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A118 recombinase

Abstract

A118 is a serine recombinase that mediates a reversible site-specific recombination system first discovered in bacteriophages. This site-specific recombination system is used by bacteriophages to integrate their genes into the host genomic DNA for proliferation and to transfer genes from the host genome to daughter phages by expressing recombination directionality factor (RDF) before the host is lysed and released. Specifically, A118 can mediate the excision or inversion of genes between a pair of specific recognition sites (attB and attP) and simultaneously convert this pair of sites into another pair of recognition sites (attL and attR). This site-specific recombination is reversible, and when the auxiliary protein RDF is present, the serine recombinase can reduce attL and attR to attB and attP.

It is worth noting that a recombinase can have multiple pairs of mutually orthogonal attB and attP recombination sites. For example, A118B-AA (BBa_K5276017) and A118P-AA (BBa_K5276018) are a pair of recombination sites of A118, while A118B-GG (BBa_K5276019) and A118P-GG (BBa_K5276020) are another pair of recombination sites orthogonal to that.

Molecular mechanism

Each recognition site consists of two incompletely symmetrical sequences on the left and right and a 2bp linker sequence between them. We named the left and right sequences of attB (attP) as B (P) type arms.

In the free state, recombinase exists as a dimer (a), and the N-terminal domains (NTD) of the two integrases bind to each other; when attB & attP are present, the C-terminal domain (CTD) of the integrase will bind to the two arms of the binding site respectively (b); after CTD binding, the conformation changes, so that the dimers bound to attB & attP respectively form a tetramer (c); the tetramer conformation activates NTD, causing DNA being cut from the 2bp linker region in the middle of the binding site (e); after cutting, the two integrases on one side will rotate 180° relative to each other (f); and finally form a new attL & attR binding site (g). Therefore, as shown in Figure (g), the newly formed attL and attR are both composed of a B arm and a P arm. Since the two arms connecting the dimer are different, tetramers cannot form without RDF; thus, attL and attR cannot be reconstituted. If RDF is present, similar recombination will occur between the attL and attR sites, transforming back into attB and attP.

The recognition site of the recombinase is directional. If a pair of attB and attP sites are in the same direction, the sequence between them will be removed; if the attB and attP sites are in opposite directions, the sequence between them will be inversed.

Results

We experimentally verified the orthogonality between A118 and other integrases and found that they have good orthogonality.

We designed twelve plasmids, divided them into six 'recombinase plasmids' and six 'verification plasmids,' and double-transformed them into bl21(DE3). Finally, we used colony PCR to verify our results.

Figure 1: Recombinase Plasmid

The six "recombinase plasmids" comprise six different recombinases and Plac promoters, which express the recombinases we need in the presence of inducer IPTG. At both ends of the recombinase gene of these plasmids, we designed a pair of primers (PET_28_T7_term and PET_28_T7, see Figure 1) for colony PCR to verify whether the recombinase plasmids had been successfully transferred.

Figure 2: Verification Plasmid

These six "verification plasmids" comprise recombination sites with opposite directions for six recombinases. Theoretically, if a recombinase matches the site, the DNA sequence between the recombination sites will inverse. We designed a pair of primers (Test_F1_sk and Test_R1_sk) upstream and downstream of the recombination site and designed a reverse primer (Test_R_sk, see Figure 2) on the DNA sequence between the recombination sites. We first used Test_F1_sk and Test_R1_sk to check whether the verification plasmid was successfully transferred. Then, we used Test_F1_sk & Test_R_sk, and Test_R_sk & Test_R1_sk for colony PCR. If the sequence is not inversed, the group of Test_F1_sk & Test_R_sk will have obvious fragments; if the sequence is inversed, the group of Test_R_sk & Test_R1_sk will have obvious fragments.

The following are the results of our colony PCR verification using different primers, after we double-transfected the recombinase plasmid and the verification plasmid and induced them with IPTG: Each fragment on the gel image is marked with two letters and a number, where the first letter represents the recombinase corresponding to the recombinase plasmid, the second letter represents the recombination site corresponding to the verification plasmid, and the number represents the parallel repeated test. (Abbreviations of recombinases: A-A118, B-Bxb1, C-Cre, F-Fime, T-Tp901, φ-PhiC31)

According to the gel image results, except for the Cre-recombinase plasmid, all other recombinase plasmids and verification plasmids were correctly transferred. The group double-transformed with the Cre-recombinase plasmid had no fragments, which requires our subsequent further verification.

After we confirmed that the plasmids had been successfully transferred, we used the two pairs of primers Test_F1_sk & Test_R_sk and Test_R_sk & Test_R1_sk for colony PCR to check whether our "verification plasmid" was inversed. The results are below.

Figure 5(a)

Figure 5(b)

Figure 5(c)

Figure 5(d)

Figure 5(e)

Figure 5(f)
The white marked fragments are the results of PCR using Test_F1_sk and Test_R_sk.
The red coded fragments are the results of PCR using Test_R_sk and Test_R1_sk.

Theoretically, if the "verification plasmid" has not inversed, the length of the white-marked fragments should be 587bp, and there should be no fragment marked in red-marked groups; if the "verification plasmid" has inversed, there should be no fragment marked in white-marked groups, and the length of the white-marked fragments should be 968bp.

The gel image shows that the orthogonality between the binding sites of different recombinases is good. Specifically, for the non-AA BB CC FF TT φφ group, the white-marked fragments except Aφ2 have clear fragments of the correct length; the red-marked ones have no clear fragments, and even if there are fragments, the length is not 968bp. Only AB2 FB2 Aφ2 Fφ2 TF2 have possible non-orthogonal recombinase site interactions. However, since these fragments only appear in one of the two parallel repeats, we suspect this may be related to the impurity of the plasmid we used.

Directed Evolution

Sequence and Features