Difference between revisions of "Part:BBa K243000:Design"

Revision as of 09:19, 21 October 2009

Protein domain (active) of the restriction endonuclease FokI

Design Notes

Planning the design of two different FokI-heterodimers
For the catalytic active Fok partner, named Fok_a, the first 1158 nucleotides, i.e. the recognition domain, were deleted and glutamate 490 was switched to lysine (GAA->AAA) as well as isoleucine 538 to lysine (ATC->AAA) for the heterodimer formation

• extract
• delete the first 1158 nucleotides/386 aa (recognition domain)
  
• switch Cystein 541/463-465 to Ser (TGT->TCT)
  
  

Modifications of the single vectors to introduce heterodimeric modifications according to [http://www.ncbi.nlm.nih.gov/pubmed/17603475 Miller J, Rebar E Nature biotech 2007]

Modifications of the first vector (catalytic active heterodimer)
-heterodimeric aminio acids

• switch Glutamate 490/310-312 to Lysin (GAA->AAA)
  
• switch isoleucin 538/454-456 to Lysin (ATC->AAA)
  

Modifications of the second vector (catalytic inactive heterodimer)
-heterodimeric amino acids

• switch Glutamin 486/298-300 to Glutamate (CAA->GAA)
  
• switch Isoleucin 499/337-339 to Leucin (ATC->CTG)
  

-catalytic amino acids

• switch Aspartate 450/190-192 to Alanin (GAC->GCG)
  
• switch Aspartate 467/243-245 to Alanin (GAT->GCG)
  

Annotations:

• The notation e.g. for Cystein 541/463-465 means the amino acid 541 in literature which correspond to the codons 463-465 in our vector.
  
• For exchanging the amino acids we used the Codon usage table in E.coli from Hénaut and Danchin. [http://www.faculty.ucr.edu/~mmaduro/codonusage/codontable.htm E.coli Codon Usage]
  
  

Designed according the assembly standard 25 RFC 25

Source

Source of the protein was the coding region of FokI from the restriction-modification genes of the chromosomal DNA of Flavobacterium okeanokoites [http://www.ncbi.nlm.nih.gov/nuccore/148723?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum F.okeanokoites fokIR and fokIM genes]
Part synthesized by Mr.Gene

References

Mary C. Looneya, Laurie S. Morana, William E. Jacka, George R. Feeherya, Jack S. Bennera, Barton E. Slatkoa and Geoffrey G. Wilson;(1989)
Nucleotide sequence of the FokI restriction-modification system: separate strand-specificity domains in the methyltransferase; Gene Vol.80 Issue:2 Pages:193-208

Jeffrey C Miller1, Michael C Holmes1, Jianbin Wang1, Dmitry Y Guschin1, Ya-Li Lee1, Igor Rupniewski1, Christian M Beausejour1,2, Adam J Waite1, Nathaniel S Wang1, Kenneth A Kim1, Philip D Gregory1, Carl O Pabo1,2 & Edward J Rebar (2007);
An improved zinc-finger nuclease architecture for highly specific genome editing; Nature Biotechnology 25, 778 - 785