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.

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

Another kind of recombinases - excisionases are able to recognize and bind attL and attR sequences. With the help of excisionases, the state transitions become reversible. Recombination Directionality Factors (RDF) are a kind of protein that achieves reverse flipping to recover the sequence flipped by recombinases. By co-expression of the integrase with the corresponding RDF, or expression of the integrase-RDF fusion protein, recombination between the attL and attR sites can be induced. Thus, the flip-flop can restore to the previous state.

Fig2. Schematic drawing of RDF mechanism. (Olorunniji et al. 2017)

Experience

Using TP901-1 integrases and its corresponding excisionases, we designed the bio-flip-flop device to store state information. Because our final goal is to realize the state transition of our Bio-Flip-Flop, it is significant to characterize the efficiency of TP901-1 recombinase. For expression vector with p15A replication origin, proper RBS for TP901-1 was picked out.

Functional Parameters

Figure 3. TP901-1 recombination efficiency with variety of RBS from iGEM (B0030~B0035). T.I.R = Translation Initiation Rate

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

Figure 4. TP901-1 recombination efficiency with variety of RBS from iGEM (B0030~B0035). T.I.R = Translation Initiation Rate

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.