Difference between revisions of "Part:BBa K243000:Design"

(→‎Design Notes)
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'''Planning the design of two different FokI-heterodimers'''  
 
'''Planning the design of two different FokI-heterodimers'''  
 
<br>
 
<br>
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*extract coding region of Fok 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]<br>
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*delete the first 1158 nucleotides/386 aa (recognition domain)  <br>
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For the catalytically active Fok component named Fok_a, the first 1158 nucleotides naturally 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.<br>
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*switch Cystein 541/463-465 to Ser (TGT->TCT)<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><br>
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Modifications of the first vector (catalytic active heterodimer) <br>
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-heterodimeric aminio acids
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*switch Glutamate 490/310-312 to Lysin (GAA->AAA)<br>
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*switch isoleucin 538/454-456 to Lysin (ATC->AAA)<br>
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<br>
 
<br>
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Modifications of the second vector (catalytic inactive heterodimer) <br>
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[[Image:Freiburg09 fokmodel fig2.png]]
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-heterodimeric 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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*switch Glutamin 486/298-300 to Glutamate (CAA->GAA)<br>
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Red: Catalytically active FokI cleavage domain (Fok_a); Cyan:
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*switch Isoleucin 499/337-339 to Leucin (ATC->CTG)<br>
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Catalytically inactive FokI cleavage domain (Fok_i); Green: aminoacids naturally involved in catalysis (in FokI);
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-catalytic amino acids
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Pink: residues glutamate 490 and isoleucine 538; Blue: residues glutamine 486 and isoleucine
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*switch Aspartate 450/190-192 to Alanin (GAC->GCG)<br>
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499.<br>
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*switch Aspartate 467/243-245 to Alanin (GAT->GCG)<br>
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<br>
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Annotations:
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*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. <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]<br><br>
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<br>
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Designed according the assembly standard 25 [https://parts.igem.org/Assembly_standard_25 RFC 25]
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===Source===
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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>
 +
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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extract coding region of Fok from the restriction-modification genes of the chromosomal DNA of Flavobacterium okeanokoites. Part synthesized by Mr.Gene
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Designed with Biobrick pre-and suffix for the creation of fusion proteins according to the [https://parts.igem.org/Assembly_standard_25 RFC 25]
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<br>[https://static.igem.org/mediawiki/parts/5/5f/Freiburg09_fok_a.txt  Commented GenBank file]
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===Source===
  
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Source of the protein was the coding region of FokI from the restriction-modification genes of the chromosomal DNA of<br>[http://www.ncbi.nlm.nih.gov/nuccore/148723?ordinalpos=1&itool=EntrezSystem2.PEntrez.Sequence.Sequence_ResultsPanel.Sequence_RVDocSum Flavobacterium okeanokoites fokIR and fokIM genes]<br>
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<br>Planed and designed by Team Freiburg Bioware and synthesized by Mr.Gene.
  
 
===References===
 
===References===

Latest revision as of 02:46, 22 October 2009

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 naturally 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 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 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