Composite
406

Part:BBa_K398406:Design

Designed by: Hugo Federico Cueto Rojas and Ramon van der Valk   Group: iGEM10_TU_Delft   (2010-10-12)

Solvent tolerance cluster


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 37
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 37
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 37
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The original sequences can be found in [http://www.ncbi.nlm.nih.gov/nuccore THIS LINK], these sequences were enhanced for their expression on E. coli K12. Additionally, the restriction sites for the enzymes XbaI, PstI, EcoRI and SpeI were removed from the CDS and the stop codon was replaced by the codon TAA in order to avoid the formation of possible ORF's. For this process we used the [http://www.jcat.de/ jcat] software tool.

An extra XbaI site can be found between RBS and Promoter so you can increase the expression level of the protein by changing the promoter.


The expected size for Prefoldin alpha-subunit is 16.97 kDa, its aminoacid sequence can be checked below: 

         1 mirmaqnnke leklayeyqv lqaqaqilaq nlellnlaka evqtvretle nlkkieeekp
       61 eilvpigags flkgvivdkn naivsvgsgy aversideai sflekrlkey deaikktqga
     121 laelekrige varkaqevqq kqsmtsfkvk k

The expected size for Prefoldin beta-subunit is 13.28 kDa, its aminoacid sequence can be checked below: 

        1 mqnippqvqa mlgqldtyqq qlqlviqqkq kvqadlneak kaleeietlp ddaqiyktvg
      61 tlivkttkek avqelkekie tlevrlnaln rqeqkinekv keltqkiqaa lrpptag

Source

Genomic sequence obtained from NCBI.

Prefoldin alpha accession number: [http://www.ncbi.nlm.nih.gov/gene/1444414 PH0527]

Prefoldin beta accession number: [http://www.ncbi.nlm.nih.gov/gene/1444421 PH0532]

References

  1. Okochi M. et al., "Overexpression of prefoldin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 endowed Escherichia coli with organic solvent tolerance", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, Volume 79, Number 3, 443-449, DOI: 10.1007/s00253-008-1450-1.
  2. Tanaka T. et al., Direct submission of the sequence. (11-JUN-1998) Biotechnology Center, National Institute of Technology and Evaluation, 2-49-10 Nishihara, Shibuyaku, Tokyo 151-0066, Japan.