Protein_Domain
Fok_a

Part:BBa_K243000:Design

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-08)
Revision as of 09:18, 21 October 2009 by Timo (Talk | contribs) (→‎Design Notes)

Protein domain (active) of the restriction endonuclease FokI


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 487


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

extract coding region of Fok from the restriction-modification genes of the chromosomal DNA of Flavobacterium okeanokoites. 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