Protein_Domain
Fok_a

Part:BBa_K243000:Design

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-08)
Revision as of 10:03, 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

Freiburg09 fokmodel fig2.png

  • 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