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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. Figure 15. 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 15. TP901-1 recombination efficiency with variety of RBS from iGEM (B0030~B0035). T.I.R = Translation Initiation Rate
Reference
Roquet, Nathaniel et al. "Synthetic recombinase-based state machines in living cells." Science 353.6297 (2016): aad8559.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 28
Illegal AgeI site found at 1486 - 1000COMPATIBLE WITH RFC[1000]
None |