Revision as of 11:26, 19 October 2019

TG1 attB

This is a recombination site attB for TG1 integrase (BBa_K2460007). When used with phiBT1 attP (BBa_K2460009) and TG1 integrase, it can flip a DNA sequence flanked by attB and attP sites, as well as integrating a circular DNA with a attB/P site into a linear/circular DNA with a attP/B site. For more information about kinetic characterization and the orthogonality between the integrase and some other serine recombinase, go to TG1 integrase.

Characterization by GENAS_China 2019

• Group: GENAS_China 2019

We use this part and BBa_K2460009(placed in the same deriction) as funcitional elements in BBa_K3254005, and the DNA between BBa_K2460008 and BBa_K2460009 was excised via recombination by TG1 integrase as expected, see below.

Genetic design

The composition and principle of the experimental system are indicated below. More details can be seen on the pages of BBa_K3254013 and BBa_K3254005.

Experimental Setup

The reporter plasmid contained this part and BBa_K2460009 were co-transferred into E.coli DH5α host with 6 integrase generator plasmids. 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.

Results

IBR-G22 was the plasmid with TG1 attB and attP sites. The result indicates that the TG1 att sites can only be recombined by TG1 integrase. The sequence after recombination (attL or attR) is GATCAGCTCCGCGGGCAAGACCGTGCTCTTACCCAGTTGGGCGGGA.

Sequence and Features