Composite

Part:BBa_K1937005:Design

Designed by: Oumnia KARIM   Group: iGEM16_Toulouse_France   (2016-10-13)
Revision as of 19:31, 17 October 2016 by Brice (Talk | contribs) (→‎Sequence:)

Part: BBa_K1937005 (1035 bp) (Chassis E. coli, carrier plasmid pSB1C3, part destined for use in Bacillus subtilis)

Background: pNagP is the promoter of the NagP gene, which expresses a transporter of N-acetylglucosamine-6-phosphate in Bacillus subtilis (http://www.biocyc.org/gene?orgid=BSUB&id=BSU07700). 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_K1937005 part was designed to demonstrate the induction of pNagP 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 pNagP promoter and its RBS (position 840,455 to 840,655 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 K1937005-map.png

Validation: The part was sub-cloned in the pSBBsOK-mini plasmid (BBa_K1937001) for validation (resulting in part BBa_K1937006). 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 pNagP promoter growing on NAG (as well as with the pNagA promoter, see part BBa_K1937004), 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:

TCTAATTCTTGATCGTTTGTTACAATCATCTGTCATTCCCGCTTTCTTTTTAAATAATGTAGATTAAGCTTACCACAACTGTCTTAAAAATAGGAAACACACGGACCTGGGAAAAAAGAAATACCCCCGGGAAAATTGGTATAGATCACTAGATATCTTATATGGTATATTTGAAAAAAAAGGGTATGAGGGGGATGGGTGCTAGCatggcttcctccgaagacgttatcaaagagttcatgcgtttcaaagttcgtatggaaggttccgttaacggtcacgagttcgaaatcgaaggtgaaggtgaaggtcgtccgtacgaaggtacccagaccgctaaactgaaagttaccaaaggtggtccgctgccgttcgcttgggacatcctgtccccgcagttccagtacggttccaaagcttacgttaaacacccggctgacatcccggactacctgaaactgtccttcccggaaggtttcaaatgggaacgtgttatgaacttcgaagacggtggtgttgttaccgttacccaggactcctccctgcaagacggtgagttcatctacaaagttaaactgcgtggtaccaacttcccgtccgacggtccggttatgcagaaaaaaaccatgggttgggaagcttccaccgaacgtatgtacccggaagacggtgctctgaaaggtgaaatcaaaatgcgtctgaaactgaaagacggtggtcactacgacgctgaagttaaaaccacctacatggctaaaaaaccggttcagctgccgggtgcttacaaaaccgacatcaaactggacatcacctcccacaacgaagactacaccatcgttgaacagtacgaacgtgctgaaggtcgtcactccaccggtgcttaataacgctgatagtgctagtgtagatcgcccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctc

Annotation:
Promoter NagP+RBS: 1-200
RFP : 207-912
Terminator : 913-992