Difference between revisions of "Part:BBa K1742008"

 
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<partinfo>BBa_K1742008 short</partinfo>
 
<partinfo>BBa_K1742008 short</partinfo>
  
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The part consists of a T35S terminator flanked by BxbI recognition sites. When the integrase is functionally expressed, it makes double strand breaks in the recognition sites so that the terminator is removed.
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PhiC31 is a site-specific serine recombinase derived from a ''Streptomyces'' phage (Keravala et al.,2008). 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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== Biology and Usage ==
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In order to see the activity of the integrase, Valencia_UPV 2015 designed the present 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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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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With a binary GoldenBraid assembly step we obtained a 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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Revision as of 01:52, 17 September 2015

PhiC31 Reporter attP:T35S:attB:omegaUTR

PhiC31 is a site-specific serine recombinase derived from a Streptomyces phage (Keravala et al.,2008). 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.

Biology and Usage

In order to see the activity of the integrase, Valencia_UPV 2015 designed the present 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. 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). With a binary GoldenBraid assembly step we obtained a 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.


Sequence and Features


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]