Protein_Domain
Fok_a

Part:BBa_K243000:Design

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-08)
Revision as of 10:43, 21 October 2009 by Timo (Talk | contribs) (→‎Source)

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

Freiburg09 fokmodel fig2.png

Fig.2 two FokI cleavage domains aminoacid 387 to 579. Red: Catalytically active FokI cleavage domain; Cyan: Catalytically inactive FokI cleavage domain; Green: catalytically active aminoacids; Pink: glutamate 490 and isoleucin 538; Blue: glutamin 486 and isoleucin 499.


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]
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 with Biobrick pre-and suffix for fusion proteins according the RFC 25
Commented GenBank file

Source

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

Planed by Team Freiburg Bioware and 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