind in the product. Protocol is shown here: http://2010.igem.org/Team:Edinburgh/Project/Protocol false false _441_ 0 6225 9 It's complicated true Note: MABEL was used to mutate an internal EcoRI site to GAGTTC (underlined). Features: coding sequence runs from 13 to 1434, RBS 1 to 3, EcoRI site mutated 211 to 216. false Chris French and Maria Kowal annotation2098480 1 Coding sequence range2098480 1 13 1434 annotation2098481 1 Removed EcoRI site range2098481 1 211 216 annotation2098459 1 RBS range2098459 1 1 3 BBa_K322921_sequence 1 gagacatgaacgatgaacatcaaaaagtttgcaaaacaagcaacagtattaacctttactaccgcactgctggcaggaggcgcaactcaagcgtttgcgaaagaaacgaaccaaaagccatataaggaaacatacggcatttcccatattacacgccatgatatgctgcaaatccctgaacagcaaaaaaatgaaaaatatcaagttcctgagttcgattcgtccacaattaaaaatatctcttctgcaaaaggcctggacgtttgggacagctggccattacaaaacgctgacggcactgtcgcaaactatcacggctaccacatcgtctttgcattagccggagatcctaaaaatgcggatgacacatcgatttacatgttctatcaaaaagtcggcgaaacttctattgacagctggaaaaacgctggccgcgtctttaaagacagcgacaaattcgatgcaaatgattctatcctaaaagaccaaacacaagaatggtcaggttcagccacatttacatctgacggaaaaatccgtttattctacactgatttctccggtaaacattacggcaaacaaacactgacaactgcacaagttaacgtatcagcatcagacagctctttgaacatcaacggtgtagaggattataaatcaatctttgacggtgacggaaaaacgtatcaaaatgtacagcagttcatcgatgaaggcaactacagctcaggcgacaaccatacgctgagagatcctcactacgtagaagataaaggccacaaatacttagtatttgaagcaaacactggaactgaagatggctaccaaggcgaagaatctttatttaacaaagcatactatggcaaaagcacatcattcttccgtcaagaaagtcaaaaacttctgcaaagcgataaaaaacgcacggctgagttagcaaacggcgctctcggtatgattgagctaaacgatgattacacactgaaaaaagtgatgaaaccgctgattgcatctaacacagtaacagatgaaattgaacgcgcgaacgtctttaaaatgaacggcaaatggtacctgttcactgactcccgcggatcaaaaatgacgattgacggcattacgtctaacgatatttacatgcttggttatgtttctaattctttaactggcccatacaagccgctgaacaaaactggccttgtgttaaaaatggatcttgatcctaacgatgtaacctttacttactcacacttcgctgtacctcaagcgaaaggaaacaatgtcgtgattacaagctatatgacaaacagaggattctacgcagacaaacaatcaacgtttgcgccaagcttcctgctgaacatcaaaggcaagaaaacatctgttgtcaaagacagcatccttgaacaaggacaattaacagttaacaaataataa BBa_K322210_sequence 1 tctatcccggcaatagttacccttattatcaagataagaaagaaaaggatttttcgctacgctcaaatcctttaaaaaaacacaaaagaccacattttttaatgtggtctttattcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatatttttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattgggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtactttttacagtcggttttctaaaacgatacattaataggtacgaaaaagcaactttttttgcgcttaaaaccagtcataccaataacttaagggtaactagcctcgccggaaagag BBa_K322922_sequence 1 tctatcccggcaatagttacccttattatcaagataagaaagaaaaggatttttcgctacgctcaaatcctttaaaaaaacacaaaagaccacattttttaatgtggtctttattcttcaactaaagcacccattagttcaacaaacgaaaattggataaagtgggatatttttaaaatatatatttatgttacagtaatattgacttttaaaaaaggattgattctaatgaagaaagcagacaagtaagcctcctaaattcactttagataaaaatttaggaggcatatcaaatgaactttaataaaattgatttagacaattggaagagaaaagagatatttaatcattatttgaaccaacaaacgacttttagtataaccacagaaattgatattagtgttttataccgaaacataaaacaagaaggatataaattttaccctgcatttattttcttagtgacaagggtgataaactcaaatacagcttttagaactggttacaatagcgacggagagttaggttattgggataagttagagccactttatacaatttttgatggtgtatctaaaacattctctggtatttggactcctgtaaagaatgacttcaaagagttttatgatttatacctttctgatgtagagaaatataatggttcggggaaattgtttcccaaaacacctatacctgaaaatgctttttctctttctattattccatggacttcatttactgggtttaacttaaatatcaataataatagtaattaccttctacccattattacagcaggaaaattcattaataaaggtaattcaatatatttaccgctatctttacaggtacatcattctgtttgtgatggttatcatgcaggattgtttatgaactctattcaggaattgtcagataggcctaatgactggcttttataatatgagataatgccgactgtactttttacagtcggttttctaaaacgatacattaataggtacgaaaaagcaactttttttgcgcttaaaaccagtcataccaataacttaagggtaactagcctcgccggaaagagtactagaggagacatgaacgatgaacatcaaaaagtttgcaaaacaagcaacagtattaacctttactaccgcactgctggcaggaggcgcaactcaagcgtttgcgaaagaaacgaaccaaaagccatataaggaaacatacggcatttcccatattacacgccatgatatgctgcaaatccctgaacagcaaaaaaatgaaaaatatcaagttcctgagttcgattcgtccacaattaaaaatatctcttctgcaaaaggcctggacgtttgggacagctggccattacaaaacgctgacggcactgtcgcaaactatcacggctaccacatcgtctttgcattagccggagatcctaaaaatgcggatgacacatcgatttacatgttctatcaaaaagtcggcgaaacttctattgacagctggaaaaacgctggccgcgtctttaaagacagcgacaaattcgatgcaaatgattctatcctaaaagaccaaacacaagaatggtcaggttcagccacatttacatctgacggaaaaatccgtttattctacactgatttctccggtaaacattacggcaaacaaacactgacaactgcacaagttaacgtatcagcatcagacagctctttgaacatcaacggtgtagaggattataaatcaatctttgacggtgacggaaaaacgtatcaaaatgtacagcagttcatcgatgaaggcaactacagctcaggcgacaaccatacgctgagagatcctcactacgtagaagataaaggccacaaatacttagtatttgaagcaaacactggaactgaagatggctaccaaggcgaagaatctttatttaacaaagcatactatggcaaaagcacatcattcttccgtcaagaaagtcaaaaacttctgcaaagcgataaaaaacgcacggctgagttagcaaacggcgctctcggtatgattgagctaaacgatgattacacactgaaaaaagtgatgaaaccgctgattgcatctaacacagtaacagatgaaattgaacgcgcgaacgtctttaaaatgaacggcaaatggtacctgttcactgactcccgcggatcaaaaatgacgattgacggcattacgtctaacgatatttacatgcttggttatgtttctaattctttaactggcccatacaagccgctgaacaaaactggccttgtgttaaaaatggatcttgatcctaacgatgtaacctttacttactcacacttcgctgtacctcaagcgaaaggaaacaatgtcgtgattacaagctatatgacaaacagaggattctacgcagacaaacaatcaacgtttgcgccaagcttcctgctgaacatcaaaggcaagaaaacatctgttgtcaaagacagcatccttgaacaaggacaattaacagttaacaaataataa igem2sbol 1 iGEM to SBOL conversion Conversion of the iGEM parts registry to SBOL2.1 Chris J. Myers James Alastair McLaughlin 2017-03-06T15:00:00.000Z