Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure

Construction of strains with different locations of the φ80-attBsite

The native φ80-attB-site is located at ~28 min of E. coli MG1655 chromosome (http://www.genetics.wisc.edu/ and http://mol.genes.nig.ac.jp/ecoli/). Insertion of the artificial φ80-attB into the chromosome of strain with deleted φ80-attB native site will allow us to introduce the DNA cassette into the new loci by φ80-Int-dependent system. If cassettes, integrated in the set of strains in different points, possess excisable selective markers, it will be possible to bring them in one strain by P1vir-mediated generalized transduction (P1-duction) due to removing the marker from the recipient genome before the next step of transduction.

The desired locations of φ80-attB sites can be spread in non-essential parts of bacterial genome at different distances from oriC. In this case, the level of expression of the same integrated cassette will depend on its position due to the θ-like structure of replicating chromosome and gene-dosage effect: the cassettes closer to oriC will be expressed at higher level than those located near the terminus of the chromosome replication [31].

We consider as good candidates for the further integration points the positions of the "native" IS elements (in our case IS5.7–IS5.11) since insertions into the genes already disrupted by IS elements will not be detrimental to cell viability and IS elements are almost randomly distributed along the chromosome of MG1655 [32]. Accordingly, the set of genes disrupted by IS5-elements insertion was chosen as the future locations of the artificial φ80-attB sites. These points are rather far from each other, so the future integrated cassettes can be combined in one strain by independent acts of P1-duction.

Realization of this strategy includes several steps. At first, the deletion of the native φ80-attB was carried out by Recombineering between E. coli MG1655 chromosome and the constructed "λ-excisable" Cm^R-marker amplified by PCR from pMW118-(λattL- Cm^R-λattR) [19]. After Red recombination, the DNA locus modification was verified by PCR, and followed by λ-Int/Xis-mediated excision of the marker from the chromosome. The marker-less strain, MG-Δ(φ80-attB), was used as the recipient for the insertion of the artificial φ80-attB. The latter includes:

1) construction and cloning of the cassette [(φ80-attL) - Km^R - (φ80-attR)] in the plasmid;

2) using the obtained plasmid, pMWattphi, as the template for PCR amplification of φ80-removable Km^R-marker flanked by the 36 bp arms homologous to the desired loci on the MG1655 chromosome;

3) Red integration of the markers into the chromosome of MG-Δ(φ80-attB) for construction of the desired library of "marked" strains;

4) each obtained strain was cured from the marker by φ80-Int/Xis-system [30]. In this way the "unmarked" part of the library was constructed;

5) new strains and MG1655 (as a control) were tested for φ80-driven integration/excision of the "conditionally-replicated integrative and modular (CRIM) plasmid" pAH162 carrying φ80-attP site [30], using φ80-Int- or φ80-Int/Xis-helper plasmids, respectively. Here, the efficiencies of integration/excision In or Out of the artificial points were practically the same as for In/Out of the native site.

Although the efficiency of CRIM plasmids excision from all tested sites in our experiments was 15–30% (not 100%, as reported in [30]), nevertheless, selection the marker-less clones was not the problem. We noticed, as well that, in our hands, the efficiency of λ-Int/Xis-driven excision under the same conditions was significantly higher and exceeded 80%.

Construction of φ80-integrative CRIM plasmid with λ-removable "vector part"

The φ80-cognate CRIM plasmids with different selective markers can be used for integration of cassettes in the obtained strains that differ in location of φ80-attB. A recombinant strain that contains multiple insertions can be constructed by P1 transduction. However the presence of plasmids' markers in the bacterial chromosome cannot satisfy "marker-less" criteria for the practical application of the engineered strain. CRIM plasmids constructed by Haldimann and Wanner [30], only allow the site-specific excision of the entire recombinant structure initially inserted in the chromosome. It is useful to modify CRIM plasmids to allow excising of the vector part after site-specific integration of recombinant DNA. In this way, the new φ80-cognate CRIM plasmid (Fig. 2) with a λ-removable vector part was obtained (for details see Methods).

The new plasmid pAH162-λattL-Tc^R-λattR (Fig. 2) retains from its progenitor pAH162 the non-modified fragment carrying φ80-attP and the multi-cloning site flanked by bacterial (rgnB) and phage λ (tL3) transcription terminators. This fragment is bracketed by λ-attL/R sites which allow λ-Int/Xis excision of the vector part, including conditionally-replicative origin of R6K (oriRγ) and the selective marker, Tc^R (from Tn10). We have confirmed the expected properties of pAH162-λattL-Tc^R-λattR by its integration into the native φ80-attB of MG1655 chromosome, followed by λ-Int/Xis excision of the marked vector part flanked by λ-attL/attR. Of 80 tested clones cures from λ-Int/Xis-helper plasmid, more than 80% have lost the Tc^R marker of integrated CRIM plasmid, as well.

Exploiting the new system for multiple integration of cat-gene into E. colichromosome

To test the Dual-In/Out strategy of marker-less strain construction, we used the efficiently expressed variant of cat-gene as the model cassette. This experiment includes several steps:

1) cloning of cat-gene into pAH162-λattL-Tc^R-λattR, using E. coli CC118 (λpir⁺) strain as the recipient;

2) φ80-driven integration of the recombinant CRIM plasmid into the strains that differ in location of φ80-attB;

3) construction of "marker-less" strains with single cat-cassette by λ-Int/Xis excision of Tc^R-containing DNA fragment flanked by λ-attL/R-sites, followed by determination of cat expression levels in these strains;

4) combining the set of cat-cassettes in one "marker-less" strain by sequential P1-duction of the "marked" cassettes followed by curing the Tc^R-marker from the recipient strain before the next stage of transduction.

The previously constructed [19] pMW118-(λattL-Cm^R-λattR) plasmid was chosen as a template for PCR amplification of cat-gene. In this plasmid the structural part of cat-gene with its native RBS from E. coli Tn9 is under the transcriptional control of rather strong phage T7 A2 promoter [33] that is recognized by E. coli RNA polymerase with σ70 in a constitutive manner. This cat-gene was amplified by PCR with the primers that carry restriction sites for cloning in pAH162-λattL-Tc^R-λattR. The transformants of E. coli CC118 (λpir⁺) carrying the recombinant plasmid of interest, were selected on the medium supplemented with Cm. The expected structure of the plasmid was verified by restriction analysis and PCR.

At the next stage this plasmid was integrated by φ80-Int system into MG1655-derived strains with a different location of φ80-attB, using Tc^R-marker for selection (Fig. 3). The correct integration was confirmed by PCR, and the corresponding strains were entitled MG-Tc^R-cat-(i), where (i) varied from 1 to 6 depending on the number of φ80-attB sites. Subsequently, the vector part of the integrated plasmids in all MG-Tc^R-cat-(i) strains was excised by λ-Int/Xis site-specific recombination. After checking by PCR, the corresponding recombinant strains retaining the cat-gene in their chromosomes, was entitled MG-cat-(i).

The efficiency of cat-gene expression in MG-cat-(i)-strains was evaluated by determination of Cat enzymatic activity (Fig. 4, Table 1). As expected (see explanation above and reference [31]), the level of cat-gene expression correlates with the distance between the integration point and oriC: the strains with the cat-gene position closer to oriC have higher Cat activity.

strain MG-cat-(i)	IS element	Activity, nmol/minxmg
MG-cat-(1)	IS 5.7	180 ± 15
MG-cat-(2)	IS 5.8	180 ± 15
MG-cat-(3)	IS 5.9	240 ± 30
MG-cat-(4)	IS 5.10	280 ± 15
MG-cat-(5)	IS 5.11	270 ± 15
MG-cat-(6)	native φ80-attB	200 ± 30
MG- cat- (i+j)
MG-cat-(1+2)	IS 5.7 IS 5.8	330 ± 15
MG-cat-(1+3)	IS 5.7 IS 5.9	420 ± 30
MG-cat-(2+3)	IS 5.8 IS 5.9	420 ± 30
MG-cat-(1+2+3)	IS 5.7 IS 5.8 IS 5.9	520 ± 15
MG-cat-(4+5)	IS 5.10 IS 5.11	500 ± 30
MG-cat-(4+6)	IS 5.10 native φ80-attB	370 ± 15
MG-cat-(5+6)	IS 5.11 native φ80-attB	400 ± 15
MG-cat-(4+5+6)	IS 5.10 IS 5.11 native φ80-attB	540 ± 30

Several cat-cassettes were combined in one strain by sequential P1-duction of Tc-marked fragments from MG-Tc^R-cat-(i) strains into MG-cat-(j) (where i ≠ j), followed by λ-Int/Xis-driven curing of Tc^R-marker that gives an MG-cat-(i + j) "marker-less" strain carrying two cat-cassettes in (i) and (j) positions; a third cassette was added by an analogous procedure. In addition, all steps used in increasing the number of cat-cassettes in the chromosome of recombinant "marker-less" strain were controlled by determination of Cat activity. The data presented in Table 1 show that the total Cat activity of multi-integrants correlates with the expected sum of the Cat activities of the corresponding single-integrants.

Strains and plasmids

Escherichia coli K-12 MG1655, a wild-type strain with sequenced genome [32], was used as the recipient for the insertion of the artificial φ80-attB. CRIM plasmids were propagated in CC118 λpir⁺ strain [37]. E. coli W3350(80) lysogenic for phage φ80 was obtained from GosNIIGenetika collection and used as a template for PCR-driven amplification of φ80-attL/R sites.

pKD46 was used as a donor of λRed-genes for providing Red-dependent recombination according to the described procedure [8].

pAH123 and pAH129-helper plasmids [30], GenBank accession numbers AY048726 and AY048727 respectively. These helper plasmids were used for φ80-dependent integration/excision procedures.

pMWts-λInt/Xis-helper plasmid is similar to the plasmid pMP955A described in [1]. It has low-copy-number thermo-sensitive replicon pSC101, genes xis and int of phage λ under the control of λP_R, thermo-sensitive repressor cIts857. It is used for λ-Int/Xis-mediated excision of the DNA fragments flanked by λattL/R.

pMWattphi – this recombinant plasmid was constructed on the base of pMW118 (GenBank accession number AB005475). This plasmid is used as the template for PCR amplification of fragment (φ80-attL) - Km^R - (φ80-attR) flanked with 36 bp arms homologous to targeted site in MG1655 DNA. Hybrid φ80-attL and φ80-attR sites were obtained by PCR amplification from purified chromosome of E. coli W3350(80) using primers: P1 – P2 for attL and P3 – P4 for attR. Amplified fragments were restricted with EcoRI-BamHI and XbaI-PstI restrictases and cloned into corresponding sites flanking kan on plasmid to give pMWattphi (Fig. 3).

P1 5'-atagaattcgaaaggtcatttttcctgaatatgc-3'

P2 5'-ataggatccatcattgaatgggtacacatttttg-3'

P3 5'-atattctagagatttgaatagcgagcgtaccttag-3'

P4 5'-atactgcagtcgtttgttgacagctggtccaatg-3'

pAH162-λattL-Tc^R-λattR – integrative plasmid. Construction of this plasmid included several steps with isolation and analysis of recombinant DNA intermediates. The structure of this plasmid is shown in Fig. 2. Sequence landmarks: 1) from 6 to 1031 – fragment from pAH162 (GenBank accession number AY048738) which contains conditional-replication origin oriRγ; 2) from 1038 to1145 – fragment contains attL of phage λ from plasmid pMW118-(λattL-tetA-tetR-λattR) which structural similar to the plasmid pMW118-(λattL-Cm^R-λattR) [19]; 3) from 1153 to 2274 – fragment from pAH162 which contains bacterial terminator rgnB, multiple cloning sites MCS, phage λ terminator tL3, phage attachment attP phi80; 4) from 2281 to 2462 – fragment contains attR of phage λ from plasmid pMW118-(λattL-tetA-tetR-λattR); 5) from 2456 to 4463 – fragment from plasmid pMW118-(λattL-tetA-tetR-λattR) which contains the Tn10-encoded tetracycline resistance gene tetA and the repressor gene tetR.

pAH162-λattL-Tc^R-λattR-Cm^R was constructed by cloning of the SphI-SacI cat-carrier DNA fragment amplified from pMW118-(λattL-Cm^R-λattR) [19] with primers P5 – P6.

P5 5'-cagtaagcatgcgcggccgcccggataagtagacagcctgataag-3'

P6 5'-cagtaagagctcgcggccgcttacgccccgccctgccactc-3'

Molecular biology methods

Restriction analysis of the recombinant plasmids and Ca²⁺-dependent transformation of E. coli cells were performed in accordance with the routine experimental protocols [38]. Commercially available preparations of restrictases, T4 DNA ligase and the Klenow fragment of E. coli DNA polymerase I (Fermentas, Lithuania) were used. PCR fragment for cloning were generated by using AccuTaq DNA polymerase (Sigma, USA). Sigma (USA) products were used for the isolation of plasmid DNA, extraction of DNA fragments from agarose gels.

Construction of strains with different locations of the φ80-attBsite

The deletion of the native φ80-attB was carried out by Red-dependent integration of "λ-excisable" Cm^R-marker which has been amplified by PCR from pMW118-(λattL-Cm^R-λattR) [19] with primers P7 – P8. DNA locus modification was verified by PCR with primers P9 – P10.

P7 5'-gtaatcaaaggatttgagcgagcaactgtacctcagcgctcaagttagtataaaaaagctgaac-3'

P8 5'-acatttagcacgtttacagttactgcatgatgaaggtgaagcctgcttttttatactaagttgg-3'

P9 5'-tgcagcgcgtgaatgtgtta-3'

P10 5'-ctcaagacaaagctgatagcc-3'

The obtained marker-less strain, MG-Δ(φ80-attB), was used as the recipient for the insertion of the artificial φ80-attB. pMWattphi was used as the template for PCR amplification of fragment (φ80-attL) - Km^R - (φ80-attR) which integrated into the chromosome of MG-Δ(φ80-attB) to the desired loci – the set of genes disrupted by IS5-elements insertion. For these purposes the following primers were used:

IS5.7 P11 5'-tcctaaagaaagtatctattctgatacggttgttgagaaaggtcatttttcctgaatatg-3'

P12 5'-aagccatttacacgcacaaaatctgaaaaacgtacctcgtttgttgacagctggtccaatg-3'

P13 5'-gtcttctcacgggaacggtt-3'

IS5.8 P14 5'-gagggtatcagtacattgaaatgaatggcgccgcaggaaaggtcatttttcctgaatatg-3'

P15 5'-tctggtttgccgcgccacccatttgaacaatttgattcgtttgttgacagctggtccaatg-3'

P16 5'-cctcccttttcgatagcgacaa-3'

IS5.9 P17 5'-gggcgtattaccgcgcaaatagataccttgcaccgcgaaaggtcatttttcctgaatatg-3'

P18 5'-ctgcggatcatcaatggcgtcaatcatgccgaaatg-tcgtttgttgacagctggtccaatg-3'

P19 5'-gttcaatatgcgcggcatacca-3'

IS5.10 P20 5'-tatcaattgacgttaaggtgactctggaagctgcaggaaaggtcatttttcctgaatatg-3'

P21 5'-tattgactgaatgactaccgaagttaacaactccgctcgtttgttgacagctggtccaatg-3'

P22 5'-ttccggtggtcatactatccattc-3'

IS5.11 P23 5'-attattaaccattaatgacaaccttttacgagcaaagaaaggtcatttttcctgaatatg-3'

P24 5'-tatgaaagattggttatcctggcctctaaaaatttatcgtttgttgacagctggtccaatg-3'

P25 5'-ctttttcattaggcagtggcctc-3'

The integration of fragment was verified by PCR with primers P13, 16 – P26 and P19, 22, 25 – P27.

P26 5'-tgtttcgggcggaccaaatgata-3'

P27 5'-gccatggcagaatctgctccatgcggg-3'

Plasmid integration and excision of the vector part of integrated plasmid

For testing the new MG1655 strains with artificial φ80-attB sites and CRIM plasmid integration/excision, procedures were driven by standard protocols using helper plasmids pAH123 (φ80-Int) and pAH129 (φ80-Int/Xis) respectively [30]. The vector part of CRIM plasmids was excised by λInt/Xis – site-specific recombination using helper plasmids pMWts-λInt/Xis by standard protocols [1]. The integration of plasmid was verified by PCR with primers P13, 16, 19, 22, 25, 28 – P30 and P 26, 27, 29 – P31. P28 and P29 are primers for native φ80-attB [19]; P30 is a primer annealing on vector part of integrated plasmid [19] and P31 is annealing on tetR gene.

P28 5'-taaggcaagacgatcagg-3'

P29 5'-ctgcttgtggtggtgaat-3'

P30 5'-acgagtatcgagatggca-3'

P31 5'-gtaaactcgcccagaagctagg-3'

Cat activity assays

The Cat activity was assayed using a spectrophotometric method (UVmini 1240; Shumadzu, Japan). Log-phase cells harvested at OD₅₉₅ = 0.8 were resuspended in potassium phosphate buffer (50 mM; pH 7.5). Cell lysates were prepared by sonication. The quantity of protein was determined by the Bradford method [39]. Assays were performed in 1 ml (1 cm light path) cuvettes at room temperature. The reaction mixture in each cuvette contained 100 μl of 1 M Tris-hydrochloride, pH 7.5, 100 μl of 1 mM acetyl CoA (Sigma, USA), 100 μl of 10 mM 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB; Sigma, USA), 0.05 mg of protein, H₂O for a total volume of 0.99 ml. 10 μl of 10 mM Cm (Sigma, USA) was added to start the reaction, and thionitrobenzoic acid (TNB) production was followed at 412 nm. Enzyme activity was calculated in terms of nmol of thionitrobenzoic acid produced per min per mg of protein. The results were averaged over three independently grown cultures for each clone; the scatter was no more than 15%.