This part was improved from BBa_K1132010. http://parts.igem.org/Part:BBa_K1132010
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.
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.
Fig 1. Site-specific recombination either integrates, deletes or reverses a DNA sequence
Many researchers have paid attention to recombinases because of their ability of changing genetic circuits. TP901-1 is one of the recombinases with outstanding performance. In existence of recombinase TP901-1, different orientation of attB and attP allows the sequence to be flipped, excised, or inserted between recognition sites, which makes it useful for gene editing. In our project, we selected TP901-1 to flip the sequence flanked by attB and attP site for Bio-Flip-Flop construction.
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 T[901 have an optimal performance. We used the standard testing system, consisting of the integrase expression plasmid and the recombination reporter plasmid (BBa_K2243010). By choosing 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.The expression vector and reporter of a recombinase were used to co-transform E. coli Top10 and samples were prepared for testing. We picked out our optimal RBS with low leakage and high efficiency for both backbone.
Figure 2. The standard testing system used to characterize the recombinases.
Fig 3. Integrase expression vectors with different replication origins. The one shown on top has a re-laxed ColE1 replication origin and recombinase expression is induced by arabinose via a pBad induc-tion system. The one shown in the bottom picture has a relaxed p15A replication origin and recom-binase expression is induced by IPTG.
We used a microplate reader to roughly measure the efficiency of the selected integrases. We used flow cytometry to conduct a more accurate characterization.
Microplate readers are instruments used to detect biological, chemical or physical events in samples in microtiter plates. We used a microplate reader to detect optical density and fluorescence intensity follwing the procedure as below:
1.Pick and inoculate single colony into 1ml of LB media with antibiotics in a V-bottom 96-well plate. Grow the culture overnight (about 12 hours) at 37°C, 220 rpm in a Thermo VARIOSKAN FLASH shaker.
2. Then, an aliquot comprising 2 μL of the culture was transferred into 1ml LB media with antibiotics and inducer (1mM IPTG or 10mM arabinose). Culture overnight (about 12 hours) at 37°C and 220 rpm.
3. Aliquot 1 μl of the cultures in the deep-well plates, then copy them to the 96-well plate filled with 200μl PBS. Don’t forget to leave at least 12 wells with only PBS in it as your negative conctrol.
4. Measure the samples (OD and Fluorescence). I The former represents the density of bacteria, and the latter implies the efficiency of recombination.
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).
For more detailed measurements,flow cytometry were used to evaluate the recombination efficiency. Our procedures are as follows:
1.Single colonies were picked and used to inoculate into 1ml of LB media with antibiotics in a V-bottom 96-well plate.
2.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.
3.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.
Fig 5.A example of Gating the RFP and GFP subsets. Change of fluorescence after induction was seen. Left: no inducer. Right: 10 mM Ara for 15h.
For the vector with ColE1 replication origin, we found proper RBS in a list of calculated RBSs for TP901-1.
Figure 6. TP901-1 recombination efficiency with various RBS. T.I.R = Translation Initiation Rate
Besides, we construct integrase-RDF fusion protein to recognize and bind attL and attR sequences,so the state transitions become reversible.click here to read more. http://parts.igem.org/Part:BBa_K2243014
Furthermore, we co-transformed the system containing the terminator flanked by TP901-1 attB/P sites and the system expressing the TP901-1 recombinase to test the inversion efficiency using the plate reader. And the results are show in the third bar group in the graph below.
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3.Bonnet, J., Yin, P., Ortiz, M. E., Subsoontorn, P., & Endy, D. (2013). Amplifying genetic logic gates. Science, 340(6132), 599-603.