Difference between revisions of "Part:BBa K1132009"

Revision as of 02:56, 18 September 2015

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 (https://parts.igem.org/Part:BBa_K1132003, https://parts.igem.org/Part:BBa_K1132004).

attB + attP + integrase → attR + attL + integrase

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
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 1689
Illegal SapI.rc site found at 1380
Illegal SapI.rc site found at 1438
Illegal SapI.rc site found at 1543

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 <!-- Add more about the biology of this part here
 ===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 ([https://parts.igem.org/Part:BBa_K1742008 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.
+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 ([https://parts.igem.org/Part:BBa_K1742013 BBa_K1742013]). B) Plant leaf transformed with the PhiC31 reporter element assembled with the promoter, GFP and the terminator.</b></p></small>
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