This is a serine-type phage (LSTP) integrases. The original organism was the Streptomyces phage PhiK38-1. The product protein of this part can catalyze the recombination between the corresponding attB and attP site (see part BBa_K3254001).
We conducted orthogonality tests to see the compatibility between the 6 integrase used in our project. The other integrases included phiC31, Int7, Int8, Int10 and TG1.
The plasmid containing the Int5 expressive unit was co-transferred into E.coli DH5α host with 6 reporter plasmid containing different attP-attB sites sequences. Then single colonies were inoculated into M9 supplemented medium for overnight growth. Then, the cell cultures were diluted 1000-fold with M9 supplemented medium with 500 μM IPTG inducer and growth for another 20 hours. All incubations were carried out using a Digital Thermostatic Shaker maintained at 37 °C and 1000 rpm, using flat-bottom 96-well plates sealed with sealing film. Finally, the cultures were sampled for genotype PCR testing. The principle of genotype identification was shown on the right of results image.
- M9 medium (supplemented）: 6.8 g/L Na2HPO4, 3 g/L KH2PO4, 0.5 g/L NaCl, 1 g/L NH4Cl, 0.34 g/L thiamine, 0.2% casamino acids, 0.4% glucose, 2mM MgSO4, and 100 μM CaCl2.
The result indicate that Int5 integrase has a good orthogonality with the other 5 att sites and compatible with other integrases. IBR-C35/F55/S37/E21/T25/G22 were the plasmids with phiC31/Int5/Int7/Int8/Int10/TG1 att sites.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 30
- 23COMPATIBLE WITH RFC
- 25Illegal AgeI site found at 22
Illegal AgeI site found at 406
Illegal AgeI site found at 781
- 1000COMPATIBLE WITH RFC