Difference between revisions of "Part:BBa K243001:Design"
(→‎Design Notes)
 
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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).<br>
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For the catalytically inactive Fok partner named Fok_i, the amino acid glutamine 486 was switched to glutamate (CAA->GAA) and isoleucine 499 was switched to leucine (ATC->CTG). The catalytic amino acid aspartate 450 was switched to alanine (GAC->GCG) and aspartate 467 to alanine (GAT->GCG) to inactivate the DNA cleavage activity of the protein domain.<br>
 
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[[Image:Freiburg09 fokmodel fig2.png]]
 
[[Image:Freiburg09 fokmodel fig2.png]]
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<p class=MsoNormal><span class=itatext><b>Fig.2 two FokI cleavage domains amino acids
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<p class=MsoNormal><span class=itatext><b>Fig.2 The two cleavage domains derived from the FokI protein.<br></b>
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387 to 579. </b>Red:<b> </b>Catalytically active FokI cleavage domain; Cyan:
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Red: Catalytically active FokI cleavage domain (Fok_a); Cyan:
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Catalytically inactive FokI cleavage domain; Green: catalytically active amino acids;
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Catalytically inactive FokI cleavage domain (Fok_i); Green: aminoacids naturally involved in catalysis (in FokI);
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Pink: </span>glutamate 490 and isoleucin 538; Blue: glutamin 486 and isoleucin
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Pink: residues glutamate 490 and isoleucine 538; Blue: residues glutamine 486 and isoleucine
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499.</p>
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499.<br>
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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.<br>
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For exchanging the individual amino acids we used the E. coli codon usage table from [http://www.faculty.ucr.edu/~mmaduro/codonusage/codontable.htm Hénaut and Danchin].<br><br>
  
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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] <br>
 
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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]
 
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<br>
 
 
Designed with Biobrick pre-and suffix for fusion proteins according to the [https://parts.igem.org/Assembly_standard_25 RFC 25]
 
Designed with Biobrick pre-and suffix for fusion proteins according to the [https://parts.igem.org/Assembly_standard_25 RFC 25]
 
<br> [https://static.igem.org/mediawiki/parts/3/3f/Freiburg_Fok_i.txt Commented GenBank file]
 
<br> [https://static.igem.org/mediawiki/parts/3/3f/Freiburg_Fok_i.txt Commented GenBank file]

Latest revision as of 02:45, 22 October 2009

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 catalytically inactive Fok partner named Fok_i, the amino acid glutamine 486 was switched to glutamate (CAA->GAA) and isoleucine 499 was switched to leucine (ATC->CTG). The catalytic amino acid aspartate 450 was switched to alanine (GAC->GCG) and aspartate 467 to alanine (GAT->GCG) to inactivate the DNA cleavage activity of the protein domain.

Freiburg09 fokmodel fig2.png

Fig.2 The two cleavage domains derived from the FokI protein.
 Red: Catalytically active FokI cleavage domain (Fok_a); Cyan: Catalytically inactive FokI cleavage domain (Fok_i); Green: aminoacids naturally involved in catalysis (in FokI); Pink: residues glutamate 490 and isoleucine 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 [http://www.faculty.ucr.edu/~mmaduro/codonusage/codontable.htm Hénaut and Danchin].

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