ATTP MV4-DERIVED SITE-SPECIFIC RECOMBINATION AND ITS USE FOR INTEGRATION OF SEQUENCE OF INTEREST
20250354175 ยท 2025-11-20
Inventors
Cpc classification
C12N15/70
CHEMISTRY; METALLURGY
C12N2800/30
CHEMISTRY; METALLURGY
C12N15/90
CHEMISTRY; METALLURGY
C12N9/12
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure relates to a method for preparing a site-specific recombination polynucleotide molecule derived from the attP site of the bacteriophage mv4 and to a kit for such site-specific recombination. The kit can be used to transform procaryote hosts to integrate any polynucleotide sequence of interest.
Claims
1. A method for preparing a site-specific recombination polynucleotide molecule comprising the steps of: aselecting a DNA target site in a genome of a bacterial host cell having a sequence of BOB wherein: B is 5-X1-X1-X2-X3-X3-X3-X4-3 wherein at most 1 of the nucleic acids of B may be N; O is 5-NNNNNNN-3 and B is 5-X1-X5-X5-X5-X6-X7-X2-3 wherein at most 1 of the nucleic acids of B may be N; wherein X1 to X7 and N have independently the following definitions: X1 is A or G or T; X2 is C or G or T; X3 is A or G; X4 is A or T; X5 is C or T; X6 is A or C or G; X7 is A or C or T; and N is A or C or G or T; bproviding the site-specific recombination polynucleotide molecule having a sequence of COC wherein: C is 5-X1-X1-X2-X1-X3-X1-X4-3 wherein at most 1 of the nucleic acids of C may be N; O is 5-NNNNNNN-3; and C is 5-X1-X5-X5-X5-X6-X7-X5-3 wherein at most 1 of the nucleic acids of C may be N; and wherein X1, X2, X3, X4, X5, X6, X7 and N are as defined previously; and wherein O of COC is identical to O of BOB of the bacterial host cell.
2. The method for preparing a site-specific recombination polynucleotide molecule according to claim 1, wherein B is 5-X4-X5-X5-X5-X6-X7-X2-3 and wherein at most 1 of the nucleic acids of B may be N; C is 5-X1-X1-X8-X1-X3-X1-X4-3 wherein at most 1 of the nucleic acids of C may be N; and C is 5-X4-X5-X5-X5-X9-X7-X5-3 wherein at most 1 of the nucleic acids of C may be N; and wherein X8 is T or G and X9 is A or C.
3. A kit for site-specific recombination of at least one polynucleotide sequence of interest into a genome of a bacterial host cell comprising: Aa polynucleotide molecule A comprising: (i) a sequence of between 220 to 250 pb comprising polynucleotide fragments P1-P2, COC and P 1-P2 wherein: TABLE-US-00014 P1-P2is (SEQIDNo2) 5-ATCAACTAGATTTTTAACTAGAA-3; COC is the site-specific recombination polynucleotide molecule according to claim 1 or 2; and TABLE-US-00015 P1-P2is (SEQIDNo3) 5-TTTAACTAGAAAATAACTAGAA-3; the sequence interacting with the DNA target site according to claim 1 or 2 in the bacterial host cell for integrating the polynucleotide sequence of interest; and (ii) at least one polynucleotide sequence of interest; Ba polynucleotide molecule int having at least 80%, preferably at least 85%, 90%, 95% or 100% identity with the sequence of SEQ ID No 4 coding for .sup.mv4Int or the .sup.mv4Int of SEQ ID No 5.
4. The kit of claim 3, wherein the polynucleotide molecule A is inserted in a first vector.
5. The kit of claim 4, wherein the polynucleotide molecule int coding for .sup.mv4Int is inserted in the first vector or in a second vector.
6. A method for integrating a polynucleotide sequence of interest into a genome of a genetically modified bacterial host cell comprising: apreparing a vector comprising a polynucleotide molecule A comprising: (i) a sequence of between 220 to 250 pb comprising the following polynucleotide fragments P1-P2, COC and P1-P2 wherein: TABLE-US-00016 P1-P2is (SEQIDNo2) 5-ATCAACTAGATTTTTAACTAGAA-3; COC is the site-specific recombination polynucleotide molecule according to claim 1 or 2; and TABLE-US-00017 P1-P2is (SEQIDNo3) 5-TTTAACTAGAAAATAACTAGAA-3; (ii) at least one polynucleotide sequence of interest; btransforming the bacterial host cell with the vector obtained at step (a) and the polynucleotide molecule int of SEQ ID No 4 coding for .sup.mv4Int; cmaintaining the transformed host cell under conditions that allow integration of the polynucleotide sequence of interest into the genome of the host cell.
7. A genetically modified bacterial host cell obtained by the method of claim 6, wherein the genetically modified bacterial host cell comprises a vector comprising a polynucleotide molecule A comprising: (i) a sequence of between 220 to 250 pb comprising the following polynucleotide fragments P1-P2, COC and P1-P2 wherein: TABLE-US-00018 P1-P2is (SEQIDNo2) 5-ATCAACTAGATTTTTAACTAGAA-3; COC is the site-specific recombination polynucleotide molecule according to claim 1 or 2; and TABLE-US-00019 P1-P2is (SEQIDNo3) 5-TTTAACTAGAAAATAACTAGAA-3; (ii) at least one polynucleotide sequence of interest; and the polynucleotide molecule INT of SEQ ID No 4 coding for .sup.mv4Int.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
Examples
Material and Methods
Strains, Plasmids, Primers, and Media
[0113] The different strains and plasmids used in this study are listed in Table 5 and 6. All sequences of primers that were used are available in Table 7A, 7B, 7C and 7D. The E. coli strain NEB5- repA+ was built by using the protocol from Datsenko and Warner (Datsenko and Wanner, 2000). It was constructed by replacing the glgB gene with the glgB::Kan-repA region from E. coli strain EC1000. E. coli strains were grown in Lysogenic Broth (LB) at 37 C. L. lactis were grown on GM17 at 28 C. Antibiotics were used at the following concentration: carbenicillin, 100 g/ml; chloramphenicol, 12.5 g/ml; erythromycin, 150 g/ml (1 g/ml for L. lactis); kanamycin, 50 g/ml.
DNA Procedures
[0114] Standard techniques were used for DNA manipulation and cloning. Polymerase chain reaction (PCR) was performed with Q5-HF polymerase (New England Biolabs) or with CloneAmp Hifi polymerase (Takara Bio), according to the manufacturer's instructions. PCR products were purified using the QIAquick PCR purification kit (Qiagen). Plasmids were constructed using Gibson assembly (42) with NEBuilder HIFI DNA Assembly (New England Biolabs) or blunt-end cloning with T4 PNK (New England Biolabs) and T4 DNA ligase (New England Biolabs), according to the manufacturer's instructions. Plasmid DNA was extracted using QIAprep Spin Miniprep kit (Qiagen) or Nucleobond Xtra Midi (Macherey-Nagel) and their sequence was verified by Sanger sequencing (Mix2seq, Eurofins).
Constructing a Randomized attB Library and Core-attP Library
[0115] The randomized oligonucleotides (109 bp, attB library; 184 bp, core-attP library) were obtained by chemical synthesis (IDT, USA). PCR was used to create double-stranded DNA using primers attBlibrary-F and attBlibrary-R for attB and attPlibrary-F and attPlibrary-R for attP (Table 7B). Each PCR product was separately cloned either into pCC1Fos (Lucigen, USA) for attB libraries, or plasmid pMET359 (Table 6) for attP libraries by DNA assembly (Gibson et al., 2009). Clones were propagated in E. coli EP1300 (Lucigen, USA) under chloramphenicol selection for attB libraries and NEB5- repA+(Table 5) under carbenicillin selection for attP libraries.
Purification of .sup.mv4Int
[0116] For .sup.mv4Int purification, the pET-Int plasmid (Table 6) was transferred into E. coli strain BL21(DE3) (New England Biolabs). The resulting strain was grown in LB at 42 C. up to an OD600 of 0.6. Integrase gene expression was induced by addition of 0.1 mM of IPTG, and the culture was incubated at 22 C. for 3 h. Cells were recovered by centrifugation, resuspended in buffer A (50 mM Tris pH 8, 500 mM NaCl, 20 mM imidazole, 10% glycerol, 1 mg/ml lysozyme, and one tablet of SIGMAFAST Protease Inhibitor Cocktail Tablets EDTA-Free [Merck, Germany]), and disrupted by sonication (10 cycles of 30 sec at 40% intensity in ice, followed by 45 sec of rest between each cycle). The lysate was cleared by centrifugation (20000 g, 4 C., 20 min). .sup.mv4Int was first purified on nickel-nitrilotriacetic acid affinity resin (1 ml His-trap HP, GE Healthcare). Column equilibration was performed by injecting 10 column volumes of buffer B (50 mM Tris pH 8, 500 mM NaCl, 20 mM imidazole, 10% glycerol). After equilibration, the lysate was injected and unbound protein were washed using 10 column volumes of buffer B. .sup.mv4Int was eluted using a buffer C gradient of 0 to 30% (50 mM Tris pH 8, 500 mM NaCl, 500 mM imidazole, 10% glycerol). Eluted fractions were then injected in a gel filtration column (HiLoad 16/60 Superdex 200, GE Healthcare, USA). This column was equilibrated using 2 column volumes of buffer D (50 mM Tris pH 8, 500 mM NaCl, 10% glycerol, 1 mM DTT, 1 mM EDTA) and the fractions containing .sup.mv4Int were injected and eluted using the same buffer. Eluted fractions containing .sup.mv4Int were then 2-fold diluted in buffer E (50 mM Tris pH 8, 10% glycerol, 1 mM DTT, 1 mM EDTA). A heparin column (1 ml HiTrap Heparin HP, GE Healthcare, USA) was equilibrated using 10 column volumes of buffer F (50 mM Tris pH 8, 250 mM NaCl, 20% glycerol, 1 mM DTT, 1 mM EDTA). Eluted fractions containing .sup.mv4Int were then injected and unbound protein were removed using 10 column volumes of buffer F. .sup.mv4Int was eluted using a buffer G gradient of 0 to 100% (50 mM Tris pH 8, 1 M NaCl, 20% glycerol, 1 mM DTT, 1 mM EDTA). Purified integrase was aliquoted, snap-frozen in liquid N.sub.2 and stored at 80 C. in buffer containing 50 mM Tris pH 8, 500 mM NaCl, 20% glycerol, 1 mM DTT, 1 mM EDTA.
In Vitro Fluorescent Assay
[0117] Reaction mixtures (20 l) contained 0.08 pmol (300 ng) of supercoiled plasmid carrying the attP site, 0.08 pmol (15 ng) of linear fluorescent (Cy3) 308-bp attB fragment, 7.2 pmol (300 ng) of .sup.mv4Int and 40 g of a crude-extract from E. coli BL21(DE3) heated at 95 C. for 10 min, in 25 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM DTT, and 10% PEG8000 (TENDP 1 buffer). The reaction was incubated at 42 C. either 1 h 30 or 16 h and was stopped by addition of 0.1% SDS. Samples were analysed by electrophoresis in 0.8% agarose gels. Fluorescence was revealed using the ChemidocMP imaging system (Biorad).
In Vitro Recombination Assay Using Libraries
[0118] Reaction (20 l) containing 0.08 pmol (450 ng) of attB plasmid, 0.08 pmol of attP plasmid, 7.2 pmol of .sup.mv4Int and 40 g of crude-extract from E. coli BL21(DE3), heated at 95 C. for 10 min, in TENDP 1 buffer (25 mM Tris pH 7.5, 1 mM EDTA, 150 mM NaCl, 1 mM DTT, and 10% PEG8000) and incubated 1 h 30 at 42 C. The attB, attP, attL and attR sites were amplified by PCR using SeqbanqueattB-F/SeqbanqueattB-R (Table 7B) primers for attB; SeqbanqueattP-F/SeqbanqueattP-R (Table 7B) primers for attP; SeqbanqueattB-F/SeqbanqueattL-R (Table 7B) primers for attL and SeqbanqueattR-F/seqbanqueattP-R (Table 7B) primers for attR. PCR products were purified and analysed by Sanger sequencing (Mix2seq, Eurofins).
NGS Sequencing
[0119] PCR products (attL from recombination attB Lib6attPWT; attBWTattP Lib1 and attR from recombination attBWTattP Lib3; attB Lib8attPWT) used for Sanger sequencing were also used for NGS sequencing (Eurofins). Data were uploaded on the public server at usegalaxy.org (Afgan et al., 2018) for various analyses. Sequence Logo were generated using Weblogo3 (Crooks et al., 2004) and the occurrence of each word was characterized using the Wordcount program (Rice et al., 2000).
Electrophoretic Mobility Shift Assay
[0120] 5 Cy3 end-labelled synthetic oligonucleotides (HPLC purified) were obtained from Eurofins. Labelled double strand DNA substrates were prepared by hybridization of complementary oligonucleotides (Table 7C) in 10 mM Tris pH 7.5, 50 mM NaCl by incubating the samples 5 min at 95 C. in a thermal cycler (Biorad) and decreasing the temperature of 1.5 C./min until it reaches 25 C. Binding reactions (20 l) were performed with 0.87 pmol of labelled core- or arm-type DNA and 4.48 pmol of unlabelled arm- or core-type DNA in buffer containing 25 mM Tris pH 8, 75 mM NaCl, 10% glycerol, 0.5 mM DTT, 0.5 mM EDTA, 1 g polydIdC (Sigma), 0.1 mg/ml BSA. The protein was added, the reaction performed at room temperature for 20 min and samples were loaded onto a non-denaturing 7.5% polyacrylamide gel (Mini-PROTEAN TGX, Biorad). The gels were run at 4 C., 75V for 2 h. Fluorescence was revealed using the ChemidocMP imaging system (Biorad).
In Vivo Recombination
[0121] L. lactis strain MG1363 was transformed as described by Le Bourgeois et al., (Le Bourgeois et al., 2000) by using 1 g of plasmid pMET306 (Table 6). Cells were incubated 3 h at 28 C. and selected for erythromycin resistance on M17 plates supplemented with 5 g/L of glucose. For E. coli, commercially electrocompetent EPI300 cells (Lucigen) were used and transformed with 300 ng of plasmid pMET376 (Table 6). Cells were incubated for 5 h at 37 C. and selected for carbenicillin resistance on LB agar plates. Genomic DNA of antibiotic resistant cells was extracted using the DNeasy Blood and Tissue kit (Qiagen). Site-specific recombination into the targeted tRNA.sup.SER(CGA) was verified by amplifying the attB and attL sites by PCR. PCR amplification was performed using 1 ng of genomic DNA in 25 L of 1Q5 buffer (New England Biolabs), containing 800 M of dNTP, 0.5 U of Q5 polymerase (New England Biolabs) and 0.5 M of each primer. For attB amplification, the thermal cycle program consisted of a 5 min denaturation period at 98 C., followed by 30 cycles of a three-steps thermal profile (10 s at 98 C., 30 s at 60 C., and 3 min at 72 C.) ended with one cycle at 72 C. for 2 min. For attL amplification, the thermal cycle program consisted of a 5 min denaturation period at 98 C., followed by 30 cycles of a three-steps thermal profile (10 s at 98 C., 30 s at 60 C., and 30 s at 72 C.) ended with one cycle at 72 C. for 2 min. PCR products were analysed after electrophoresis in 0.8% agarose.
Results
The .sup.mv4Int is a 369-Aminoacids Tyrosine Integrase
[0122] The original analysis of the integration region of mv4 bacteriophage described the .sup.mv4Int as a 427-aminoacids (AA) protein with significant similarity with the .sup.Int integrase (Dupont et al., 1995a). This result was confirmed through its comparison with other Y recombinases (Nunes-Diby et al., 1998), although .sup.mv4Int contains only six from the seven conserved residues defining the Int family of SSR, with the structurally important D215 residue of .sup.Int (E176 in P1 Cre) missing (
Global Strategy of the Use of Randomized Libraries
[0123] Due to the originality of the core region of attP and of the attB site, these regions were reanalysed by an approach based on the use of randomized DNA libraries. Those DNA libraries, corresponding either to the attB site or to the core-attP region, contained 7 to 10 randomized positions (
Redefining the Minimal attB.sub.Mv4 Site
[0124] The attB minimal site has been previously characterized by Auvray et al., and resulted in a 16-bp sequence, the shortest attB described in the literature (Auvray et al., 1999b). In order to validate the particular size of this site, different sizes of attB.sub.WT were amplified by PCR (
Redefining the Size of the attB and attP Overlap Regions
[0125] A previous study based on the use of DNA suicide substrates determined the length of the attB.sub.mv4 and attP.sub.mv4 overlap regions to 8 bp (Coddeville et al., 2014a), a size-range typical of YR recombination systems (Grindley et al., 2006). However, the Inventors noticed that all heterobivalent recombination systems currently characterised, such as phages (Craig and Nash, 1983), HK022 (Kolot and Yagil, 1994), HP1 (Hauser and Scocca, 1992), L5 (Pea et al., 1996), P22 (Smith-Mungo et al., 1994) or the ICE CTnDOT (Malanowska et al., 2006), display a 7-bp overlap region. As the knowledge of the strand exchange mechanism used by tyrosine recombinases make it possible to determine indirectly the length of the overlap region (Grindley et al., 2006), it has been taken advantage of this property to reanalyse the overlap region of the .sup.mv4Int/attP/attB system using random DNA libraries. Indeed, as the two overlap regions of attP/attB from phage systems must contain identical DNA sequence to promote a full recombination reaction, only one of the four nucleotides present on the attP or attB random libraries should be recovered on both attL and attR sites if the position is included into the overlap region (
Characterization of the Nucleotide Constraints Existing on attB and attP Overlap Regions
[0126] For the model integrase .sup.Int, it has been soon observed that the nature of the bases in the overlap region was not important for recombination but that sequence identity between attB and attP overlap was mandatory (Weisberg et al., 1983; Bauer et al., 1985). A similar feature has been observed for the phage HK022 recombination module (Kolot et al., 2015) and for Flp (McLeod et al., 1986) and Cre (Hoess et al., 1986), although it has been shown that presence of heteroduplex in the overlap of loxP sites can sometimes be functional (Lee and Saito, 1998; Sheren et al., 2007). In order to characterize the constraints exerted in the overlap region, two libraries composed of 7 randomized positions in the overlap region were constructed (attB Lib7 and attP Lib2,
Characterization of the Nucleotide Constraints Exerted on the DNA Regions Surrounding the Overlap Sequence of attB Site and Core-attP Region
[0127] The attB Lib6 library (
[0128] Each library has the tendency to contain A or C slightly underrepresented compared to T and G, with a minimum of 15% of C and 20% of A for attP Lib3 library (
Characterization of the B, B, C and C Motifs Most Permissive for .sup.mv4Int-Mediated In Vitro Recombination
[0129] By determining the individual sequence of each molecule from a DNA population, NGS sequencing not only allows high resolution in the nucleotide composition of the randomized part of the libraries, but also to determine the occurrence of each 7-bp motif into the recombined DNA population. As it is highly unlikely that the thousands of motifs from the libraries will all be functional because of uncharacterized constraints, the Inventors assumed that comparing motifs occurrence in the randomized libraries before and after recombination by NGS sequencing will give relevant information about their ability to form a productive recombination complex with the mv4 integrase. Once constructed, each attB or attP library was sequenced and found to contains from 16312 to 16384 motifs (Table 8), corresponding from 99.5 to 100% of the theoretical number for 7-bp random library (4.sup.7). Depending on the library and the experimental repetition, one-third (33.32%) to two-thirds (71.91%) of the motifs were recovered after .sup.mv4Int-mediated in vitro recombination (Table 8). In addition, their occurrence was highly biased, with a factor ranging from 2,000 to 70,000, depending on the experiment, between the least and the most represented motif (Table 8), with a rapid drop in read counts relative to the rank (
B, B, C and C Sites are the Core-Binding Sites for the Mv4 Integrase
[0130] To experimentally demonstrate that imperfect inverted-repeats B, B, C and C correspond to the .sup.mv4Int core-binding sites, the Inventors used a gel shift assay (EMSA) based on the protocol developed on the COC region of .sup.Int recombination system (Sarkar et al., 2001). In this study, Sarkar et al., demonstrated that the N-ter domain exerts an inhibitory effect on the C-ter domain when not bound to the P arm-binding sites. However, if DNA containing the P1-P2 arm-binding sites was added to the reaction, the inhibition was removed and .sup.Int was able to stably bind to the core-binding sites. To determine if .sup.mv4Int N-ter domain also exerts such inhibitory effect on its C-ter domain, they compared the .sup.mv4Int binding to a labelled 35-bp dsDNA containing the 21-bp core-attP region (Table 7C), in the presence or absence of unlabelled dsDNA containing the P1 and P2 arm-type binding sites (Table 7C). .sup.mv4Int binding to the COC sequence appeared quite unstable, as only faint bands and strong background smear can be observed (lane 3,
attP.sub.mv4 contains two pairs of direct repeats of .sup.mv4Int Arm-Binding Sites.
[0131] As the EMSA experiments allowed the Inventors to indirectly observe .sup.mv4Int binding to the arm-binding sites by stabilizing the binding of .sup.mv4Int to the core-type sites, they reanalysed the number and locations of the five arm-binding sites previously described (Auvray et al., 1999a), by studying eleven sets of .sup.mv4Int arm-binding sites (Table 7C). Among all the combinations used, only those containing either the P1-P2 pair or P1-P2 pair allow the stabilization of .sup.mv4Int binding to COC (
The .sup.mv4Int/attP system can be reprogrammed to target tRNA.sup.SER of Other Bacterial Species
[0132] The characterization of the high degeneracy of mv4 attB site and core-attP region led us to postulate that .sup.mv4Int may be able to recombine DNA targets other than its cognate site by reprogramming the core-attP region, as long as these targets belong to the consensus pattern defined in this study (
Reprogramming the Core-attP Site Allows In Vivo Recombination at the tRNA.sup.SER of E. coli and L. lactis
[0133] As .sup.mv4Int promote site-specific recombination in vitro into the L. lactis tRNA.sup.SER when using the adapted attP site, the Inventors tested if this retargeted recombination could be performed in vivo. In addition, as the tRNA.sup.SER of E. coli contains only one nucleotide that derogates the consensus pattern (
Extending the .sup.mv4Int/attP system reprogramming to sequences other than tRNA.sup.SER
[0134] The Inventors performed an in-silico analysis of the E. coli MG1655 genome in order to identify putative recombination sites that obey the consensus pattern of attB.sub.mv4. After analysis, 7959 putative sites have been identified, with three of them located into the lacZ gene. To test if the reaction can be reprogrammed to target these three sites in vitro, the overlap region of the mv4 core-attP was replaced by the overlap sequence of either lacZ1, lacZ2 and lacZ3 (
[0135] At last, the Inventors determined if targeted sequences containing one nucleotide that did not belong to the consensus patterns would still be productive in recombination, as two nucleotides excluded from the patterns seemed detrimental (see above). For that purpose, two nucleotide positions at the B site were independently replaced by forbidden nucleotides (
TABLE-US-00009 TABLE 5 Strains. Strain Relevant characteristics Reference E. coli B F.sup. ompT gal dcm lon hsdSB(r.sub.Bm.sub.B) (DE3 [lacI (Studier et al., BL21(DE3) lacUV5-T7p07 ind1 sam7 nin5]) [malB.sup.+].sub.K-12(.sup.S) 1990) E. coli E. coli EC101, repA(pWV01) glgB::repA(pWV01), Kan.sup.R (Leenhouts et al., EC1000 1996) E. coli F endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR New England Neb5 nupG purB20 80dlacZM15 (lacZYA- Biolabs argF)U169, hsdR17(r.sub.Km.sub.K+), E. coli F mcrA (mrr-hsdRMS-mcrBC) Epicentre EPI300 80dlacZM15 (lac)X74 recA1 endA1 araD139 (ara, leu)7697 galU galK rpsL (Str.sup.R) nupG trfA dhfr E. coli Strain NEB5 glgB::repA(pWV01), Kan.sup.R This study NEB5-RepA L. lactis Wild Type strain. Plasmid-free derivative (Gasson, 1983) MG1363 strain of NCDO712
TABLE-US-00010 TABLE 6 Plasmids Name Relevant properties Reference pBSattB pBS::attB.sub.mv4; Amp.sup.R (Dupont et al., 1995) pMC1 pRC1::.sup.mv4Int-attP.sub.mv4; Erm.sup.R (Dupont et al., 1995) pET-Int pET15b::.sup.mv4Int; Amp.sup.R (Auvray et al., 1999a) pCC1Fos ori F-factor and oriV; Cm.sup.R Epicentre pMET302 pMC1::core attP tRNA.sup.SER L. acidophilus; Erm.sup.R This study pMET303 pMC1::core attP artificial 2; Erm.sup.R This study pMET304 pMC1::core attP tRNA.sup.SER L. mesenteroides; Erm.sup.R This study pMET305 pMC1::core attP tRNA.sup.SER E. faecalis; Erm.sup.R This study pMET306 pMC1::core attP tRNA.sup.SER L. lactis; Erm.sup.R This study pMET307 pMC1::core attP tRNA.sup.SER S. mutans; Erm.sup.R This study pMET308 pMC1::core attP tRNA.sup.SER L. reuteri; Erm.sup.R This study pMET309 pMC1::core attP artificial 1; Erm.sup.R This study pMET310 pMC1::core attP L. sakei; Erm.sup.R This study pMET311 pMC1 IS10::.sup.mv4Int; Erm.sup.R This study pMET320 pBSattB::tRNA.sup.SER L. acidophilus; Amp.sup.R This study pMET321 pBSattB::artificial 2; Amp.sup.R This study pMET322 pBSattB::tRNA.sup.SER E. faecalis; Amp.sup.R This study pMET323 pBSattB::tRNA.sup.SER L. lactis; Amp.sup.R This study pMET324 pBSattB::tRNA.sup.SER S. mutans; Amp.sup.R This study pMET325 pBSattB::tRNA.sup.SER L. mesenteroides; Amp.sup.R This study pMET326 pBSattB::tRNA.sup.SER L. reuteri; Amp.sup.R This study pMET327 pBSattB::tRNA.sup.SER L. sakei; Amp.sup.R This study pMET328 pBSattB::artificial 1; Amp.sup.R This study pMET340 pMC1 colE1::ori pWV01; Erm.sup.R This study pMET348 pCC1Fos::attB.sub.mv4; Cm.sup.R This study pMET349 pCC1Fos::attB.sub.mv4 23 bp; Cm.sup.R This study pMET357 pCC1Fos::attB.sub.mv4 16 bp; Cm.sup.R This study pMET359 pMET340 + bla; Erm.sup.R; Amp.sup.R This study pMET364 pMET348::overlap lacZ.sub.1; Cm.sup.R This study pMET365 pMET348::overlap lacZ.sub.3; Cm.sup.R This study pMET366 pMET348::attB lacZ.sub.1; Cm.sup.R This study pMET367 pMET348::attB lacZ.sub.2; Cm.sup.R This study pMET368 pMET348::attB lacZ.sub.3; Cm.sup.R This study pMET370 pMET348::overlap lacZ.sub.2; Cm.sup.R This study pMET371 pMET359::overlap lacZ.sub.1; Erm.sup.R, Amp.sup.R This study pMET372 pMET359::overlap lacZ.sub.2; Erm.sup.R, Amp.sup.R This study pMET373 pMET359::overlap lacZ.sub.3; Erm.sup.R, Amp.sup.R This study pMET376 pMET359::core attP tRNA.sup.SER E. coli; Erm.sup.R, Amp.sup.R This study
TABLE-US-00011 TABLE7 Listofoligonucleotides A.Oligonucleotidesusedforcloning. Primername Sequence(5-3) SDM-attB4-F GACCTGTACTCTCCTTAATAAGGTCAAATG(SEQIDNo7) SDM-attB4-R GACCTTATTAAGGAGAGTACAGGTCTTGAAC(SEQIDNo8) SDM-attB5-F CCTGTACTCTCCTGAATAAGGTCAAATGGTATC(SEQIDNo9) SDM-attB5-R GACCTTATTAAGGAGAGTACAGGTCTTGAAC(SEQIDNo10) SDM-LbsakattB-F CCTGTACTCTCCTTTTAAAGGTCAAATGGTATC(SEQIDNo11) SDM-LbsakattB-R CATTTGACCTTTAAAAGGAGAGTACAGGATTTG(SEQID No12) SDM-LbacidoattB-F CCTGTACACTCCTTTTTAAGGTCAAATGGTATC(SEQIDNo13) SDM-LbacidoattB-R CATTTGACCTTAAAAAGGAGTGTACAGGATTTG(SEQID No14) SDM-LbreutattB-F CCCCTTGTCTCCTTAGAAAGGTCAAATGGTATC(SEQIDNo15) SDM-LbreutattB-R CTTTCTAAGGAGACAAGGGGATTTGAACCTGCG(SEQID No16) SDM-LlactisattB-F CCCCTTGACTCCTTTTAAAGGTCAAATGGTATCC(SEQID No17) SDM-LlactisattB-R CTTTAAAAGGAGTCAAGGGGATTTGAACCTGCG(SEQID No18) SDM-SmutansattB-F CCCGTCCTTTCCTTAACAAGGTCAAATGGTATC(SEQIDNo19) SDM-SmutansattB-R CTTGTTAAGGAAAGGACGGGATTTGAACCTGCG(SEQID No20) SDM-EfaecattB-F CCCTTATCCTCCGTACCAAGGTCAAATGGTATCC(SEQID No21) SDM-EfaecattB-R CTTGGTACGGAGGATAAGGGATTTGAACCTGCG(SEQID No22) SDM-LnmesentattB-F CCCGCTAGCTCCTTTATAAGGTCAAATGGTATC(SEQIDNo23) SDM-LnmesentattB-R CTTATAAAGGAGCTAGCGGGATTTGAACCTGCG(SEQID No24) SDM-EcoliattB-F CCTCTCTCCGCCACTTTAAGGTCAAATGGTATCC(SEQID No25) SDM-EcoliattB-R CTTAAAGTGGCGGAGAGAGGATTTGAACCTGCG(SEQID No26) SDMgibsonermAM-F GAGAATATCGTCAACTGTTTACTAAAAATC(SEQIDNo27) SDMgibsonermAM-R GTAAACAGTTGACGATATTCTCGATTG(SEQIDNo28) SDMgibsonattP5-F AAAGAACCTGTACTCTCCTGAATCAAAGCAATAATC(SEQID No29) SDMgibsonattP5-R TTCAGGAGAGTACAGGTTCTTTCAACCATGTTTC(SEQID No30) SDMgibsonattPsakei- AAAGAACCTGTACTCTCCTTTTACAAAGCAATAATC(SEQID F No31) SDMgibsonattPsakei- AAAAGGAGAGTACAGGTTCTTTCAACCATGTTTC(SEQID R No32) SDMgibsonattPacido- AAAGAACCTGTACACTCCTTTTTCAAAGCAATAATC(SEQID F No33) SDMgibsonattPacido- AAAAGGAGTGTACAGGTTCTTTCAACCATGTTTC(SEQID R No34) SDMgibsonattPmutans- AAAGAACCCGTCCTTTCCTTAACCAAAGCAATAATCCC(SEQ F IDNo35) SDMgibsonattPmutans- TTAAGGAAAGGACGGGTTCTTTCAACCATGTTTC(SEQID R No36) SDMgibsonattPreuteri- AAAGAACCCCTTGTCTCCTTAGACAAAGCAATAATCCC(SEQ F IDNo37) SDMgibsonattPreuteri- CTAAGGAGACAAGGGGTTCTTTCAACCATGTTTC(SEQID R No38) SDMgibsonattPlactis- AAAGAACCCCTTGACTCCTTTTACAAAGCAATAATCCC(SEQ F IDNo39) SDMgibsonattPlactis- AAAAGGAGTCAAGGGGTTCTTTCAACCATGTTTC(SEQID R No40) SDMgibsonattPfaeca- AAAGAACCCTTATCCTCCGTACCCAAAGCAATAATCCC(SEQ F IDNo41) SDMgibsonattPfaeca- GTACGGAGGATAAGGGTTCTTTCAACCATGTTTC(SEQID R No42) SDMgibsonattPcoli-F AAAGAACCTCTCTCCGCCACTTTCAAAGCAATAATCCC(SEQ IDNo43) SDMgibsonattPcoli-R AAGTGGCGGAGAGAGGTTCTTTCAACCATGTTTC(SEQID No44) SDMgibsonattPmesent- AAAGAACCCGCTAGCTCCTTTATCAAAGCAATAATCCC(SEQ F IDNo45) SDMgibsonattPmesent- TAAAGGAGCTAGCGGGTTCTTTCAACCATGTTTC(SEQID R No46) pBS-antiattB-F GCTTGGGCTGCAGGA(SEQIDNo47) pBS-antiattB-R GGAAAGGACATCTAAATCAAATGG(SEQIDNo48) SDM-Lbreuteri2attB-F TCCATAGAAAGGTCAAATGGTATC(SEQIDNo49) SDM-Lbreuteri2attB-R GACAAGGGGATTTGAACCTG(SEQIDNo50) oripGh9-F TTAAATTTATACTGCAATCGGATGC(SEQIDNo51) oripGh9-R GTATTTTTAATAGCCATGATATAATTACCTTATC(SEQID No52) pMC1-sans-colE1-F TGGAACGAAAACTCACGTTAAG(SEQIDNo53) pMC1-sans-colE1-R TGATTCTGTGGATAACCGTATTAC(SEQIDNo54) pCC1FOS-F GTGGGATCCCCGGGTAC(SEQIDNo55) pCC1FOS-R GTGGGATCCTCTAGAGTCGAC(SEQIDNo56) attB-gibsonpCC1Fos- GCAGGTCGACTCTAGAGGATCCCACGAATTCCTGCAGCCCAA F GC(SEQIDNo57) attB-gibsonpCC1Fos- GAGCTCGGTACCCGGGGATCCCACCCCCATTTGATTTAGATG R TCCTTTC(SEQIDNo58) attBWT-F ATCCTGTACTCTCCTTAAT(SEQIDNo59) attBWT-R ATTAAGGAGAGTACAGGAT(SEQIDNo60) attB-WTGGTT-F GGTTCAAATCCTGTACTCTCCTTAAT(SEQIDNo61) attB-WTGGTT-R ATTAAGGAGAGTACAGGATTTGAACC(SEQIDNo62) pMC1delTOPO-F AAATCGAAACAGCAAAGAATGG(SEQIDNo63) Bla-F TGGCACTTTTCGGGGAAATG(SEQIDNo64) Bla-R GTGCTACAGAGTTCTTGAAGTG(SEQIDNo65) Site1LacZ-Gib-F AAAGAATTTCGGCTCTCCTTAATCAAAGCAATAATCCC(SEQ IDNo66) Site2LacZ-Gib-F AAAGAATGCCAACTCTCCTTAATCAAAGCAATAATCCC(SEQ IDNo67) Site3LacZ-Gib-F AAAGAAAAGGTTTTCTCCTTAATCAAAGCAATAATCCC(SEQ IDNo68) Site1LacZ-Gib-R TTAAGGAGAGCCGAAATTCTTTCAACCATGTTTCTGGAG (SEQIDNo69) Site2LacZ-Gib-R TTAAGGAGAGTTGGCATTCTTTCAACCATGTTTCTGGAG(SEQ IDNo70) Site3LacZ-Gib-R TTAAGGAGAAAACCTTTTCTTTCAACCATGTTTCTGGAG(SEQ IDNo71) AmpGib-F GAATTATGCAGTGCTGCCATAAC(SEQIDNo72) AmpGib-R GTTATGGCAGCACTGCATAATTC(SEQIDNo73) OverlapSite1lacZ-R AAGGAGAGCCGAAAATTTGAACCTGCGCACC(SEQIDNo74) OverlapSite2lacZ-R AAGGAGAGTTGGCAATTTGAACCTGCGCACC(SEQIDNo75) OverlapSite3lacZ-R AAGGAGAAAACCTTATTTGAACCTGCGCACC(SEQIDNo76) OverlapSite1lacZ-F TCAAATTTTCGGCTCTCCTTAATAAGGTCAAATGGTATC(SEQ IDNo77) OverlapSite2LacZ-F TCAAATTGCCAACTCTCCTTAATAAGGTCAAATGGTATC(SEQ IDNo78) OverlapSite3lacZ-F TCAAATAAGGTTTTCTCCTTAATAAGGTCAAATGGTATC(SEQ IDNo79) attBsite1LacZ-F GAAATCCCGAACCTGCGCACCAATTCAACATTG(SEQID No80) attBsite1LacZ-R GGCGCTCCACAATAAGGTCAAATGGTATCCCTATAGG(SEQ IDNo81) attBsite2LacZ-F GGCAATTTAACCCTGCGCACCAATTCAACATTG(SEQIDNo82) attBsite2LacZ-R AACGCTTATTAATAAGGTCAAATGGTATCCCTATAGG(SEQ IDNo83) attBsite3LacZ-F CCTTATTTATCCCTGCGCACCAATTCAACATTG(SEQIDNo84) attBsite3LacZ-R TTTTCCCCTGAATAAGGTCAAATGGTATCCCTATAGG(SEQID No85) B.OligonucleotidesusedforRandomised DNAlibrariesconstructionandsequencing. Primername Sequence(5-3) SeqbanqueattB-F TGCCTGCAGGTCGACTCTAG(SEQIDNo86) SeqbanqueattL-R ACGCTAATGCCATCTATTAACTAGC(SEQIDNo87) SeqbanqueattR-F GAAACAACCAGAAACGCTTTTTAG(SEQIDNo88) SeqbanqueattB-R GGCGAATTCGAGCTCGGTAC(SEQIDNo89) SeqbanqueattP-R GGTCGACGGTATCGATAAGC(SEQIDNo90) SeqbanqueattP-F GCAGGCGGAATGTTGAAAGAG(SEQIDNo91) attP-N10-N19-R GTTCTGGAGGTTTCGAATCTTG(SEQIDNo92) attP-N10-N19-Vf-F GTGATAATCGCCTGCCCGTTTGAC(SEQIDNo93) attPlibrary-F CAAGATTCGAAACCTCCAGAAC(SEQIDNo94) attPlibrary-R GTCAAACGGGCAGGCGATTATCAC(SEQIDNo95) attBlibrary-F GCAGGTCGACTCTAGAGGATCCCACAAGCTTCGAAATCCGCCGA ACCAATG(SEQIDNo96) attBlibrary-R GAGCTCGGTACCCGGGGATCCCACGAATTCACTACTGGCTACTT TGAAATACTTCC(SEQIDNo97) attB-gibsonpCC1Fos-F GCAGGTCGACTCTAGAGGATCCCACGAATTCCTGCAGCCCAAGC (SEQIDNo57) attB-gibsonpCC1Fos-R GAGCTCGGTACCCGGGGATCCCACCCCCATTTGATTTAGATGTC CTTTC(SEQIDNo58) attBLib9 CGAAATCCGCCGAACCAATGTTGANNNNNNNNGCAGGTTCANN NNNTGTACTCTCCNNNNNAAGGTCAAATNNNNNNNNTATAGGA AGTATTTCAAAGTAGCCAGTAGT(SEQIDNo98) attBLib7 CGAAATCCGCCGAACCAATGTTGANNNNNNNCGCAGGTTCAAA TNNNNNNNTCTCCTTAATAAGGTCAAATGNNNNNNNTATAGGA AGTATTTCAAAGTAGCCAGTAGT(SEQIDNo99) attBLib8 CGAAATCCGCCGAACCAATGTTGANNNNNNNCGCAGGTTCAAA TCCTGTACNNNNNNNAATAAGGTCAAATGNNNNNNNTATAGGA AGTATTTCAAAGTAGCCAGTAGT(SEQIDNo100) attPLib2 CAAGATTCGAAACCTCCAGAACCCTCCAGAAACATGGTTGAAAG AANNNNNNNTCTCCTTAATCAAAGCAATAATCCCCGAGAAATCA ACATTCTCGGGGATTATTTTTGTTTTTAACTAGAAAATAACTAGA AAGAGCTAGTTAATAGATGGCATTAGCGTGATAATCGCCTGCCC GTTTGAC(SEQIDNo101) attPLib1 CAAGATTCGAAACCTCCAGAACCCTCCAGAAACATGGTTGAAAG AACCTGTACNNNNNNNAATCAAAGCAATAATCCCCGAGAAATC AACATTCTCGGGGATTATTTTTGTTTTTAACTAGAAAATAACTAG AAAGAGCTAGTTAATAGATGGCATTAGCGTGATAATCGCCTGCC CGTTTGAC(SEQIDNo102) attBLib5 CGAAATCCGCCGAACCAATGTTGAATTGGTGNNNNNNNNNNAA TCCTGTACTCTCCTTAATAAGGTCAAATGGTATCCCTATAGGAAG TATTTCAAAGTAGCCAGTAGT(SEQIDNo103) attPLib4 CAAGATTCGAAACCTCCAGAACCCTCCAGAAACNNNNNNNNNN GAACCTGTACTCTCCTTAATCAAAGCAATAATCCCCGAGAAATC AACATTCTCGGGGATTATTTTTGTTTTTAACTAGAAAATAACTAG AAAGAGCTAGTTAATAGATGGCATTAGCGTGATAATCGCCTGCC CGTTTGAC(SEQIDNo104) attBLib6 CGAAATCCGCCGAACCAATGTTGAATTGGTGCGCNNNNNNNNN NCCTGTACTCTCCTTAATAAGGTCAAATGGTATCCCTATAGGAA GTATTTCAAAGTAGCCAGTAGT(SEQIDNo105) attPLib3 CAAGATTCGAAACCTCCAGAACCCTCCAGAAACATGGTTNNNNN NNCCTGTACTCTCCTTAATCAAAGCAATAATCCCCGAGAAATCA ACATTCTCGGGGATTATTTTTGTTTTTAACTAGAAAATAACTAGA AAGAGCTAGTTAATAGATGGCATTAGCGTGATAATCGCCTGCCC GTTTGAC(SEQIDNo106) C.OligonucleotidesusedEMSA. Primername Sequence(5-3) COCWT-F GGTATTGGAAAGAACCTGTACTCTCCTTGCGTAAC(SEQID No107) COCWT-Cy3-R GTTACGCAAGGAGAGTACAGGTTCTTTCCAATACC(SEQID No108) P12WT-28bp-F GTTTTTAACTAGAAAATAACTAGAATTC(SEQIDNo109) P12WT-28bp-R GAATTCTAGTTATTTTCTAGTTAAAAAC(SEQIDNo110) P12WT-40bp-F CACGTCGTTTTTAACTAGAAAATAACTAGAATTCCACGTC (SEQIDNo111) P12WT-40bp-R GACGTGGAATTCTAGTTATTTTCTAGTTAAAAACGACGTG (SEQIDNo112) P12WTdelP3-F TTTTTAACTAGAAAATAACTAGAAAGCGACCGAGCGGTCG (SEQIDNo113) P12WTdelP3-R CGACCGCTCGGTCGCTTTCTAGTTATTTTCTAGTTAAAAA (SEQIDNo114) P123WT-F TTTTTAACTAGAAAATAACTAGAAAGAGCTAGTTAATAGA (SEQIDNo115) P123WT-R TCTATTAACTAGCTCTTTCTAGTTATTTTCTAGTTAAAAA (SEQIDNo116) P1WT-F TTTTTAACTAGAACGACCGAGCGGAGGCGGCACGCTGTCG (SEQIDNo117) P1WT-R CGACAGCGTGCCGCCTCCGCTCGGTCGTTCTAGTTAAAAA (SEQIDNo118) P2WT-F GCCGACCGAGCGGAATAACTAGAAAGGCGGCACGCTGTCG (SEQIDNo119) P2WT-R CGACAGCGTGCCGCCTTTCTAGTTATTCCGCTCGGTCGGC (SEQIDNo120) P3WT-F GCCGACCGAGCGGGCGGCACGCTGAGAGCTAGTTAATAGA (SEQIDNo121) P3WT-R TCTATTAACTAGCTCTCAGCGTGCCGCCCGCTCGGTCGGC (SEQIDNo122) P23WT-F GCCGACCGAGCGGAATAACTAGAAAGAGCTAGTTAATAGA (SEQIDNo123) P23WT-R TCTATTAACTAGCTCTTTCTAGTTATTCCGCTCGGTCGGC (SEQIDNo124) P13WT-F TTTTTAACTAGAACGACCGAGCGGAGAGCTAGTTAATAGA (SEQIDNo125) P13WT-R TCTATTAACTAGCTCTCCGCTCGGTCGTTCTAGTTAAAAA (SEQIDNo126) P12WT-F CACGTCGTGATCAACTAGATTTTTAACTAGAAACCACGTC (SEQIDNo127) P12WT-R GACGTGGTTTCTAGTTAAAAATCTAGTTGATCACGACGTG (SEQIDNo128) P1WT-F CACGTCGTGATCAACTAGATTCGACCGAGCGGACCACGTC (SEQIDNo129) P1WT-R GACGTGGTCCGCTCGGTCGAATCTAGTTGATCACGACGTG (SEQIDNo130) P2WT-F CACGTCGTGCGACCGAGCGGTTTTAACTAGAAACCACGTC (SEQIDNo131) P2WT-R GACGTGGTTTCTAGTTAAAACCGCTCGGTCGCACGACGTG (SEQIDNo132) Nositebras-F CACGTCGTGCGACCGAGCGGTGCGGCACGCTGACCACGTC (SEQIDNo133) Nositebras-R GACGTGGTCAGCGTGCCGCACCGCTCGGTCGCACGACGTG (SEQIDNo134) P12WT-40bp-R- CACGTCGTTTTTAACTAGAAAATAACTAGAATTCCACGTC Cy3 (SEQIDNo135) COCWT-R GTTACGCAAGGAGAGTACAGGTTCTTTCCAATACC(SEQID No136) BOBWT-F GGTATTGTTCAAATCCTGTACTCTCCTTGCGTAAC(SEQID No137) BOBWT-R GTTACGCAAGGAGAGTACAGGATTTGAACAATACC(SEQ IDNo138) BOBWT-R-Cy3 GTTACGCAAGGAGAGTACAGGATTTGAACAATACC(SEQ IDNo139) XOC-F GGTATTGGAACCCGCCTGTACTCTCCTTGCGTAAC(SEQID No140) XOC-R-Cy3 GTTACGCAAGGAGAGTACAGGCGGGTTCCAATACC(SEQID No141) COX-F GGTATTGGAAAGAACCTGTACCGGGCTTGCGTAAC(SEQID No142) COX-R-Cy3 GTTACGCAAGCCCGGTACAGGTTCTTTCCAATACC(SEQID No143) BOX-F GGTATTGTTCAAATCCTGTACCGGGCTTGCGTAAC(SEQID No144) BOX-R-Cy3 GTTACGCAAGCCCGGTACAGGATTTGAACAATACC(SEQID No145) P12-9pb-F CACGTCGTGCTCAACTAGAGTCTTAACTAGAGACCACGTC (SEQIDNo146) P12-9pb-R GACGTGGTCTCTAGTTAAGACTCTAGTTGAGCACGACGTG (SEQIDNo147) P12-6pb-F CACGTCGTGCCGAACTAGGGTCCGAACTAGGGACCACGTC (SEQIDNo148) P12-6pb-R GACGTGGTCCCTAGTTCGGACCCTAGTTCGGCACGACGTG (SEQIDNo149) P23forward-F GCCGACCGAGCGGAATAACTAGAAAGATTAACTAGCTAGA (SEQIDNo150) P23forward-R TCTAGCTAGTTAATCTTTCTAGTTATTCCGCTCGGTCGGC (SEQIDNo151) P12reverse-F TTTTTAACTAGAATTCTAGTTATTAGCGACCGAGCGGTCG (SEQIDNo152) P12reverse-R CGACCGCTCGGTCGCTAATAACTAGAATTCTAGTTAAAAA (SEQIDNo153) D.Oligonucleotidesusedfor invitrofluorescentrecombination. Primername Sequence LbbulgattB-F GAATTCCTGCAGCCCAAGC(SEQIDNo154) Cy3-New-attB-R GATGTAGATAATTTTTGGGCCAAGG(SEQIDNo155) E.Oligonucleotidesusedtovalidate invivointegrationintotRNASER Primername Sequence(5-3) ARNtSERcoli-F ACAGTGACGATCTAACCCTTC(SEQIDNo156) ARNtSERcoli-R TGACTAATTTGCTTTGTTCCTG(SEQIDNo157) ARNtSERlactis-F CATCATTTTTCTTCTTTCAAATTAATATAAATGC(SEQID No158) ARNtSERlactis-R CAGGAGGAAAAGGAGTAAGC(SEQIDNo159) attL-R ACGCTAATGCCATCTATTAACTAGC(SEQIDNo160) Bold nucleotides indicate the localization of arm or core-binding sites as well as the overlap sequence. Underlined nucleotides are nucleotides that differ from the WT sites.
TABLE-US-00012 TABLE 8 NGS data of the randomized DNA libraries. DNA Motif number Motif number Library Motif number after Motif occurrence representing 50% (core- before recombination after recombination of the read count binding recombination (attL or attR) Rep1 Rep2 after recombination region) (attB or attP) Rep1 Rep2 min max min max Rep1 Rep2 attB 16384 5459 6955 1 14405 1 11737 196 199 Lib6 (100%) (33.32%) (42.45%) (3.59%) (2.86%) (B) attB 16312 9497 10085 1 49310 1 69525 371 460 Lib8 (99.56%) (57.97%) (61.55%) (3.91%) (4.56%) (B) attP 16340 10416 11781 1 2139 1 3925 784 799 Lib3 (99.73%) (63.57%) (71.91%) (7.53%) (6.78%) (C) attP 16315 7549 7247 1 16157 1 46672 254 167 Lib1 (99.58%) (46.08%) (44.23%) (3.36%) (2.30%) (C)
REFERENCES
[0136] Afgan, E., Baker, D., Batut, B., van den Beek, M., Bouvier, D., Cech, M., et al. (2018) The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Research 46: W537-W544 https://doi.org/10.1093/nar/gky379. Accessed Feb. 2, 2022. [0137] Aihara, H., Kwon, H. J., Nunes-Diby, S. E., Landy, A., and Ellenberger, T. (2003) A conformational switch controls the DNA cleavage activity of lambda integrase. Mol Cell 12: 187-198. [0138] Auvray, F., Coddeville, M., Espagno, G., and Ritzenthaler, P. (1999a) Integrative recombination of Lactobacillus delbrueckii bacteriophage mv4: functional analysis of the reaction and structure of the attP site. Mol Gen Genet 262: 355-366. [0139] Auvray, F., Coddeville, M., Ordonez, R. C., and Ritzenthaler, P. (1999b) Unusual structure of the attB site of the site-specific recombination system of Lactobacillus delbrueckii bacteriophage mv4. J Bacteriol 181: 7385-7389. [0140] Auvray, F., Coddeville, M., Ritzenthaler, P., and Dupont, L. (1997) Plasmid integration in a wide range of bacteria mediated by the integrase of Lactobacillus delbrueckii bacteriophage mv4. J Bacteriol 179: 1837-1845. [0141] Bauer, C. E., Gardner, J. F., and Gumport, R. I. (1985) Extent of sequence homology required for bacteriophage lambda site-specific recombination. J Mol Biol 181: 187-197. [0142] Biswas, T., Aihara, H., Radman-Livaja, M., Filman, D., Landy, A., and Ellenberger, T. (2005) A structural basis for allosteric control of DNA recombination by k integrase. Nature 435: 1059-1066 https://www.nature.com/articles/nature03657. Accessed Aug. 10, 2021. [0143] Cluzel, P.-J., Veaux, M., Rousseau, M., and Accolas, J.-P. (1987) Evidence for temperate bacteriophages in two strains of Lactobacillus bulgaricus. J Dairy Res 54: 397-405. [0144] Coddeville, M., and Ritzenthaler, P. (2010) Control of directionality in bacteriophage mv4 site-specific recombination: functional analysis of the Xis factor. J Bacteriol 192: 624-635. [0145] Coddeville, M., Spinella, J.-F., Cassart, P., Girault, G., Daveran-Mingot, M.-L., Le Bourgeois, P., and Ritzenthaler, P. (2014a) Bacteriophage mv4 site-specific recombination: the central role of the P2 mv4Int-binding site. J Virol 88: 1839-1842. [0146] Coddeville, M., Spinella, J. F., Cassart, P., Girault, G., Daveran-Mingot, M. L., Le Bourgeois, P., and Ritzenthaler, P. (2014b) Bacteriophage mv4 site-specific recombination: the central role of the P2 .sup.mv4Int-binding site. J Virol 88: 1839-1842. [0147] Craig, N. L., and Nash, H. A. (1983) The mechanism of phage lambda site-specific recombination: site-specific breakage of DNA by Int topoisomerase. Cell 35: 795-803. [0148] Crooks, G. E., Hon, G., Chandonia, J.-M., and Brenner, S. E. (2004) WebLogo: a sequence logo generator. Genome Res 14: 1188-1190. [0149] Datsenko, K. A., and Wanner, B. L. (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97: 6640-6645. [0150] Dupont, L., Boizet-Bonhoure, B., Coddeville, M., Auvray, F., and Ritzenthaler, P. (1995a) Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum. J Bacteriol 177: 586-595. [0151] Dupont, L., Boizet-Bonhoure, B., Coddeville, M., Auvray, F., and Ritzenthaler, P. (1995b) Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum. J Bacteriol 177: 586-595. [0152] Gasson, M. J. (1983) Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J Bacteriol 154: 1-9. [0153] Gibb, B., Gupta, K., Ghosh, K., Sharp, R., Chen, J., and Van Duyne, G. D. (2010) Requirements for catalysis in the Cre recombinase active site. Nucleic Acids Res 38: 5817-5832. [0154] Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., and Smith, H. O. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods 6: 343-345. [0155] Grindley, N. D. F., Whiteson, K. L., and Rice, P. A. (2006) Mechanisms of site-specific recombination. Annu Rev Biochem 75: 567-605. [0156] Hauser, M. A., and Scocca, J. J. (1992) Site-specific integration of the Haemophilus influenzae bacteriophage HP1. Identification of the points of recombinational strand exchange and the limits of the host attachment site. J Biol Chem 267: 6859-6864. [0157] Hoess, R. H., Wierzbicki, A., and Abremski, K. (1986) The role of the loxP spacer region in P1 site-specific recombination. Nucleic Acids Res 14: 2287-2300. [0158] Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., et al. (2021a) Highly accurate protein structure prediction with AlphaFold. Nature 596: 583-589 http://www.nature.com/articles/s41586-021-03819-2. Accessed Jan. 16, 2022. [0159] Jumper, J., Evans, R., Pritzel, A., Green, T., Figurnov, M., Ronneberger, O., et al. (2021b) Highly accurate protein structure prediction with AlphaFold. Nature 596: 583-589. [0160] Kolot, M., Malchin, N., Elias, A., Gritsenko, N., and Yagil, E. (2015) Site promiscuity of coliphage HK022 integrase as a tool for gene therapy. Gene Ther 22: 521-527. [0161] Kolot, M., and Yagil, E. (1994) Position and direction of strand exchange in bacteriophage HK022 integration. Mol Gen Genet 245: 623-627. [0162] Le Bourgeois, P., Langella, P., and Ritzenthaler, P. (2000) Electrotransformation of Lactococcus lactis. In Electrotransformation of Bacteria. Eynard, N., and Teissi, J. (eds). Springer, Berlin, Heidelberg. pp. 56-65 https://doi.org/10.1007/978-3-662-04305-9_6. Accessed Sep. 7, 2021. [0163] Lee, G., and Saito, I. (1998) Role of nucleotide sequences of loxP spacer region in Cre-mediated recombination. Gene 216: 55-65. [0164] Leenhouts, K., Buist, G., Bolhuis, A., Berge, A. ten, Kiel, J., Mierau, I., et al. (1996) A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet 253: 217-224 https://doi.org/10.1007/s004380050315. Accessed Sep. 7, 2021. [0165] Malanowska, K., Salyers, A. A., and Gardner, J. F. (2006) Characterization of a conjugative transposon integrase, IntDOT. Mol Microbiol 60: 1228-1240. [0166] Mata, M., Trautwetter, A., Luthaud, G., and Ritzenthaler, P. (1986) Thirteen virulent and temperate bacteriophages of Lactobacillus bulgaricus and Lactobacillus lactis belong to a single DNA homology group. Appl Environ Microbiol 52: 812-818. [0167] McLeod, M., Craft, S., and Broach, J. R. (1986) Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 microns circle. Mol Cell Biol 6: 3357-3367. [0168] Nunes-Duby, S. E., Kwon, H. J., Tirumalai, R. S., Ellenberger, T., and Landy, A. (1998) Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26: 391-406. [0169] Nunes-Duby, S. E., Kwon, H. J., Tirumalai, R. S., Ellenberger, T., and Landy, A. (1998) Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26: 391-406. [0170] Pea, C. E., Stoner, J. E., and Hatfull, G. F. (1996) Positions of strand exchange in mycobacteriophage L5 integration and characterization of the attB site. J Bacteriol 178: 5533-5536. [0171] Rice, P., Longden, I., and Bleasby, A. (2000) EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics 16: 276-277 http://www.cell.com/trends/genetics/abstract/S0168-9525(00)02024-2. Accessed Feb. 2, 2022. [0172] Sarkar, D., Radman-Livaja, M., and Landy, A. (2001) The small DNA binding domain of lambda integrase is a context-sensitive modulator of recombinase functions. EMBO J 20: 1203-1212. [0173] Schneider, T. D., and Stephens, R. M. (1990) Sequence logos: a new way to display consensus sequences. Nucleic Acids Res 18: 6097-6100. [0174] Sheren, J., Langer, S. J., and Leinwand, L. A. (2007) A randomized library approach to identifying functional lox site domains for the Cre recombinase. Nucleic Acids Res 35: 5464-5473. [0175] Smith-Mungo, L., Chan, I. T., and Landy, A. (1994) Structure of the P22 att site. Conservation and divergence in the lambda motif of recombinogenic complexes. J Biol Chem 269: 20798-20805. [0176] Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990) Use of T7 RNA polymerase to direct expression of cloned genes. In Methods in Enzymology. Academic Press, pp. 60-89 https://www.sciencedirect.com/science/article/pii/007668799085008C. Accessed May 30, 2022. [0177] Weisberg, R. A., Enquist, L. W., Foeller, C., and Landy, A. (1983) Role for DNA homology in site-specific recombination. The isolation and characterization of a site affinity mutant of coliphage lambda. J Mol Biol 170: 319-342. [0178] Wojciak, J. M., Sarkar, D., Landy, A., and Clubb, R. T. (2002) Arm-site binding by lambda-integrase: solution structure and functional characterization of its amino-terminal domain. Proc Natl Acad Sci USA 99: 3434-3439. [0179] Zhu, B., Cai, G., Hall, E. O., and Freeman, G. J. (2007) In-Fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques 43: 354-359 https://www.future-science.com/doi/10.2144/000112536. Accessed Jun. 3, 2022.
Sequences
TABLE-US-00013 SEQIDNo1:nucleotidesequenceof tRNASER(CGA)oftheLactobacillusdelbrueckii subsp.bulgaricus GGAGAGTTGGCAGAGCGGTAATGCAGCGGACTCGAAATCCGCCGA ACCAATGTTGAATTGGTGCGCAGGTTCAAATCCTGTACTCTCCTT AAT SEQIDNo2:polynucleotidefragmentP1-P2 5-ATCAACTAGATTTTTAACTAGAA-3 SEQIDNo3:polynucleotidefragmentP1-P2 5-TTTAACTAGAAAATAACTAGAA-3 SEQIDNo4:nucleotidesequenceof.sup.mv4Int ATGCCAAAGCGTAATCCTGCAATCAAAAAATACACCAGCCGGGGC CAAACAAAATACAAATTCCAGATTTACCTGGGCCAGGACGAAAGC GGAAAATCAATCAACACGACCCGGAGTGGTTTTAAATCTTACTCC CAAGCATCAGCAGCTTACAACAAGCTTAAGGCCCAAGGATTGGCC GCCAAAGCACCCAAAAAAGCGACCACCGATGAGGTGTGGTCGCTT TGGTTTGATAGCTATAAAGGCGGAGTTAAAGAGTCGTCAGCAAAC AAAACGCTGACTAGTTACAGAGTCCACATCAAGCCTGCTTTTGGT GATAAAATGATCAGCTCGATCAAGACGGCCACCGTACAACTCTGG GCAAACAATTTGGCCACCAAGCTGGTCAACTACAAGGTGGTTGTG CGCCTGCTAGGGACTCTTTTTGAATTTGCCAAGCGCCTGGACTAT TGCAAGGACAACCCGGTCAAGCAGATCATCATGCCAAAAGCTACC TCCAGGCCTCGCAGAGACATCAGCACCAACTACTATAACCGTGAT GAGCTTCAGCAGTTCCTGCAGGCCGCTAAAGAAGTAGGATCCCGG ACTTATGTCTTCTTTCTACTCCTTGCTACCACGGGCCTCCGAAAA GGCGAAGCACTAGCCCTGGATTGGTCGGACATCGACTACGATCAA GGAAAAATCTCCGTCACTAAGACTCTTGCCTATGGCCTGGGTGGC AAGTACGGGATCCAGCCACCTAAGACTAAGGCAGGGATCCGCACG GTGCCACTGACTGATCAGATGGCAGCCGTTTTAAAAGACTACCAT AGTGATCTCTGCCCGCACCTTTTTCACACGCTTGATGGTGATTAT CTCCGTCTTAGTAAGCCAGATCAGTGGCTTCAGGCTGTTTATAAA CACGACCCAGACCTCCGACAAATTAGAATCCATGGCTTCCGTCAT ACTTTTGCGTCCCTGCTCATCACTGCGGATCCGTCAATCAAGCCA ACAGACGTGCAAGCAATCCTGGGTCATGAATCAATCGATATTACC ATGGAGATTTACATGCACGCCACTCAAGAAGGCAGGCGGAATGTT GAAAGAGTTCTAAATCAACTAGATTTTTAA SEQIDNo5:peptidesequenceof.sup.mv4Int MPKRNPAIKKYTSRGQTKYKFQIYLGQDESGKSINTTRSGFKSYS QASAAYNKLKAQGLAAKAPKKATTDEVWSLWFDSYKGGVKESSAN KTLTSYRVHIKPAFGDKMISSIKTATVQLWANNLATKLVNYKVVV RLLGTLFEFAKRLDYCKDNPVKQIIMPKATSRPRRDISTNYYNRD ELQQFLQAAKEVGSRTYVFFLLLATTGLRKGEALALDWSDIDYDQ GKISVTKTLAYGLGGKYGIQPPKTKAGIRTVPLTDQMAAVLKDYH SDLCPHLFHTLDGDYLRLSKPDQWLQAVYKHDPDLRQIRIHGFRH TFASLLITADPSIKPTDVQAILGHESIDITMEIYMHATQEGRRNV ERVLNQLDF