Difference between revisions of "Part:BBa K1742004"

Revision as of 00:18, 17 September 2015

PhiC31 Plant codon optimized

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

The plant codon-optimized PhiC31 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 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.

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 421
Illegal NheI site found at 591
Illegal NheI site found at 1633
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 79
Illegal BamHI site found at 816
Illegal BamHI site found at 846
Illegal BamHI site found at 932
Illegal BamHI site found at 942
Illegal XhoI site found at 773
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 13
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 1303
Illegal SapI.rc site found at 1628

@@ Line 12: / Line 12: @@
 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.
 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.
+https://static.igem.org/mediawiki/2015/thumb/4/43/BBa_1742004_GFP_PhiC31.png/800px-BBa_1742004_GFP_PhiC31.png
+<small><p><b>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.</b></p></small>