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 λPR, 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) - KmR - (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-TcR-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-CmR-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-TcR-λattR-CmR was constructed by cloning of the SphI-SacI cat-carrier DNA fragment amplified from pMW118-(λattL-CmR-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" CmR-marker which has been amplified by PCR from pMW118-(λattL-CmR-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) - KmR - (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%.