Part:BBa_K3697004

Recombination-based Detection System for B. Subtilis (manP)

This system, once incorporated into the B. Subtilis will act as a detection system for a customizable nucleic acid sequence corresponding to the sequence with homology to the "homology arms" of the system. It acts a detection system, as when exposed to the target sequence a recombination will be triggered causing the excision of the negative selection marker that is flanked by the homology arms. More information about the specific negative selection marker used in this system can be found in the documentation for part BBa_K3697002 and more information about the specific set of homology arms used in this system can be found in the documentation for part BBa_K3697003.

Usage and Biology

This system works in B. subtilis because of the way that it integrates with the competence and genomic recombination systems in B. subtilis. A brief overview of both of these systems is given below, but more information about how the homology arms of the system trigger site specific recombination events can be found in the documentation for part BBa_K3697003.

The competence system in B. subtilis is driven by the expression of the ComK transcription factor[1]. This system naturally occurs in B. Subtilis when they are put on environmental stress, but strains of B. subtilis have been engineered to have inducible competence. Some examples of strains with inducible competence systems that were successfully used by the 2020 Stanford iGEM team are 1A976 and 1A1276. Both of these strains were obtained through the Bacillus Genetic Stock Center (http://www.bgsc.org/). Once DNA is taken in through B. subtilis' competence system, B. subtilis will allow it to be recombined into the genome if there is sufficient levels of homology to the sequences already in the genome. In general, ~1000 base pairs of homology split between two homology arms flanking the site of integration into the genome are generally used to trigger a recombination event [2], but recombination can be triggered with less homology though with less efficiency.

This detection system was used in conjunction with the mannitol inducible competence system of Bacillus subtilis strain 1A1276. The first step of using this detection system in B. subtilis was assembling this system into PBS1C. This was done via a Gibson Assembly. Next, once the this system was put into PBS1C it was transformed into 1A1276 and selected for on media with chloramphenicol. Next, the cells with the detection system were transformed with a sample of DNA. In the event that the cells were exposed to a sample with the target sequence of interest, a second recombination even will be triggered causing the excision of the manP negative selection marker. If the target sequence was not in the sample, then this second recombination event will not be triggered and the negative selection marker will remain in the cells. The manP negative selection marker can selected for by exposing these cells to mannose. If the cells live and grow when exposed to media with mannose in it, then the second recombination event occurred and the target sequence was in the sample. If the cells die and/or don't grow then this second recombination event has not occurred and the target sequence was not detected in the sample.

A model for the effectiveness of this system is described and illustrated below:

[1] Turgay K, Hahn J, Burghoorn J, Dubnau D. 1998. Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor. EMBO J 17:6730–6738. [2] Dubnau D. Sonenshein AL, Hoch JA, Losick R. Genetic exchange and homologous recombination, Bacillus subtilis and Other Gram-positive Bacteria: Biochemistry, Physiology, and Molecular Genetics, 1993Washington, DCASM(pg. 555-584) Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 679
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1272
  Illegal AgeI site found at 922
  Illegal AgeI site found at 1016
  Illegal AgeI site found at 2682
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 822
  Illegal SapI site found at 1844