Difference between revisions of "Part:BBa K1321340:Design"

Revision as of 09:58, 13 October 2014

Double CBD (dCBD) with N-terminal linker

Design Notes

Nucleotide sequence was first obtained from the relevant portions of [http://www.ncbi.nlm.nih.gov/nuccore/E00389.1 NCBI E00389.1] and [http://www.ebi.ac.uk/ena/data/view/M16190 ENI M16190.1] including removing introns where necessary.

The DNA sequence was then run through the codon optimisation tool [http://www.entelechon.com/bttool/bttool.html entelechon] with settings: standard genetic code, codon usage table imported for E.coli, forbidden sites [AgeI] ACCGGT, [NotI] GCGGCCGC, [XbaI] TCTAGA, [NgoMIV] GCCGGC, [AgeI] ACCGGT, [SpeI] ACTAGT, [PstI] CTGCAG, and avoiding the anti-Shine-Dalgarno sequence TAAGGAGGT. The DNA sequence for the linkers was then aligned using ClustalW2.1 (see alignment above) to check they were not too similar. Since there was a stretch of >17bp identical, codons were manually changed to those that are reasonably similar in frequency using a [http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/in-vitro-genetics/codon-usage.html codon usage table]. Finally, DNA sequence for RFC25 prefix and suffix was appeneded to the sequence, with an additional 4 basepairs (gatc) at the beginning and end of the sequence to allow for space for the restriction enzymes to bind to the EcoRI and PstI sites at the ends of the sequence for cloning purposes.

Source

The designed construct was ordered from IDT as a GeneString

References

As explained previously, the protein sequence for this part was based on:

1. Linder, M.; Salovuori, I.; Ruohonen, L.; Teeri, T.T., 1996. Characterization of a Double Cellulose-binding Domain. SYNERGISTIC HIGH AFFINITY BINDING TO CRYSTALLINE CELLULOSE. Journal of Biological Chemistry, 271(35), pp.21268–21272. Available at: http://www.jbc.org/content/271/35/21268.full