Protein_Domain
Fok_a

Part:BBa_K243000:Design

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-08)
Revision as of 02:14, 22 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 catalytically active Fok component named Fok_a, the first 1158 nucleotides forming the DNA recognition domain were deleted. The residue glutamate 490 was switched to lysine (GAA->AAA) and isoleucine 538 to lysine (ATC->AAA) to abolish the formation of homodimers.

Freiburg09 fokmodel fig2.png

Fig.2 The two cleavage domains of the FokI protein.
 Red: Catalytically active FokI cleavage domain; Cyan: Catalytically inactive FokI cleavage domain; Green: aminoacids involved in catalysis; Pink: residues glutamate 490 and isoleucin 538; Blue: residues glutamine 486 and isoleucine 499.
Modifications of the genes were created according to [http://www.ncbi.nlm.nih.gov/pubmed/17603475 Miller J et al. Nature Biotechnology 2007] to abolish the formation of homodimers and to enable heterodimerization.
For exchanging the individual amino acids we used the E. coli codon usage table from Hénaut and Danchin. [http://www.faculty.ucr.edu/~mmaduro/codonusage/codontable.htm E.coli Codon Usage]
Designed with Biobrick pre-and suffix for the creation of 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 and designed 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