Part:BBa_K365004

ClpX from E.coli (aa 61-425)

Background

In wild type E. coli, ClpX forms a ring of hexamer and binds to a double heptamer of ClpP. ClpX recognizes a specific C-terminal degradation tag called SsrA and starts to unfold the tagged protein. The denatured polypeptide is translocated into the ClpP subunit, which breaks peptide bonds. The N terminal end of ClpX contains a domain which is responsible for interaction with its natural adaptator protein (SspB) that we would avoid. As this N-terminal domain is also partially responsible for ClpX subunits complexation into an hexamer, fusing three C-terminal end of ClpX together with appropriate linkers increases the stability of the system (T. Baker et al.) in the absence of this N-terminal domain.
ClpX has two internal restriction sides for EcoRI and two restriction sides for AgeI.


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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 699
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Conception

ClpX has two internal restriction sides for EcoRI and two restriction sides for AgeI. The purpose of this first experimental part was to extract the ClpX gene out of the E.coli genome, to alter the internal EcoRI and AgeI sides in the ClpX gene and to fuse iGEM fusion pre- and suffixes to the ClpX sequence in order to get an iGEM Biobrick with standard prefix and suffix standard without internal EcoRI, Not, XbaI, AgeI, SpeI and PstI sides.

The sequence was obtained from the following database for DH5α E.coli cells:

1- http://www.ncbi.nlm.nih.gov/gene/945083

2- http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=456650&to=457924&report=gbwithparts