Protein_Domain
Fok_i

Part:BBa_K243001:Design

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-08)
Revision as of 20:44, 21 October 2009 by Isabel (Talk | contribs)

Protein domain (inactive) 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
    COMPATIBLE WITH RFC[1000]


Design Notes

Planning the design of two different FokI-heterodimers

For the catalytic inactive Fok partner named Fok_i, the heterodimeric amino acid glutamine 486 was switched to glutamate (CAA->GAA) and isoleucine 499 to leucine (ATC->CTG) and the catalytic amino acid aspartate 450 was switched to alanine (GAC->GCG) and aspartate 467 to alanine (GAT->GCG).

Freiburg09 fokmodel fig2.png

Fig.2 two FokI cleavage domains amino acids 387 to 579. Red: Catalytically active FokI cleavage domain; Cyan: Catalytically inactive FokI cleavage domain; Green: catalytically active amino acids; 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 to 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