Part:BBa_K3254006

Integrase Int5

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).

Orthogonality Characterization

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.

Genetic design

The composition and principle of the experimental system are indicated below. More sequence details can be seen on the pages of BBa_K3254026 and BBa_K3254005.

File:T--GENAS China--excision with backbone. PNG

The composition and principle of the experimental system

Experimental Setup

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.

Results

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 30
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 22
  Illegal AgeI site found at 406
  Illegal AgeI site found at 781
  
• 1000
  COMPATIBLE WITH RFC[1000]