Revision as of 17:47, 1 November 2017

φBT1 integrase

This ORF codes for the steptomyces phage φRv1 integrase, which catalyzes a site specific recombination between φRV1 attB (BBa_K2460002) and attP (BBa_K2460003) sequences. This recombinase can be used to 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.

Biological Characterisation

φRv1 integrase is a member of the large serine recombinases(LSRs) family. LSRs are recombinases encoded by temperate phage genomes, which precisely cut and recombine DNA in a highly controllable and predictable way. The recombinases recognize and bind to attB/P sites as dimers, and cut and recombinate on the attB/P sites, with two dimers forming a tetramer. The recombinated attB/P sites form attL and attR sites.

Kinetic Characterization

We conducted qPCR to record the ratio of attL/R to the sum of attL/R and attB/P, to characterize the efficiency varying with time.

Genetic design

• The integrase is expressed under a PBad promoter (BBa_I13453). Considering the leakage of PBad promoter would lead to flipping，the promoter is followed by a weaker RBS (BBa_B0033). Ahead of the promoter,an attB and attP are opposite to each other,flanking a 50 bp random sequence. This construct was cloned on a medium copy plasmid with p15A replication of origin and kanamycin resistance.
• To ensure a low leakage when not induced, AraC is expressed under a constitutive promoter, cloned on a high copy plasmid with pUC19 replication of origin and Ampicillin resistance.

Experimental Setup

• Since transforming the two plasmid above at a time was unable to prevent the flipping, the plasmid with AraC was first transformed into E.coli DH10b, then the plasmid with the integrase was transformed into the competent cell with the AraC plasmid.
• After culturing for over 2 hours in 2×YT borth, the cells were induced in 1％ Arabinose for 8 hours. We sampled the culture at 0 hour, the 4th hour and the 8th hour.
• We calculated flipped and unflipped random sequence by qPCR.

Results

Before inducing, the LR rate remained a relatively low level, and after inducing for 8 hours, the LR rate reached a high level of over 0.6, showing high efficiency and low leakage.

File:Fudan China phiRV1.jpg

Orthogonality Characterization

Since our circuit are using multiple integrases, it's important that the attB/P site of one integrase won't be flipped by one another integrase. We conducted orthogonality tests to see the compatibility between our integrases, including phiBT1, Bxb1, phiC31, phiRv1 and TG1 integrase.

Genetic design

• 5 pairs of attB/P sites are each flanking a 50 bp random sequence, lining up ahead of the PBad promoter (BBa_I13453), an RBS (BBa_B0033) and an integrase. This construct was cloned on a medium copy plasmid with p15A replication of origin and kanamycin resistance.
• The same AraC plasmid in Kinetic Characterization was used in orthogonality Characterization.

Experimental Setup

• The same method of transformation in Kinetic Characterization was used in Kinetic Characterization.
• After culturing for over 2 hours in 2×YT borth, the cells were induced in 1％ Arabinose 16 hours.
• Conduct PCR for each integrase's test and control group with primers from 5 pairs of attB/P sites, 2 pairs of primers for each pair of attB/P site.

Results

Expect for the phiRv1 attB/P site, none of the attB/P sites flipped, indicating that phiRv1 integrase has a good orthogonality with other attB/P sites.

Sequence and Features