Difference between revisions of "Part:BBa K2243000"
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Fig1. Site-specific recombination either integrates, deletes or reverses a DNA sequence | Fig1. Site-specific recombination either integrates, deletes or reverses a DNA sequence | ||
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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. | 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. | 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. | ||
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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. | 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. | For expression vector with p15A replication origin, proper RBS for TP901-1 was picked out. | ||
| − | Figure | + | |
| + | ===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. | For expression vector with p15A replication origin, proper RBS for TP901-1 was picked out. | ||
| − | Figure | + | Figure 4. TP901-1 recombination efficiency with variety of RBS from iGEM (B0030~B0035). T.I.R = Translation Initiation Rate |
===Reference=== | ===Reference=== | ||
| − | Roquet, Nathaniel et al. "Synthetic recombinase-based state machines in living cells." Science 353.6297 (2016): aad8559. | + | 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. | |
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Revision as of 14:07, 31 October 2017
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.