Difference between revisions of "Part:BBa K1937007:Design"

 
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<b>Part: BBa_K1937007 (AF_A)</b>
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(Chassis <i>E. coli</i>, carrier plasmid pSB1C3, part destined for use in <i>Bacillus subtilis</i>)
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Length: 1006 bp<br>
  
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__NOTOC__
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<b>Background:</b>
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<partinfo>BBa_K1937007 short</partinfo>
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AF-A is a part designed to be combined with AF-B to form an operon of 5 antifungal peptides. The interest of using several antifungals is to have a broad spectrum of fungi target, because each antifungal has its own characteristics. The peptides were designed to be expressed and secreted by Bacillus subtilis. This operon was also initially designed to be expressed under the control of the pNagA or pNagP promoter (BBa_K1937003 and BBa_K1937005 respectively). In spite of several cloning strategies, the AF-B construction was not obtained, likely due to the toxicity of one of the peptides for Bacillus subtilis.
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This BioBrick is a part developed by the Toulouse 2016 iGEM team (http://2016.igem.org/Team:Toulouse_France)
  
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<partinfo>BBa_K1937007 SequenceAndFeatures</partinfo>
 
  
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<b>This part:</b>
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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).
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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.
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<br>
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[[File:BBa_K1937003_-construction.png]]
  
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===Design Notes===
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<b>Validation:</b>
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This part encodes two genes encoding antifungal peptides: a cutted Metchnikowin and the D4E1. We created it with the end of the Metchnikowin gene because we found out that only this part of the gene had the antifungal property(http://www.ebi.ac.uk/interpro/entry/IPR012513). The beginning of the gene translates for a propeptide. This gene comes from drosophila and translates a proline-rich peptide whose expression is immune-inducible. The peptide is active against fungi.The D4E1 gene is synthetic and translates a peptide that complexes with cholesterol, current in non-germinated conidia. This synthetic peptide is more resistant than the natural peptide CecroprineA because proteases from fungi have difficulties to degrade the synthetic one (http://www.nrcresearchpress.com/doi/abs/10.1139/w98-032?journalCode=cjm#.V_pRgJOLT6Y).  
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The part was sub-cloned in the pSBBsOK-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 this substrate Nag.<br>
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So that each antifungal can be secreted out of the cell, it was necessary to add a sequence called AmyE that enables the transport of molecules outside of the cell. As AmyE is used twice in this Biobrick, we modified the AmyE sequences to avoid homologous recombination while keeping the same protein sequence. The NheI restriction site was added after the RBS to allow promoter swapping with biobrick BAAXXXX or BAAXXXX. SacII and SalI restriction site were added to built the full 5 peptides operon.
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[[File:igemtoulouse2016red.jpg]]<br>
  
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We indeed observed intense red stains on the 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) , 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).
  
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=<b>Sequence:</b>=
  
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===Source===
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<html>
  
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This gene comes from drosophila and translates a proline-rich peptide whose expression is immune-inducible. The peptide is active against fungi.The D4E1 gene is synthetic and translates a peptide that complexes with cholesterol, current in non-germinated conidia. This synthetic peptide is more resistant than the natural peptide CecroprineA because proteases from fungi have difficulties to degrade the synthetic one (http://www.nrcresearchpress.com/doi/abs/10.1139/w98-032?journalCode=cjm#.V_pRgJOLT6Y).
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<style>
  
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===References===
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/*LAYOUT */
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.column  {
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padding: 10px 0px;
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float:left;
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background-color:white;
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}
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.full_size {
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width:100%;
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word-wrap: break-word;
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}
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</style>
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<div class="column full_size" style="font-size:10px;">
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GAATTCGCGGCCGCTTCTAGAGTTGTGCGAACCTTTGCCACGATATGTTCCTCCTGTTCCGGGCTGCCCCGAGCTTGCTCACAATACTTTCATTTTATCACTTTCGGGCTTGAACCTAAAACAGATTTTATAAAAGGGGGGAAAACACCTCAGCTGGTATAGATCACTAATCTGAAAAAGAGTAAAATAAAGGTATTCAAATTCCAGAAAGGCGGATCATCTGCTAGCatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacgctgatagtgctagtgtagatcgcccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcTACTAGTAGCGGCCGCTGCAG<br><br>
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</div>
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</html>
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<b>Annotation:</b><br>
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Promoter NagP+RBS: 23-222<br>
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RFP : 229-934<br>
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Terminator : 935-1014
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<br><br>

Revision as of 14:06, 17 October 2016

Part: BBa_K1937007 (AF_A) (Chassis E. coli, carrier plasmid pSB1C3, part destined for use in Bacillus subtilis) Length: 1006 bp

Background: AF-A is a part designed to be combined with AF-B to form an operon of 5 antifungal peptides. The interest of using several antifungals is to have a broad spectrum of fungi target, because each antifungal has its own characteristics. The peptides were designed to be expressed and secreted by Bacillus subtilis. This operon was also initially designed to be expressed under the control of the pNagA or pNagP promoter (BBa_K1937003 and BBa_K1937005 respectively). In spite of several cloning strategies, the AF-B construction was not obtained, likely due to the toxicity of one of the peptides for Bacillus subtilis. 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.
BBa K1937003 -construction.png

Validation: The part was sub-cloned in the pSBBsOK-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 this substrate Nag.

Igemtoulouse2016red.jpg

We indeed observed intense red stains on the 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:

GAATTCGCGGCCGCTTCTAGAGTTGTGCGAACCTTTGCCACGATATGTTCCTCCTGTTCCGGGCTGCCCCGAGCTTGCTCACAATACTTTCATTTTATCACTTTCGGGCTTGAACCTAAAACAGATTTTATAAAAGGGGGGAAAACACCTCAGCTGGTATAGATCACTAATCTGAAAAAGAGTAAAATAAAGGTATTCAAATTCCAGAAAGGCGGATCATCTGCTAGCatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacgctgatagtgctagtgtagatcgcccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcTACTAGTAGCGGCCGCTGCAG

Annotation:
Promoter NagP+RBS: 23-222
RFP : 229-934
Terminator : 935-1014