Team:Newcastle/End of crack & signalling system

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End of crack cell-signalling system

We aim to use a subtilin-based cell signalling system to trigger calcium carbonate precipitation and filament formation once our bacteria have reached a sufficient density inside a microcrack.

Subtilin is a lantibiotic peptide produced naturally by Bacillus subtilis ATCC 6633 but not by Bacillus subtilis 168. We aim to produce two BioBricks: one for production of subtilin, and, since subtilin is a lantibiotic, one for subtilin immunity. A subtilin-sensing BioBrick was produced by Newcastle's 2008 iGEM team.



Subtilin immunity BioBrick

The spaIFEG gene cluster encodes two separate subtilin immunity systems, both present in ATCC 6633. Together they provide a high degree of subtilin resistance.

Cloning strategy

Subtilin production BioBrick

2008 Subtilin-sensing BioBrick

  • A type 1 antimicrobial peptide [AMP] or lantibiotic produced by B. subtilis
    • NB. Lantibiotic = peptide-derived antibiotics with high antimicrobial activity against various Gram-positive bacteria, including pathogenic bacteria such as propionibacteria, staphylococci, clostridia, enterococci and streptococci.
    • NB. Subtilin not same as Subtilisin!!!!!
  • Production of subtilin involves the spa gene cluster encompassing 10 genes, spaBTCSIFEGRK

NCL - Biobrick for Subtilin Sensor.PNG

The construct above contains:

  • spaRK promotor
  • spaR (subtilin peptide antibiotic Regulation) - the 220 amino acid product of this gene usually regulates the downstream production of subtilin antibiotic. It has an N-terminal domain that can be phosphorylated and a C-terminal domian that has DNA binding properties [1]
  • spaK (subtilin peptide antibiotic Kinase) - this gene codes for a 325 amino acid histadine kinase peptide that phosphorylates the N-terminus of SpaR [2]. This activates the DNA binding ability of the C-terminus of SpaR, which in turn initiates transcription of the downstream gene. In the case of our construct, this gene is gfp.
  • rrnO transcriptional terminator
  • spaS promotor - a strong promotor inducible by upstream activation of spaRK. It can be placed in front any gene to regulate its activity.

Production and Immunity Bricks

  • spaBTCS genes are required for production.
  • spaIFEG genes are required for immunity.
  • spaB and C are modifiers of the subtilin precursor.
  • spaT is a transporter.
  • spaS is the gene coding for the subtilin precursor peptide.
  • spaI 's gene product sequesters subtilin.
  • spaFEG genes code for transporters.

Spa.png

Image from Modeling Subtilin production in Bacillus subtilis Using Stochastic Hybrid Systems. Hu et al.

Biochemical pathway

Subtilin.jpeg

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