Difference between revisions of "Part:BBa K1742013"

 
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__NOTOC__
 
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<partinfo>BBa_K1742013 short</partinfo>
 
<partinfo>BBa_K1742013 short</partinfo>
  
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Composite composed of the recombinase Bxb1 optimize for Nicothiana Benthamiana and a reporter to prove its action. The reporter composite consist in a terminator flankeated by the recognition sequence of Bxb1 between the promoter and the CDS. Only if the recombinase is present, the GFP will be observed.
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PhiC31 is a site-specific serine recombinase derived from a ''Streptomyces'' phage [1]. The enzyme recognizes two different attachments sites called also attB and attP, and also excise a sequence flanked with attB and attP sites close to the promoter.
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https://static.igem.org/mediawiki/2015/a/af/Valencia_UPVPhiC31.png
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== Biology and Usage ==
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The plant codon-optimized PhiC31 ([https://parts.igem.org/Part:BBa_K1742004 BBa_K1742004]) gene was domesticated and standardized as a GoldenBraid part and cloned into the pUPD2 entry vector.  The PhiC31 integrase gene was then assembled in a multipartite reaction with the strong promoter from the Cauliflower Mosaic Virus (P35S), and its terminator (T35S).
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In order to see the activity of the integrase, Valencia_UPV 2015 designed a reporter element ([https://parts.igem.org/Part:BBa_K1742008 BBa_K1742008]) that consists of a Cauliflower Mosaic Virus terminator (T35S) flanked with the recognition sites (attP:T35S:attB:omegaUTR) of PhiC31. The CDS was subsequently assembled in a multipartite reaction with the P35S promoter with no ATG, the GFP coding sequence and the T35S terminator.  
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With a binary GoldenBraid assembly step we obtained the present multigenic construct ([https://parts.igem.org/Part:BBa_K1742013 BBa_K1742013]) of the previously described transcriptional units. This construct was then transformed, by agroinfiltration, into ''N. benthamiana'' plants for testing the expression of GFP.  
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https://static.igem.org/mediawiki/2015/thumb/4/43/BBa_1742004_GFP_PhiC31.png/800px-BBa_1742004_GFP_PhiC31.png
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<small><p><b>Figure 1. Expression levels of GFP in ''N. benthamiana'' leaves. A) Agrobacterium-mediated transformation with the multigenic construct ([https://parts.igem.org/Part:BBa_K1742013 BBa_K1742013]). B) Plant leaf transformed with the PhiC31 reporter element assembled with the promoter, GFP and the terminator.</b></p></small>
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'''References'''
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<br>
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<small>
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1. Keravala, A., Groth, AC., Jarrahian, S., Thyagarajan, B., Hoyt, JJ., Kirby, PJ., and Calos, MP. (2006). A diversity of serine phage integrases mediate site-specific recombination in mammalian cells. Molecular Genetics and Genomics, 276(2), 135–146<br>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 02:47, 17 September 2015

35S:PhiC31:T35S - 35S:ReporterPhiC31::GFP:T35S

PhiC31 is a site-specific serine recombinase derived from a Streptomyces phage [1]. The enzyme recognizes two different attachments sites called also attB and attP, and also excise a sequence flanked with attB and attP sites close to the promoter. Valencia_UPVPhiC31.png

Biology and Usage

The plant codon-optimized PhiC31 (BBa_K1742004) gene was domesticated and standardized as a GoldenBraid part and cloned into the pUPD2 entry vector. The PhiC31 integrase gene was then assembled in a multipartite reaction with the strong promoter from the Cauliflower Mosaic Virus (P35S), and its terminator (T35S). In order to see the activity of the integrase, Valencia_UPV 2015 designed a reporter element (BBa_K1742008) that consists of a Cauliflower Mosaic Virus terminator (T35S) flanked with the recognition sites (attP:T35S:attB:omegaUTR) of PhiC31. The CDS was subsequently assembled in a multipartite reaction with the P35S promoter with no ATG, the GFP coding sequence and the T35S terminator.

With a binary GoldenBraid assembly step we obtained the present multigenic construct (BBa_K1742013) of the previously described transcriptional units. This construct was then transformed, by agroinfiltration, into N. benthamiana plants for testing the expression of GFP. 800px-BBa_1742004_GFP_PhiC31.png

Figure 1. Expression levels of GFP in N. benthamiana leaves. A) Agrobacterium-mediated transformation with the multigenic construct (BBa_K1742013). B) Plant leaf transformed with the PhiC31 reporter element assembled with the promoter, GFP and the terminator.


References
1. Keravala, A., Groth, AC., Jarrahian, S., Thyagarajan, B., Hoyt, JJ., Kirby, PJ., and Calos, MP. (2006). A diversity of serine phage integrases mediate site-specific recombination in mammalian cells. Molecular Genetics and Genomics, 276(2), 135–146


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1449
    Illegal NheI site found at 1619
    Illegal NheI site found at 2661
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1107
    Illegal BamHI site found at 1844
    Illegal BamHI site found at 1874
    Illegal BamHI site found at 1960
    Illegal BamHI site found at 1970
    Illegal XhoI site found at 1801
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1041
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 2331
    Illegal SapI.rc site found at 2656