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Part:BBa_K398029:Design

Designed by: Kira Schipper   Group: iGEM10_TU_Delft   (2010-10-07)
Revision as of 15:30, 27 October 2010 by Hugo 87 (Talk | contribs) (→‎Design Notes)

ALDH generator (low expression)


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 CDS can be found [http://www.ncbi.nlm.nih.gov/nuccore/AB047106.1 in this link], the file reports a cluster of proteins induced by alkanes. We took the ORF contained from 1213bp to 2706bp.

The Nucleotide sequence was optimized for its expression in E.coli K12. We used the software tool available in [http://www.jcat.de/ jcat] for this purpose. Additionally we deleted the restriction sites for EcoRI, PstI, SpeI and XbaI and we change the stop codon for a TAA in order to avoid the formation of unexpected ORF's.

An extra XbaI site can be found between the promoter and RBS; in case that you want to express more protein you can change the promoter BBa_J23100 for a stronger promoter.

The expected aminoacid sequence is: 

                    MTMISAQTTEADLWIDGEWRPAASGERFDVIDPATGEVTARVAN
                    AGEDDVDAAVAIAEEAFSVRRWLAISPLERGRILRRIAELIRQHHCELAQLMTRENGM
                    PINLALFIEIPLADCFDFFASLVVKPQGEVLPFSVAGSAPDYMAWTMKEPIGVAGLIT
                    PWNFPLLMPTWKIAPALAAGCTMVVKPAPETPLTALKLAEICHEAGVPEGVINVLPGL
                    DEAGKALVRHPRVPKIAFTGETETGRHILQAAAPHIKRVTLELGGKSPNIIFADADLE
                    QAAKSALFGVFYNSGQVCQAGSRILVERTVYEPFVERLAERAKKLKVGPGTNPRSDLG
                    PVISREQYEKVLRYIEIGKQEGARLAAGGRALDGGGGGYFIEPTVFADVSPSMRIACE
                    EIFGPVAAVIPFADEEEAVRIANGTMYGLAAAVWTNDIKRALRLARRVKSGTVWLNTY
                    QVLSPTVPFGGYKQSGLGRELGMQALDAYLETKTVICDLNDRPMTLF

According to the authors [1], the purified protein has an activity of dodecanal dehydrogenase equal to 22.995 U/mg protein (4e-7 kat/mg protein). This part was expressed using the promoter-rbs combination J23100-J61117 and also J13002. Thus generating the parts: BBa_K398029 and BBa_K398030, respectively.

Source

Genbank accession number: [http://www.ncbi.nlm.nih.gov/nuccore/AB047106 AB047106]

References

  1. Kato T. et al. "Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23.", Extremophiles. 2010 Jan;14(1):33-9. Epub 2009 Sep 29.
  2. National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/]