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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

Characterization

Different replication origins affect the copy number of plasmids, and it is highly probable that they also influence the expression levels of their encoded recombinases. To select integrases for the bio-flip-flop, we constructed expression vectors for different recombinases and tested their performance individu-ally. This was carried out by co-transformation of E. coli Top10 with the expression vector and a re-porter plasmid. The reporter plasmid expresses different fluorescent proteins before and after flipping of a constitutive promoter.

Figure 5. The standard genetic structure used to characterize the recombinases.

According to our design of the bio-flip-flop, the two inputs X and Y comprise a signal. Consequently, two distinct induction systems need to be constructed. For this reason, recombinases were cloned onto two different backbones, under regulation of differently induced promoters. The expression vec-tors were constructed by Gibson assembly. Plasmid type 1 is cloned from the repressor generator (RPG), which has a p15A replication origin, ampicillin resistance gene, lacI, an IPTG-induced pTac promoter, and a RiboJ insulator after pTac. The backbone of plasmid type 2 was cloned from pIn-tegrase_13, which was a gift from Christopher Voigt (Addgene plasmid # 60584). It has a ColE1 repli-cation origin, kanamycin resistance gene, araC, and an arabinose-induced pBad promoter. RiboJ was added to Plasmid2 by cloning RiboJ and the recombinase from Plasmid1, and assembled after pBad.

Fig6. 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.

For a given backbone and inducible promoter, RBS strength affects the expression level post-transcription. For this reason, we tested a series of RBS for different recombinases in different back-bones. In order to tune the RBS, an intermediate vector with a 20bp random sequence (45% GC), flanked by a pair of BsmbI recognition sites (introduced by PCR) was prepared for each recombinase. Vector construction was completed by adding RBS via Golden Gate assembly. The RBS calculator was used to generate RBS sequences with a wide range of translation initiation rates. Other sequences (B0029, B0030, B0032, B0034, B0035) were obtained from the iGEM website. The RBS oligos were obtained from the annealing of two complementary primers. Expression vectors for each recom-binase with different RBS were tested by flow cytometry after co-transformation with the reporter vec-tor. For each recombinase, an RBS correlating with low leakage (small RFP subset in the cultures with no inducer) and high recombination efficiency after induction was selected.

Construction of bio-flip-flops After having already optimized the expression systems of integrases and excisionases, we set out to construct a Bio-Flip-Flop. Since it is unknown whether the Bio-Flip-Flop is practical, we considered developing two hierarchical execution units supporting our Bio-Flip-Flop. We called these two units the forward latch and the backward latch. The state transition process of each unit consists of two stable and irreversible states. Nevertheless, the state transition process of both units consists of four stable and cyclic states. Such units support the verification of the feasibility of our Bio-Flip-Flop and further allow us to construct a complete and functional Bio-Flip-Flop.

Forward latch The structure of the forward latch consists of two plasmids with different induction systems of inte-grases (Figure 9). The state transition process of the forward latch consists of two stable and irre-versible states (Figure 10).

Fig9. The structure of the forward latch. Two plasmids constitute the forward latch. Each plasmid con-tains an integrase gene (Bxb1-gp35 or TP901) and a reporter gene (GFP or mRFP). To standardize the input, we used the pBAD promoter to regulate the transcription of Bxb1 and GFP, and the pTAC promoter to regulate the transcription of TP901 and mRFP. Moreover, each integrase recognizes and converts the attB and attP sites on the other plasmid.

Fig10. The state transition process of the Forward latch. After induction with arabinose, the expres-sion of integrase Bxb1 and GFP is initiated. The integrase Bxb1 recognizes and converts the attB and attP sites flanking the pTAC promoter, which leads to a change of orientation of the pTAC promoter. If we input IPTG next, the expression of integrase TP901 and mRFP will begin. The orientation of the pBAD promoter will change. As the GFP is degraded and mRFP is produced, the ratio of mRFP/GFP fluorescence rises.

Backward latch The structure of the Backward latch consists of two plasmids with different induction systems driving excisionases (Figure 11). The state transition process of the Backward latch consists of two stable and irreversible states (Figure 12). Because excisionases recognize attL and attR sites and convert them into attB and attP sites, the backward latch is capable of resetting the state of the Forward latch.

Fig11. The structure of the Backward latch. Two plasmids constitute the Backward latch. Each plas-mid contains an excisionase gene (Bxb1-gp47 or TP901) and a reporter gene (GFP or mRFP). To standardize the input, we used the pBAD promoter to regulate the transcription of Bxb1 and GFP, and the pTAC promoter to control the transcription of TP901 and mRFP. Moreover, each excisionase rec-ognizes and converts the attL and attR sites on the other plasmid.

Fig12. The state transition process of the Backward latch. After induction with arabinose, the expres-sion of excisionase Bxb1 and GFP is initiated. The excisionase Bxb1 recognizes and converts the attL and attR sites flanking the pTAC promoter, which leads to a change of the orientation of the pTAC promoter. If we input IPTG next, the expression of excisionase TP901 and mRFP will begin. The ori-entation of the pBAD promoter will change. As GFP is degraded and mRFP is produced, the ratio of mRFP/GFP fluorescence rises.

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.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.

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. The former represents the density of bacteria, and the latter implies the efficiency of recombination.

For more detailed measurements,flow cytometry were used to evaluate the recombination efficiency. 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.

For the vector with ColE1 replication origin, we found proper RBS in a list of calculated RBSs for TP901-1.

Figure 3. TP901-1 recombination efficiency with various RBS. 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).

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.