Difference between revisions of "Part:BBa K1937002:Design"
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=<b>Part: BBa_K1937002 (pSB1C3-OriKan)</b>= | =<b>Part: BBa_K1937002 (pSB1C3-OriKan)</b>= | ||
| − | <b>(Chassis <i>E. coli/B. subtilis</i>, carrier plasmid | + | <b>(Chassis <i>E. coli/B. subtilis</i>, carrier plasmid pSB<sub>1</sub>C<sub>3</sub>) |
Length: 2510 bp</b> | Length: 2510 bp</b> | ||
<b>Background:</b> | <b>Background:</b> | ||
| − | 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 | + | 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 pSB<sub>1</sub>C<sub>3</sub>. 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>). |
This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France). | This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France). | ||
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<b>Creation:</b> | <b>Creation:</b> | ||
| − | The BBa_K1937002 part contains the repU origin of replication of <i>B. subtilis</i> and the kanamycin resistance gene for <i>B. subtilis</i>. It was obtained by amplifying the repU-Kan region of | + | The BBa_K1937002 part contains the repU origin of replication of <i>B. subtilis</i> and the kanamycin resistance gene for <i>B. subtilis</i>. 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. |
[[file:BBa_K1937002-map.jpg]] | [[file:BBa_K1937002-map.jpg]] | ||
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<b>Validation:</b> | <b>Validation:</b> | ||
| − | 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 | + | 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 pSB<sub>1</sub>C<sub>3</sub>-OriKan plasmid was demonstrated in <i>B. subtilis</i> by PCR checking. The part sequence has been verified by sequencing. |
More information available at http://2016.igem.org/Team:Toulouse_France/Description | More information available at http://2016.igem.org/Team:Toulouse_France/Description | ||
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<b>Interest:</b> | <b>Interest:</b> | ||
| − | This part can be sub-cloned in any | + | This part can be sub-cloned in any pSB<sub>1</sub>C<sub>3</sub> 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. |
Revision as of 17:33, 18 October 2016
Part: BBa_K1937002 (pSB1C3-OriKan)
(Chassis E. coli/B. subtilis, carrier plasmid pSB1C3) Length: 2510 bp
Background: While Bacillus subtilis 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 E. coli 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 (E. coli/B. subtilis). This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France).
More information available at http://2016.igem.org/Team:Toulouse_France/Description
Creation: The BBa_K1937002 part contains the repU origin of replication of B. subtilis and the kanamycin resistance gene for B. subtilis. It was obtained by amplifying the repU-Kan region of pSBBs0K-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 pSB1C3 between the EcoRI and PstI restriction sites.
Validation: We successfully transformed E. coli and selected clones on chloramphenicol. Likewise, we successfully transformed B. subtilis and selected clones on kanamycine. Presence of the pSB1C3-OriKan plasmid was demonstrated in B. subtilis by PCR checking. The part sequence has been verified by sequencing.
More information available at http://2016.igem.org/Team:Toulouse_France/Description
Interest:
This part can be sub-cloned in any pSB1C3 plasmid to make it compatible with B. subtilis. It will therefore greatly simplified the use of Bacillus subtilis as a chassis and the registration of new Bacillus-aimed parts.
Sequence:
Annotation:
repU : 431-1435
Kan : 1595-2365