Difference between revisions of "Part:BBa K1937003:Design"
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<b>Part: BBa_K1937003 (pNagA)</b> | <b>Part: BBa_K1937003 (pNagA)</b> | ||
| − | (Chassis E. coli, carrier plasmid pSB1C3, part destined for use in <i>Bacillus subtilis</i>) | + | (Chassis <i>E. coli</i>, carrier plasmid pSB1C3, part destined for use in <i>Bacillus subtilis</i>) |
Length: 1035 bp<br> | Length: 1035 bp<br> | ||
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<b>This part:</b> | <b>This part:</b> | ||
| − | The BBa_K1937003 part was designed to demonstrate the induction of pNagA by NAG. It belongs to the antifungal module in the Paleolitis project of iGEM Toulouse 2016 (http://2016.igem.org/Team:Toulouse_France). | + | The BBa_K1937003 part was designed to demonstrate the induction of pNagA by NAG. It belongs to the antifungal module in the Paleolitis project of iGEM Toulouse 2016 (http://2016.igem.org/Team:Toulouse_France). |
The part include the pNagA promoter and its RBS (position 3,595,156 to 3,595,356 of the Bacillus genome). The promoter controls the expression of the RFP fluorescent reporter gene. A NheI restriction site has been placed before the RFP ORF to facilitate promoter swapping. | The part include the pNagA promoter and its RBS (position 3,595,156 to 3,595,356 of the Bacillus genome). The promoter controls the expression of the RFP fluorescent reporter gene. A NheI restriction site has been placed before the RFP ORF to facilitate promoter swapping. | ||
<br> | <br> | ||
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<b>Validation:</b> | <b>Validation:</b> | ||
| − | The part was sub-cloned in the | + | The part was sub-cloned in the pSB<sub>Bs</sub>0K-mini plasmid (BBa_K1937001) for validation (resulting in part BBa_K1937004). In presence of N-acetyl glucosamine in the media, the clones are expected to be red, and white without NAG.<br> |
| − | [[File:igemtoulouse2016red.jpg]] | + | [[File:igemtoulouse2016red.jpg]]<br> |
| − | We indeed observed intense red | + | |
| + | We indeed observed intense red colonies with the pNagA promoter growing on NAG (as well as with the pNagP promoter, see part BBa_K1937006), albeit the expression is late and heterogeneous during the colonies development. Control (<i>B. subtilis</i> strains transformed with pSBBs0K-mini) , pSB<sub>Bs</sub>0K-mini-NagA and pSB<sub>Bs</sub>0K-mini-NagP transformed strains were spread on minimal medium with either glucose or NAG as carbon source. Red GFP spots appeared only with pNagA or pNagP on NAG (close-ups on section B of the figure). | ||
=<b>Sequence:</b>= | =<b>Sequence:</b>= | ||
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<div class="column full_size" style="font-size:10px;"> | <div class="column full_size" style="font-size:10px;"> | ||
| − | + | TTGTGCGAACCTTTGCCACGATATGTTCCTCCTGTTCCGGGCTGCCCCGAGCTTGCTCACAATACTTTCATTTTATCACTTTCGGGCTTGAACCTAAAACAGATTTTATAAAAGGGGGGAAAACACCTCAGCTGGTATAGATCACTAATCTGAAAAAGAGTAAAATAAAGGTATTCAAATTCCAGAAAGGCGGATCATCTGCTAGCatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacgctgatagtgctagtgtagatcgcccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc<br><br> | |
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<b>Annotation:</b><br> | <b>Annotation:</b><br> | ||
| − | Promoter NagP+RBS: | + | Promoter NagP+RBS: 1-200<br> |
| − | RFP : | + | RFP : 207-912<br> |
| − | Terminator : | + | Terminator : 913-992 |
<br><br> | <br><br> | ||
Latest revision as of 17:39, 18 October 2016
Part: BBa_K1937003 (pNagA)
(Chassis E. coli, carrier plasmid pSB1C3, part destined for use in Bacillus subtilis)
Length: 1035 bp
Background: pNagA is the promoter of the NagA gene, which expresses a N-acetylglucosamine-6-phosphate deacetylasein in Bacillus subtilis (http://www.biocyc.org/gene?orgid=BSUB&id=BSU35010). In Bacillus subtilis, this promoter has been reported to induce expression in presence of N-acetylglucosamine 6-phosphate (Bertram et al., 2011: https://www.ncbi.nlm.nih.gov/pubmed/21602348). This NAG molecule is the major component of chitin, a characteristic molecule of fungi. This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France)
This part:
The BBa_K1937003 part was designed to demonstrate the induction of pNagA by NAG. It belongs to the antifungal module in the Paleolitis project of iGEM Toulouse 2016 (http://2016.igem.org/Team:Toulouse_France).
The part include the pNagA promoter and its RBS (position 3,595,156 to 3,595,356 of the Bacillus genome). The promoter controls the expression of the RFP fluorescent reporter gene. A NheI restriction site has been placed before the RFP ORF to facilitate promoter swapping.
Validation:
The part was sub-cloned in the pSBBs0K-mini plasmid (BBa_K1937001) for validation (resulting in part BBa_K1937004). In presence of N-acetyl glucosamine in the media, the clones are expected to be red, and white without NAG.
We indeed observed intense red colonies with the pNagA promoter growing on NAG (as well as with the pNagP promoter, see part BBa_K1937006), albeit the expression is late and heterogeneous during the colonies development. Control (B. subtilis strains transformed with pSBBs0K-mini) , pSBBs0K-mini-NagA and pSBBs0K-mini-NagP transformed strains were spread on minimal medium with either glucose or NAG as carbon source. Red GFP spots appeared only with pNagA or pNagP on NAG (close-ups on section B of the figure).
Sequence:
Annotation:
Promoter NagP+RBS: 1-200
RFP : 207-912
Terminator : 913-992