Part:BBa_K2243000

TP901-1 integrase

TP901-1 integrase comes from TP901-1 phage and can bind to specific attB/P sites to catalyze DNA recombination. It helps the TP901-1 phage to integrate its genome into bacterial genome naturally.

By constructing the attB/P sites in different directions, TP901-1 can catalyze the recombination of DNA between their sites, leading to inversion when attB/P are in opposite directions and excision when attB/P are in the same directions. TP901-1 is widely used to construct combinational logic gate and performs well in changing DNA sequence.

Usage and Biology

TP901-1 recombinase is a serine recombinase enzyme derived from phage TP901-1 of Lactococcus lactis subsp. cremoris. The enzyme uses a topoisomerase like mechanism to carry out site specific recombination events. It (1.5 kDa) is known to integrate DNA fragment between two DNA recognition sites (attB/P site). With the help of its specific Recombination Directionality Factor (RDF) see the tag BBa_K2243014, TP901-1 recombinase can also flip DNA between the attachment sites, which makes the process reversible.

https://static.igem.org/mediawiki/2017/0/0b/Peking_flipflop_fig1.svg

Fig1. Site-specific recombination either integrates, deletes or reverses a DNA sequence

Characterization

Since the viability of a bio-flip-flop relies on the performance of two integrases and their corresponding excisionases. To select integrases for the bio-flip-flop, we constructed expression vectors for different recombinases and tested their performance individually.

To make sure that Bxb1 have an optimal performance. We used the standard testing system, consisting of the integrase expression plasmid and the recombination reporter plasmid (BBa_K2243006). By changing the vector with different replication origins(a p15A origin with a pTac promoter, and a ColE1 origin with a pBAD promoter) and the RBS sequences upon the integrase, we measure the recombination efficiency under different conditions. We picked out our optimal RBS with low leakage and high efficiency for both backbone.

Flow cytometry were used to evaluate the recombination efficiency, the expression vector and reporter of a recombinase were used to co-transform E. coli Top10 and samples were prepared for flow cytometry reading. Single colonies were picked and used to inoculate 1ml of LB media with antibiotics in a V-bottom 96-well plate. The cultures were grown at 37°C and 1000 RPM for 12h. Subsequently, an aliquot comprising 2 μL of the culture was transferred into 1ml of M9 glucose media with antibiotics and inducer (1mM IPTG or 10mM arabinose for RBS tuning, gradient concentration for transfer curve) in a V-bottom 96-well plate. The cultures were grown at 37°C and 1000 RPM for 15h. An aliquot comprising 2μL of the cul-ture was transferred into 198 μL of phosphate buffered saline (PBS) containing 2 mg/mL kanamycin in a 96-well plate. This mixture was incubated for one hour at room temperature before testing. Two lasers were used to excite GFP and RFP simultaneously. Single-cell fluorescence distribution at both emission wavelengths was recorded.

The counted cells were gated to eliminate the population which showed no fluorescence. The remaining cells were divided into two subsets by a diagonal: RFP sub-set and GFP subset. The recombination efficiency was estimated from the proportion of the RFP subset in the total fluorescent population.

Fig2. Gating of the RFP and GFP subsets and change of fluorescence after induction. Left: no induc-er. Right: 1 mM IPTG for 15h.

Functional Parameters

For expression vector with p15A replication origin, proper RBS for TP901-1 was picked out.

Figure 3. TP901-1 recombination efficiency with various RBS. T.I.R = Translation Initiation Rate

Figure 4. TP901-1 recombination efficiency with variety of RBS from iGEM (B0030~B0035).

Reference

1.Baker, T. A., Bell, S. P., Gann, A., Levine, M., & Losick, R. (1970). Molecular biology of the gene.

2.Roquet, Nathaniel et al. "Synthetic recombinase-based state machines in living cells." Science 353.6297 (2016): aad8559.

3.Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P., & Endy, D. (2013). Amplifying genetic logic gates. Science, 340(6132), 599-603.