35S:PhiC31:T35S - 35S:ReporterPhiC31::GFP:T35S
PhiC31 is a site-specific serine recombinase derived from a Streptomyces phage . 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
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.
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.
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
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 1449
Illegal NheI site found at 1619
Illegal NheI site found at 2661
- 21Illegal 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
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 1041
- 1000Illegal SapI.rc site found at 2656
Illegal SapI site found at 2331