Coding

Part:BBa_K1132009

Designed by: iGEM Toulouse   Group: iGEM13_INSA_Toulouse   (2013-09-20)

PhiC31 integrase

Isolated from the bacteriophage PhiC31, the PhiC31 integrase (frequently also written as: ΦC31 integrase) encodes a serine-type recombinase that mediates the sequence-specific recombination between two different attachment sites, called attB and attP, which share a 3 bp central region, where the crossover occurs (Thorpe et al., 2000). Because the two sites recognized by the PhiC31 integrase differ and the recombination event leads to two different sites (attR and attL), PhiC31 based switch is unidirectional and definitive, except if the required excisionase factor is present. Recombination occurs irrespective of whether the substrate is supercoiled or linear, and does not require anything more than the integrase and attB, attP sites . (HELENA M. THORPE AND MARGARET C. M. SMITH, In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvaseyinvertase family, 1998).

The recombination sites can be designed differently (position – orientation) in order to obtain a DNA 180° inversion or an integration of the desired DNA sequence. The 180° switch permits to design a lot of regulation tools, such as logical gates that can be found here (http://parts.igem.org/Part:BBa_K1132003, http://parts.igem.org/Part:BBa_K1132004).

attB + attP + integrase → attR + attL + integrase

PhiC31.png


More Usage and Biology

This part that was request to the Registry by Valencia UPV 2015 team. PhiC31 encodes a serine-type recombinase that mediates the sequence-specific recombination between two different attachment sites, called attB and attP. Our team did characterize the functionality of this part in N. benthamiana plants. To do so, we used PhiC31 as excisionase of our PhiC31-Reporter element (BBa_K1742008) coupled with a GFP tag. After transiently Agrobacterium-mediated transformation in plants, we could observe that a huge percentage of plant cells expressed GFP. See our PhiC31 recombinase module results.

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 (BBa_K1742008) assembled with the P35S promoter without ATG, GFP coding sequence and the terminator T35S.

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]


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