Device

Part:BBa_K1742013

Designed by: Ruben Escriba Piera   Group: iGEM15_Valencia_UPV   (2015-09-10)

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. ValenciaUPV_PhiC31CodonOptimGFP.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 P35S promoter without ATG, GFP coding sequence and the terminator T35S.


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
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/eukaryote/nbenthamiana
Parameters
None