Part:BBa_K316033

Pveg-spoVG-LytC-Flexible Linker-TEV Cleavage Site-AIP-His Tag

Introduction: This part was used to link the cell wall binding domain (CWB) of LytC BBa_K316030, with the quorum sensing peptide (AIP) as well as providing a cleavage site for a protease we want to detect. This construct was built as a protease detection unit.

LytC: The part carries part of LytC on its 5’ end. This was used to ligate the linker with LytC via an internal AccI restriction site that occurs naturally in B. subtilis LytC sequence.

Flexible 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 flexible amino acid sequence that allows for better access of the cleavage site by the protease. It was specifically designed to separate protein domains2.

TEV Cleavage Site: This sequence forms the 3’ end of the linker and is directly attached to the 5’ end of the AIP. It is 18 amino acids (GGGGENLYFQGGKLGGGG) long and was designed to be efficiently cleaved by the TEV protease BBa_K316012, as well as being codon-optimised for expression in B. subtilis.

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
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1087
  
• 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

</biblio>