Part:BBa_K316037

Pveg-spoVG-LytC-Helical Linker-Elastase Cleavage Site-AIP-His Tag BBa_K316037

Introduction: This part, our detection module, was designed to anchor a signal peptide (AIP) to the cell wall of B. subtilis which is released upon cleave of the part by a protease, in this case the cercarial elastase [http://www.uniprot.org/uniprot/P12546 (UniProt,] [http://merops.sanger.ac.uk/cgi-bin/pepsum?id=S01.144 MEROPS)], to activate our output. The part is fully assembled and ready to be expressed in a prokaryotic cell as it contains a promoter we characterised, an optimised RBS and a double stop codon. The protein product includes a cell wall binding domain (CWB) of LytC, BBa_K316030 a linker that has been designed to carry a protease cleave site and the AIP BBa_K316047. This part is essential for the great modularity of our system, as a change of just 4 amino acids can be used to confer specificity to another protease see our [http://2010.igem.org/Team:Imperial_College_London/Software_Tool Software Tool]. 


Pveg-spoVG: This was build in 2008 by the Imperial College iGEM team BBa_K143053.

LytC: The CWB is on the 5’ end and anchors the detection module to the cell wall of B. subtilis. The CWB was separately submitted as biobrick BBa_K316030.


Helical Linker: The Linker separates the CWB and the AIP and creates space for the protease to access the cleavage site; it consists of two main sections. The first six amino acids (SRGSRA) were suggested to be used specifically with LytC1. The second section consists of a helical amino acid sequence that is stiff and prevents interaction of the AIP with its receptor and thus false positive activation of the output system. It was specifically designed to separate protein domains2.


Elastase Cleavage Site: This sequence forms the 3’ end of the linker and is directly attached to the 5’ end of the AIP. It is four amino acids (SWPL) long and was designed to be efficiently cleaved by the schistosoma cercarial elastase3.


AIP: The linear auto-inducing peptide (AIP) from the Streptococcus pneumonia ComCDE system, called Competence-Stimulating Peptide-1 (CSP-1 or ComC) . Upon cleavage it is free to diffuse and bind it's receptor ComD BBa_K316015. Signalling cascade from ComD is then able to activate transcription of genes under specific promoter sequences.


His-Tag: To be able to purify the protein for testing, we attached a His-Tag on our linker-AIP peptide. As it would probably interfere with recognition of the AIP by the receptor it has to be removed from the final construct. 


Stop Codon: In order to end translation a double stop codon was put in place.

For more information about this part of our project please visit our wiki [http://2010.igem.org/Team:Imperial_College_London/Strategy] or take the tour [http://2010.igem.org/Team:Imperial_College_London/Tour/Page_One] to learn more about the project.

Figure I. Graphical representation of cell wall binding protein.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

<biblio>

1. 1 pmid=14594841
2. 2 pmid=11579220
3. 3 pmid=11986325

</biblio>