Difference between revisions of "Part:BBa K398029:Design"

Revision as of 15:14, 27 October 2010

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 1213 to 2706. Nucleotide sequence optimized for expression in E.coli K12. We used [http://www.jcat.de/ jcat] for this purpose. Moreover, we deleted the restriction sites for EcoRI, PstI, SpeI and XbaI and we change the stop codon for a TAA.

The molecular weight of ALDH is around 53 kDa and its 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

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.
National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/]

@@ Line 9: / Line 9: @@
 Nucleotide sequence optimized for expression in ''E.coli'' K12. We used [http://www.jcat.de/ jcat] for this purpose. Moreover, we deleted the restriction sites for EcoRI, PstI, SpeI and XbaI and we change the stop codon for a TAA.
-The expected aminoacid sequence is:
+The molecular weight of ALDH is around '''53 kDa''' and its aminoacid sequence is:
                       MTMISAQTTEADLWIDGEWRPAASGERFDVIDPATGEVTARVAN