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| − | =<b>Part: BBa_K1937002 (pSB1C3-OriKan)</b>=
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| − | <b>(Chassis <i>E. coli/B. subtilis</i>, carrier plasmid pSB<sub>1</sub>C<sub>3</sub>)
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| − | Length: 2510 bp</b>
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| − | <b>Background:</b>
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| − | While <i>Bacillus subtilis</i> is of huge interest for a growing number of iGEM projects, it is not easy to develop new parts as it is required that they registered after sub-cloning in the <i>E. coli</i> plasmid pSB1C3. During the iGEM Toulouse 2016 project, we had the idea to create a part that could turn any pSB1C3-based plasmid in a shuttle vector (<i>E. coli/B. subtilis</i>).
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| − | This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France) from the pSB<sub>Bs</sub>0K-P (BBa_K823026) previously developed by the 2012 Munich iGEM team.
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| − | More information available at http://2016.igem.org/Team:Toulouse_France/Description
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| − | <b>Creation:</b>
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| − | The BBa_K1937002 part contains the <i>repU</i> origin of replication of <i>B. subtilis</i> and the kanamycin resistance gene for <i>B. subtilis</i> (Figure 1).<br>
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| − | [[file:BBa_K1937002-map.jpg|thumb|center|800px|<b>Figure 1:</b> scheme of the Orikan part with <i>Bacillus subtilis</i> origin <i>repU</i> and a kanamycine resistance cassette.]]
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| − | <br>It was obtained by amplifying the repU-Kan region of pSB<sub>Bs</sub>0K-P (BBa_K1351040 or BBa_K823026) using primers OriKan forward (5’ cacagaatcaggggataacgcaggaaagaaACATGTAGTTATAAGTGACTAAACAAATAACTAAATAGATGGG) and OriKan reverse (5’ gttcctggccttttgctggccttttgctcaACATGTCGCAAAATGGCCCGATTTAAG). The resulting fragment was sub-cloned in the pSB<sub>1</sub>C<sub>3</sub> between the EcoRI and PstI restriction sites (Figure 2).
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| − | [[Image:Toulouse_France_backbone2.jpg|thumb|center|800px|<b>Figure 2:</b> creation of the OriKan cassette. Position of the primers are indicated by the blue arrow on pSB<sub>Bs</sub>0K-P (left part of the figure). The resulting PCR fragment is the blue pointed line. After digestion by EcoRI and PstI and ligation in the pSB1C3 plasmid, the resulting pSB1C3-OriKan plasmid was obtained (right part of the figure)]]
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| − | <br>
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| − | <b>Validation:</b>
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| − | We successfully transformed <i>E. coli</i> and selected clones on chloramphenicol. Likewise, we successfully transformed <i>B. subtilis</i> and selected clones on kanamycine. Presence of the pSB1C3-OriKan plasmid was demonstrated in <i>B. subtilis</i> by PCR checking (Figure 3).
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| − | [[Image:Toulouse_France_backbone3.jpg|thumb|center|400px|<b>Figure 3:</b> validation of the pSB1C3-Orikan presence in <i>B. subtilis.</i> PCR on colonies was performed using primers hybridizing in the kanamycine resistance gene and in the suffix. The colonies were issued from the transformation of B. subtilis by pSB1C3-Orikan (assays), by the pSB<sub>Bs</sub>0K-P plasmid (negative control), or from the transformation of E. coli by pSB1C3-Orikan (positive control).]]
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| − | <br>The part sequence has been verified by sequencing.
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| − | More information available at http://2016.igem.org/Team:Toulouse_France/Description
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| − | <b>Interest:</b>
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| − | This part can be sub-cloned in any pSB1C3 plasmid to make it compatible with <i>B. subtilis</i>. It will therefore greatly simplified the use of <i>Bacillus subtilis</i> as a chassis and the registration of new <i>Bacillus</i>-aimed parts.
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| − | =<b>Sequence:</b>=
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| − | .column {
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| − | .full_size {
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| − | width:100%;
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| − | word-wrap: break-word;
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| − | </style>
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| − | <div class="column full_size" style="font-size:10px;">
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| − | GTCTTCAAGAGTTCCTTAAGGAACGTACAGACGGCTTAAAAGCCTTTAAAAACGTTTTTAAGGGGTTTGTAGACAAGGTAAAGGATAAAACAGCACAATTCCAAGAAAAACACGATTTAGAACCTAAAAAGAACGAATTTGAACTAACTCATAACCGAGAGGTAAAAAAAGAACGAAGTCGAGATCAGGGAATGAGTTTATAAAATAAAAAAAGCACCTGAAAAGGTGTCTTTTTTTGATGGTTTTGAACTTGTTCTTTCTTATCTTGATACATATAGAAATAACGTCATTTTTATTTTAGTTGCTGAAAGGTGCGTTGAAGTGTTGGTATGTATGTGTTTTAAAGTATTGAAAACCCTTAAAATTGGTTGCACAGAAAAACCCCATCTGTTAAAGTTATAAGTGACTAAACAAATAACTAAATAGATGGGGGTTTCTTTTAATATTATGTGTCCTAATAGTAGCATTTATTCAGATGAAAAATCAAGGGTTTTAGTGGACAAGACAAAAAGTGGAAAAGTGAGACCATGGAGAGAAAAGAAAATCGCTAATGTTGATTACTTTGAACTTCTGCATATTCTTGAATTTAAAAAGGCTGAAAGAGTAAAAGATTGTGCTGAAATATTAGAGTATAAACAAAATCGTGAAACAGGCGAAAGAAAGTTGTATCGAGTGTGGTTTTGTAAATCCAGGCTTTGTCCAATGTGCAACTGGAGGAGAGCAATGAAACATGGCATTCAGTCACAAAAGGTTGTTGCTGAAGTTATTAAACAAAAGCCAACAGTTCGTTGGTTGTTTCTCACATTAACAGTTAAAAATGTTTATGATGGCGAAGAATTAAATAAGAGTTTGTCAGATATGGCTCAAGGATTTCGCCGAATGATGCAATATAAAAAAATTAATAAAAATCTTGTTGGTTTTATGCGTGCAACGGAAGTGACAATAAATAATAAAGATAATTCTTATAATCAGCACATGCATGTATTGGTATGTGTGGAACCAACTTATTTTAAGAATACAGAAAACTACGTGAATCAAAAACAATGGATTCAATTTTGGAAAAAGGCAATGAAATTAGACTATGATCCAAATGTAAAAGTTCAAATGATTCGACCGAAAAATAAATATAAATCGGATATACAATCGGCAATTGACGAAACTGCAAAATATCCTGTAAAGGATACGGATTTTATGACCGATGATGAAGAAAAGAATTTGAAACGTTTGTCTGATTTGGAGGAAGGTTTACACCGTAAAAGGTTAATCTCCTATGGTGGTTTGTTAAAAGAAATACATAAAAAATTAAACCTTGATGACACAGAAGAAGGCGATTTGATTCATACAGATGATGACGAAAAAGCCGATGAAGATGGATTTTCTATTATTGCAATGTGGAATTGGGAACGGAAAAATTATTTTATTAAAGAGTAGTTCAACAAACGGGCCAGTTTGTTGAAGATTAGATGCTATAATTGTTATTAAAAGGATTGAAGGATGCTTAGGAAGACGAGTTATTAATAGCTGAATAAGAACGGTGCTCTCCAAATATTCTTATTTAGAAAAGCAAATCTAAAATTATCTGAAAAGGGAATGAGAATAGTGAATGGACCAATAATAATGACTAGAGAAGAAAGAATGAAGATTGTTCATGAAATTAAGGAACGAATATTGGATAAATATGGGGATGATGTTAAGGCTATTGGTGTTTATGGCTCTCTTGGTCGTCAGACTGATGGGCCCTATTCGGATATTGAGATGATGTGTGTCATGTCAACAGAGGAAGCAGAGTTCAGCCATGAATGGACAACCGGTGAGTGGAAGGTGGAAGTGAATTTTGATAGCGAAGAGATTCTACTAGATTATGCATCTCAGGTGGAATCAGATTGGCCGCTTACACATGGTCAATTTTTCTCTATTTTGCCGATTTATGATTCAGGTGGATACTTAGAGAAAGTGTATCAAACTGCTAAATCGGTAGAAGCCCAAACGTTCCACGATGCGATTTGTGCCCTTATCGTAGAAGAGCTGTTTGAATATGCAGGCAAATGGCGTAATATTCGTGTGCAAGGACCGACAACATTTCTACCATCCTTGACTGTACAGGTAGCAATGGCAGGTGCCATGTTGATTGGTCTGCATCATCGCATCTGTTATACGACGAGCGCTTCGGTCTTAACTGAAGCAGTTAAGCAATCAGATCTTCCTTCAGGTTATGACCATCTGTGCCAGTTCGTAATGTCTGGTCAACTTTCCGACTCTGAGAAACTTCTGGAATCGCTAGAGAATTTCTGGAATGGGATTCAGGAGTGGACAGAACGACACGGATATATAGTGGATGTGTCAAAACGCATACCATTTTGAACGATGACCTCTAATAATTGTTAATCATGTTGGTTACGTATTTATTAACTTCTCCTAGTATTAGTAATTATCATGGCTGTCATGGCGCATTAACGGAATAAAGGGTGTGCTTAAATCGGGCCATTTTG
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| − | </div>
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| − | </html>
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| − | <b>Annotation:</b><br>
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| − | repU : 431-1435<br>
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| − | Kan : 1595-2365
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