A PCR-free cloning method for the targeted φ80 Int-mediated integration of any long DNA fragment, bracketed with meganuclease recognition sites, into the Escherichia coli chromosome

Journal of Microbiological Methods 89 (2012) 167–173

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A PCR-free cloning method for the targeted φ80 Int-mediated integration of any long DNA fragment, bracketed with meganuclease recognition sites, into the Escherichia coli chromosome Anna A. Ublinskaya, Valeriy V. Samsonov, Sergey V. Mashko, Nataliya V. Stoynova ⁎ Ajinomoto-Genetika Research Institute, 1st Dorozhny pr., 1-1, 117545 Moscow, Russian Federation

a r t i c l e

i n f o

Article history: Received 31 January 2012 Received in revised form 14 March 2012 Accepted 14 March 2012 Available online 29 March 2012 Keywords: DNA cloning Integration Escherichia coli Recombineering I-SceI endonuclease

a b s t r a c t The genetic manipulation of cells is the most promising strategy for designing microorganisms with desired traits. The most widely used approaches for integrating specific DNA-fragments into the Escherichia coli genome are based on bacteriophage site-specific and Red/ET-mediated homologous recombination systems. Specifically, the recently developed Dual In/Out integration strategy enables the integration of DNA fragments directly into specific chromosomal loci (Minaeva et al., 2008). To develop this strategy further, we designed a method for the precise cloning of any long DNA fragments from the E. coli chromosome and their targeted insertion into the genome that does not require PCR. In this method, the region of interest is flanked by I-SceI rare-cutting restriction sites, and the I-SceI-bracketed region is cloned into the unique I-SceI site of an integrative plasmid vector that then enables its targeted insertion into the E. coli chromosome via bacteriophage φ80 Int-mediated specialized recombination. This approach allows any long specific DNA fragment from the E. coli genome to be cloned without a PCR amplification step and reproducibly inserted into any chosen chromosomal locus. The developed method could be particularly useful for the construction of marker-less and plasmid-less recombinant strains in the biotechnology industry. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Bacterial plasmids are useful tools that are widely used in microbial metabolic engineering for the introduction/amplification of artificial genetic constructs with arbitrary complexity. However, the use of plasmids is undesirable in some cases due to safety problems, plasmid instability, copy number variability and the limited size capacity of several plasmid vectors. Therefore, it is often preferable to directly integrate genetic constructs into the bacterial chromosome, where they can be stably maintained. Currently, methods for the “chromosomal editing” (Balbás and Gosset, 2001) of Gram-negative bacteria and Escherichia coli, in particular, are based primarily on site-specific (Bao et al., 1991) or random (de Lorenzo and Timmis, 1994; Akhverdyan et al., 2011) transposition mechanisms and homologous (Sawitzke et al., 2007; Sharan et al., 2009) and/or phage-mediated specialized (Haldimann and Wanner, 2001) recombination. λRed/RecET-based recombination-mediated genetic engineering (recombineering) is a highly effective and powerful method for targeted chromosomal editing and the construction of plasmid-less recombinant bacteria with predesigned genome structures (Court et al., 2002; Datsenko and Wanner, 2000; Sawitzke et al., 2007). In this method, a linear DNA fragment flanked by short (30–50 bp) sequences that are homologous to the desired target is specifically integrated into the ⁎ Corresponding author. Tel.: + 7 4957803378x515, 505; fax: + 7 4953150640. 0167-7012/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2012.03.013

chromosome via homologous recombination mediated by the expressed λRed (or RecET) enzyme system. Although it is highly efficient and easy to use for relatively short (no more than 3 kb) PCR-derived linear DNA fragments, recombineering in E. coli presents several challenges when large DNA fragments are to be inserted. In addition to the difficulty of generating these fragments with high fidelity by PCR, their size makes their transformation and λRed-dependent integration significantly less efficient (Kuhlman and Cox, 2010a). Earlier, we succeeded in integrating fragments of up to 7 kb in length using a 36 bp flanking homology for λRed recombination, but it was necessary to use the specially constructed recipient E. coli strain K-12, which did not possess any additional homologies with the insertion (unpublished data). The experimentally detected shortcomings of the original recombineering approach are the motivation for its further development and modification for the integration of large DNA fragments (Kuhlman and Cox, 2010a, 2010b; Rivero-Müller et al., 2007). An effective method for the directly targeted integration/excision of genetic constructs with practically no size limitations was developed based on phage-mediated site-specific specialized recombination. CRIM (conditional-replication, integration and modular) vector plasmids were constructed and applied for this purposes (Haldimann and Wanner, 2001). CRIM plasmids, based on the γ-replication origin of R6K, which requires the trans-acting Π protein (encoded by the pir gene) for replication (Metcalf et al., 1994), carry an antibiotic resistance gene as a

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selective marker and the phage-specific attachment site attP from the λ, HK022, φ80, P21, or P22 genomes. Thus, CRIM-based recombinant DNA fragments could be selectively integrated into or retrieved from their complementary attachment (attB) site in the bacterial chromosome by supplying the phage-specific integrase (Int), with or without a simultaneously expressed excisionase (Xis). The straightforward combination of phage specialized and homologous λRed/RecET mediated recombination methods led to the development of the Dual-In/Out strategy for the site-specific multiple integration of large fragments (Minaeva et al., 2008). This strategy includes the initial λRed-driven insertion of artificial φ80-attB sites into the desired loci of the E. coli chromosome, followed by two phage-specific specialized recombination events: the φ80 Int-mediated integration of the recombinant φ80-attP CRIM plasmid into the artificially inserted φ80-attB site, followed by the λ Xis/Int-mediated excision of the inserted CRIM DNA backbone, bracketed by λattL/R-sites and consisting of the plasmid origin of replication and the selective antibiotic resistance marker. Theoretically, the length of the fragments that can be integrated into the bacterial genome using this strategy is limited only by its accessibility for cloning. In practice, PCR-amplified DNA fragments are routinely used for cloning, even for the integration of a native region of the bacterial genome. Although new thermostable DNA polymerases offer high fidelity and processivity and are routinely used for PCR (Wang et al., 2004; Vega et al., 2010), the accuracy of the PCR amplification must be confirmed, as a rule, by sequencing at the last stage of the cloning procedure. In this work, we designed a plasmid-host E. coli system that allowed the immediate cloning of large chromosomal DNA fragments initially marked with an “excisable” antibiotic resistance gene and flanked by I-SceI rare-cutting endonuclease recognition sites via λRed-driven targeted insertions. The use of the specialized CRIM plasmid as a

cloning vector allows the ensuing integration of this I-SceI-mediated DNA fragment into the predesigned locus of a bacterial chromosome carrying an artificial attB site. This strategy provides the opportunity to avoid PCR-derived mistakes in the insertion of large DNA fragments, thus enabling simple and efficient chromosome editing. The developed method was used to successfully clone an 8 kb DNA fragment harboring the E. coli isc operon, followed by its insertion into a targeted locus in the chromosome, thus confirming its utility. In addition, we demonstrate that the cloning of different fragments of the E. coli genome could be standardized by introducing I-SceI restriction sites into the specifically dispersed “scar” sequences of single-gene knockout mutants from the Keio collection (Baba et al., 2006). 2. Materials and methods 2.1. Strains, plasmids and growth conditions All of the bacterial strains and plasmids used in this study are listed in Table 1. The following media were used for the culturing of the bacteria: lysogenic broth (LB), SOB, and SOC (Sambrook and Russell, 2001). Ampicillin (Ap, 100 mg/L), chloramphenicol (Cm, 40 mg/L), tetracycline (Tc, 20 mg/L) and kanamycin (Km, 50 mg/L), were used for selection as necessary. 2.2. DNA handling procedures Protocols for the genetic manipulation of E. coli and techniques for the isolation and manipulation of nucleic acids were described previously (Sambrook and Russell, 2001). Restriction enzymes, T4 DNA ligase, High Fidelity PCR Enzyme Mix and 1 kb DNA Ladder were purchased from Fermentas (Lithuania). Plasmids and genomic DNA were isolated using

Table 1 Bacterial strains and plasmids used in this study. Strain or plasmid Strains BW25113 ΔtrmJ::Km BW25113 ΔpepB::Km CC118 MG1655 trs5.9::φ80-attB BW25113-(M1-isc) BW25113-(isc-M2) BW25113-(M1-isc-M2) TG1 ilvD::Tn10 MG1655 trs5.9::lox71-cat-lox66-isc

Plasmids pKD46 pAH123 pMWts-λInt/Xis pAH162-λattL-tetA-tetR-λattR-2Ter pAH162- λattL-tetA-tetR-λattR-2Ter-I-SceI pAH162-λattL-tetA-tetR-λattR-2Ter-I-SceI-isc-cat pMW118-λattR-cat-λattL pMW118-λattL-tetR-tetA(Tn10)-λattR pMW118-λattL-Km-λattR pACYC184

Description

Source

lacIq rrnB3 ΔlacZ4787 hsdR514 DE(araBAD)567 DE(rhaBAD)568 rph-1 ΔtrmJ::Km — used as a recipient for λRed-mediated integration lacIq rrnB3 ΔlacZ4787 hsdR514 DE(araBAD)567 DE(rhaBAD)568 rph-1 ΔpepB::Km — used as a recipient for λRed-mediated integration λpir+ Δ(ara-leu) araD ΔlacX74 galE galK phoA20 thi-1 rpsE rpoB argE(Am) recA1 — used as a host for CRIM plasmid propagation As MG1655Δφ80-attB, but trs5.9::φ80-attB — used as a recipient for φ80-Int-mediated integration As BW25113, but ΔtrmJ::lox66-CmR-lox71 — recombinant CmR strain As BW25113, but ΔpepB::λattL-tetR-tetA-λattR — recombinant TcR strain As BW25113, but ΔtrmJ::I-SceI-lox71-cat-lox66 ΔpepB::I-SceI-λattL-tetRtetA-λattR — recombinant CmR, TcR strain supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rK– mK–) [F′ traD36 proAB lacIqZΔM15] ilvD::Tn10 As MG1655Δφ80-attB, but trs5.9:: lox71-cat-lox66-isc — contained two directly repeated isc gene copies

Baba et al., 2006

oriR101, repA101ts, araC, ParaB-[γ β exo of phage λ], ApR — used as a donor of λRed-genes to provide λRed-dependent recombination oriR101, repA101ts, λcIts857, λPR→φ80-int, ApR — used as a helper plasmid for thermoinducible expression of the φ80-int gene oriR101, repA101ts, λcIts857, λPR→λxis-int, ApR — used as a helper plasmid for thermoinducible expression of the λ xis-int genes oriRγ, φ80-attP, MCS, TcR — CRIM vector oriRγ, φ80-attP, I-SceI site in MCS, TcR — CRIM vector with unique I-SceI site for cloning oriRγ, φ80-attP, [lox66-cat-lox71, isc operon of E. coli] cloned in I-SceI site, TcR, CmR — recombinant CRIM plasmid with the desired chromosomal DNA fragment oriR101, repA, MCS, ApR, CmR — donor of λXis/Int-excisable CmR marker oriR101, repA, MCS, ApR, TcR — donor of λXis/Int-excisable TcR marker oriR101, repA, MCS, ApR, KmR — donor of λXis/Int-excisable KmR marker CmR, TcR

Baba et al., 2006 Herrero et al., 1990 Minaeva et al., 2008 This work This work This work Laboratory collection This work

Datsenko and Wanner, 2000 Haldimann and Wanner, 2001; GenBank accession number AY048726 Minaeva et al., 2008 Minaeva et al., 2008 This work This work Katashkina et al., 2005 This work Laboratory collection GenBank accession number X06403

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QIAGEN Plasmid Mini Kits (QIAGEN GmbH, Germany) and Bacterial Genomic DNA Kits (Sigma), respectively. QIAquick Gel Extraction kits (QIAGEN GmbH, Germany) were used to isolate DNA from agarose gels. Oligonucleotides were purchased from Sintol (Russia). The sequences of oligonucleotide primers are presented in Table 2. 2.3. Construction of plasmids 2.3.1. Construction of pMW118-λattL-tetR-tetA(Tn10)-λattR, harboring the “excisable” Tc R marker The plasmid pMW118-λattL-tetR-tetA(Tn10)-λattR was obtained as follows: a DNA fragment containing tetR-tetA(Tn10) was PCR amplified from strain TG1 ilvD::Tn10 using primers 1 and 2 (Table 2), which contain PstI restriction sites at their 5′ ends. Then, the PCR product was digested with PstI and ligated with the pMW118-λattL-KmλattR plasmid, which was similarly digested. Cloning was performed in the XL1Blue strain and TcR KmS clones harboring pMW118-λattL-tetRtetA(Tn10)-λattR were selected. This plasmid contains the “excisable” cassette λattL-tetR-tetA(Tn10)-λattR, one copy of which provides resistance to Tc. 2.3.2. Construction of the CRIM vector pAH162-λattL-tetA-tetR-λattR2Ter-I-SceI containing a unique I-SceI site for cloning A DNA fragment containing an I-SceI restriction site adjacent to the antibiotic resistance marker Cm R (cat) and flanked by SmaI sites was PCR amplified from pACYC184 using primers 3 and 4. The PCR product was gel-purified, digested with SmaI and ligated into pAH162λattL-tetA-tetR-λattR-2Ter, which was similarly digested. The cloning was performed in E. coli CC118 (λpir +); clones with the recombinant plasmids which harbored KpnI-flanked cat gene were selected on Cmcontaining medium. The KpnI fragment containing the CmR gene was eliminated from the obtained plasmid, resulting in pAH162-λattL-tetAtetR-λattR-2Ter-I-SceI, which harbored a unique I-SceI site for cloning.

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directly repeated sites (5′-TAGGGATAACAGGGTAAT-3′), which are recognized by the Saccharomyces cerevisiae I-SceI endonuclease. I-SceI cleaves within the above-mentioned non-palindromic target sequence, generating four-base pair 3′ overhangs (Colleaux et al., 1986; 1988; Monteilhet et al., 1990). This sequence is very rare, appearing in a random nucleotide sequence with a frequency of 1 per 418 (10 11) base pairs (Lippow et al., 2009; Thierry et al., 1991). Most importantly, this site is absent in the E. coli genome. λRed-mediated integration of linear DNA fragments harboring the I-SceI recognition site, in which the 3′ portion is linked with the M1 and M2 selective markers, is used to flank YFR with directly repeated I-SceI sites (Fig. 1A). The marker (M1 in Fig. 1) would be linked with YFR in the I-SceI-flanked chromosomal DNA fragment. This marked fragment can then be selectively cloned by I-SceI-mediated cleavage of the purified chromosome into a specialized CRIM vector plasmid carrying M3 as a selective marker (Fig. 1A). The resulting recombinant plasmid, CRIM-M1-YFR (Fig. 1B), can be specifically inserted into the corresponding chromosomal attB site(s) in the presence of the expressed phage integrase (Haldimann and Wanner, 2001; Minaeva et al., 2008), as shown in Fig. 1C. Application of the M1 and M3 “excisable” markers flanked by the lox66/71 and λattR/L sites, respectively, ensures the specific Cre-dependent excision of the M1 marker and the λXis/Int-mediated excision of the CRIM plasmid backbone, enabling the repeatability of the insertion procedure (Fig. 1D). This approach based on immediate cloning of the chromosomal DNA fragment avoids the risk of errors generated by PCR amplification. If the pre-selection of the recombinant CRIM-based plasmid harboring YFR is omitted and the φ80-Int-mediated integration is performed just after the ligation of the I-SceI-treated vector and purified bacterial chromosome, theoretically, there is no limit to the size of the I-SceI fragment that can be inserted. 3.2. Construction of linear DNA fragments containing I-SceI restriction sites adjacent to a selectable marker

2.4. Estimation of integration efficiency The efficiency of integrating pAH162-λattL-tetA-tetR-λattR-2Ter-ISceI-isc-cat into the trs5.9 locus was defined as the proportion of Cm R-colonies in the total amount of cells plated after electrotransformation of MG1655 trs5.9::φ80-attB with the above plasmid. The average data of triplicated experiments were represented. 3. Results and discussion 3.1. General scheme of chromosomal DNA fragment cloning and integration The proposed scheme of chromosomal DNA fragment cloning and integration is presented in Fig. 1. According to this strategy, your favorite region (YFR) of the bacterial chromosome must first be marked by an antibiotic resistance gene, with simultaneous bracketing by the

Two marker-carrier DNA fragments, I-SceI-lox71-cat-lox66 (1046 bp) and I-SceI-λattL-tetR-tetA-λattR (2331 bp), which harbor the M1 and M2 “excisable” antibiotic resistance markers (Cm R and TcR, respectively) linked to the 3′ portion of the I-SceI restriction site, were constructed (Fig. 2A). Both constructs were obtained by PCR amplification. For the first construct, the pMW118-λattR-cat-λattL (Katashkina et al., 2005) plasmid was used as a template with primers 5 and 6. To amplify the second DNA fragment, the pMW118-λattL-tetR-tetA(Tn10)-λattR plasmid (see Materials and methods) was used as a template with primers 7 and 8 (Fig. 2A). Both of the resulting PCR fragments contained 18-bp non-palindromic I-SceI recognition sequences immediately 5′ to the M1 (lox71-cat-lox66) or M2 (λattL-tetR-tetA-λattR) markers. The DNA-fragment I-SceI-lox71-cat-lox66 harbored a chloramphenicol resistance gene flanked with mutant lox66/lox71 sites (Albert et al., 1995; Lambert et al., 2007). The lox sites can serve as recognition sites for

Table 2 Sequences of PCR primers used in this studya. Title

Sequence

Restriction site

1 2 3 4 5 6 7 8 9 10 11 12

5′-gtgctgcagggaaaaaggttatgctgctt-3′ 5′-actctgcagaagctaaatcttctttatcgta-3′ 5′-atcccgggagcgctgatgtccggcggtgcttttgcc-3′ 5′-ctcccgggtagggataacagggtaatggtaccttacgccccgccctgccactcatcgcag-3′ 5′-ctactgcagtaccgttcgtataatgtatgctatacgaagttattggtcgaaaaaaaaagcccgcac-3′ 5′-tgcctgcagtagggataacagggtaattaccgttcgtatagcatacattatacgaagttatccggataagtagacagcctgata-3′ 5′-atggtacctagggataacagggtaattgaagcctgcttttttatactaagttgg-3′ 5′-atgagctccgctcaagttagtataaaaaagctgaac-3′ 5′-attccggggatccgtcgacctgcagttggaagttaagggataacagggtaattaccgt-3′ 5′-tgtaggctggagctccttcgaagttcctatactttaccgttcgtataatgtatgctatacga-3′ 5′-attccggggatccgtcgacctgcagttcgaagtttagggataacagggtaattgaagcctgctt-3′ 5′-tgtaggctgcagctgcttcgaagttcctatacttcgctcaagttagtataaaaaagctgaac-3′

PstI PstI SmaI SmaI, I-SceI PstI PstI, I-SceI I-SceI SacI I-SceI – I-SceI –

a

Restriction sites are underlined

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Fig. 1. The general scheme of genetic manipulations used for the cloning and integration of long DNA fragments. YFR — your favorite region; M1, M2, M3 — positive selection markers. The I-SceI site is designated by a blue arrow. A: λRed-mediated integration of linear DNA fragments harboring 18-bp non-palindromic I-SceI recognition sites adjacent to positive selection markers upstream and downstream region for cloning. B: Cloning of the I-SceI DNA-fragment containing YFR and marked with M1 into the MCS of the CRIM vector. C: φ80-Int-mediated integration of the CRIM vector with the target cassette into different φ80-attB sites. In addition to YFR, the inserted cassette harbors the “excisable” positive selection marker M1 flanked by, for example, lox66/71 sites, for the further curing and the “excisable” vector portion of the CRIM plasmid, marked with M3 and flanked, for example, by λattL/attR for further curing. The lox71 and lox66 sites are designated with the boxes shaded horizontally and vertically, respectively. The λattL and λattR sites are designated as circles shaded horizontally and vertically, respectively. These sites are omitted in A and B for simplicity. D: Excision of the vector portion of the CRIM plasmid and excision of the positive selection marker, M1.

the Cre recombinase-mediated curing of the CmR marker. Cre-mediated recombination between the lox66/71 sites forms a lox72 site, which is poorly recognized by Cre (Lambert et al., 2007); accordingly, this Crelox66/71-based system is widely used for multiple intrachromosomal genetic manipulations (Albert et al., 1995; Lambert et al., 2007). Similarly, the DNA-fragment I-SceI-λattL-tetR-tetA-λattR contained a Tc R marker flanked with λattR/L sites, which enables its λXis/Intmediated excision, if necessary (Katashkina et al., 2005). In the next step, the resulting constructs can be subjected to PCR to generate fragments for the λRed-driven insertion of the I-SceI recognition sites into any chromosomal region of interest (Fig. 2B). 3.3. Cloning of the isc operon flanked by I-SceI-sites into the CRIM plasmid vector To confirm the usability of the developed method, a DNA fragment containing the E. coli isc operon (about 8 kb in length) was chosen as a model for cloning. The enzymes encoded by this operon are involved in

the biogenesis of cellular iron–sulfur proteins (Tokumoto and Takahashi, 2001). The PCR fragment was obtained with I-SceI-lox71-cat-lox66 as the marker M1, in the scheme presented in Fig. 1, with the addition of the 36 bp upstream of the isc operon to its 5′ ends. Analogously, I-SceIλattL-tetR-tetA-λattR, based on a PCR amplicon, was inserted downstream of the isc operon as the marker M2. Generally, to insert the I-SceI-carrier markers into the desired locus of the chromosome, it is necessary to use locus-specific oligonucleotide primers to obtain the PCR-fragments for λRed integration. However, to make the cloning system universal and user-friendly, we propose to use universal primers that are homologous to the “scar” sequence of single-gene knockout mutants of E. coli K-12 from the collection of Keio University (Baba et al., 2006). In every strain from this collection, an individual coding region has been replaced with a KmR cassette flanked by specific sequences containing FRT sites that are subject to site-specific recombination mediated by the Flp recombinase of S. cerevisiae. After the Flp-driven removal of the marker from the chromosome of the “Keio”

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Fig. 2. The two-step construction of DNA fragments containing “excisable” selection markers. A: Construction of linear DNA fragments containing “excisable” selection markers adjacent to the I-SceI restriction site. B: Construction of DNA fragments for the λRed-driven insertion of the I-SceI recognition sites into any chromosomal region of interest. Regions homologous to the “scar” sequence (see text) are designated by black lines. The lox71 and lox66 sites are designated by horizontally and vertically shaded boxes, respectively. The λattL and λattR sites are designated by horizontally and vertically shaded circles, respectively.

collection strain, a “scar” sequence (105 bp), including the FRT site (34 bp), remains in genome. This “scar” sequence is the same for all of the deleted open-reading frames. In our experiments, this sequence was used as a target for the integration of the I-SceI restriction site. For example, for the cloning of the isc operon, the I-SceI-carrier markers (M1 and M2) were introduced into the ΔtrmJ::FRT-KmR-FRT and ΔpepB::FRT-KmR-FRT loci of the E. coli chromosome, respectively, with the help of universal primers targeting the “upper” and “lower” parts of the “scar” sequence. Thus, using universal primers 9 and 10, the fragment designated as “scar”-I-SceI-lox71-cat-lox66-“scar” (1114 bp) was amplified by PCR from the I-SceI-lox71-cat-lox66 fragment (see Section 3.2) (Fig. 2B). This fragment was introduced as the M1 marker into BW25113 ΔtrmJ::Km, which was pre-transformed with pKD46 according to the standard λRed-driven recombineering approach (Datsenko and Wanner, 2000); CmR-recombinants containing the ΔtrmJ::I-SceI-lox71-cat-lox66 insertion were selected (Fig. 3). Similarly, the I-SceI-λattL-tetR-tetA-λattR fragment (see Section 3.2 and Fig. 2B) was inserted into the “scar” sequence of BW25113 ΔpepB::Km/pKD46 as the M2 marker (Fig. 3). All of the insertions were verified by PCR. Then, both of the modifications were combined into one genome using P1 phage transduction (Fig. 3). In the chromosome of the resulting strain, BW25113-(M1-isc-M2), the chromosomal DNA fragment which included M1 marker and isc operon located downstream, was bracketed by directly repeated I-SceI restriction sites. The chromosomal DNA was purified, digested with I-SceI and, then, M1-isc-containing I-SceI-fragment was inserted in vitro into the specially constructed pAH162-λattL-tetA-tetR-λattR-2Ter-I-SceI digested with the same restriction endonuclease. The resulting plasmid was used as the CRIM vector and transformed into E. coli CC118 (λpir+). CmR selection was used to isolate the recombinant clones of interest. 3.4. Integration of the isc operon into the desired locus After purification from the CC118 (λpir +) strain, the recombinant CRIM plasmid carrying the φ80-attP site could be integrated into native or artificially generated φ80-attB sites in the E. coli genome in the presence of expressed φ80-Int (Haldimann and Wanner, 2001; Minaeva et al., 2008). In the present study, one strain with an artificially

inserted φ80-attB site from the earlier-obtained collection (Minaeva et al., 2008), MG1655 trs5.9::φ80-attB/pAH123, was used as the recipient for the integration of pAH162-λattL-tetA-tetR-λattR-2Ter-I-SceI-isc-cat using TcR as the selective marker. The helper pAH123 plasmid was used for the expression of the φ80-int gene. The efficiency of integration was about 5×10− 2, and the accuracy of the insertion was verified by PCR. According to our strategy, the integration process was mediated by the initially constructed, cloned and purified recombinant CRIM plasmid. It is known (Metcalf et al., 1994) that CRIM plasmids with an R6K γ-replication origin have a moderate (15 per cell) copy number in an appropriate Pir + host. Therefore, insertions that are toxic at moderate copy number may accumulate undesirable mutations at this stage of cloning. To avoid this problem, we used a strategy that omits the step of cloning the desired fragment before its integration into the chromosome. The Pir− strain, MG1655 trs5.9::φ80-attB/pAH123, was transformed with a ligation mixture of the I-SceI-linearized CRIM vector and I-SceIdigested chromosomal DNA of BW25113 ΔtrmJ::I-SceI-lox71-cat-lox66 ΔpepB::I-SceI-λattL-tetR-tetA-λattR, or BW25113-(M1-isc-M2). The subsequently selected CmR clones contained the recombinant CRIM plasmid of interest inserted into the trs5.9::φ80-attB locus of the recipient chromosome (Fig. 4). The insertion site was verified by PCR. As a matter of course, the integration efficiency was lower than that of the purified recombinant CRIM plasmid for the φ80-Int-mediated integration. In this case the integration efficiency was about 1× 10− 3. According to the previously developed strategy, the vector portion of the integrated CRIM plasmid, bracketed by λattL/R-sites, was removed via the λXis/Int recombinase expressed in the presence of the helper plasmid pMWts-λInt/Xis (Minaeva et al., 2008). The resulting strain, MG1655 trs5.9::lox71-cat-lox66-isc, contained two direct repeats of the isc operon, one in its native locus and one in the trs5.9 locus; the distance between these copies was about 10 min of the E. coli genetic map (Fig. 4). The essential genes in this region ensure the stability of the recombinant genome structure: cells in which homologous recombination between the isc operons leads to the deletion of the DNA fragment between the isc copies are inviable. The integrated isc operon was marked by the CmR gene to enable its transfer into the desired E. coli strain via to phage P1mediated generalized transduction. In its turn, the CmR marker bracketed

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Fig. 3. The introduction of I-SceI-sites upstream and downstream of the isc operon. The fragment containing the I-SceI-site, together with the lox71-cat-lox66 cassette as a marker, was inserted into the “scar” sequence of BW25113 ΔtrmJ::Km by λRed-mediated integration. The fragment containing the I-SceI-site, together with λattL-tetR-tetA-λattR as a marker, was inserted into the “scar” sequence of BW25113 ΔpepB::Km by λRed-mediated integration. Both modifications were combined into one genome by P1 phage transduction to yield the strain BW25113-(M1-isc-M2). M1 — CmR marker; M2 — TcR marker. The isc operon is schematically represented as an orange arrow. The lox71 and lox66 sites are designated by horizontally and vertically shaded boxes, respectively. The λattL and λattR sites are designated by horizontally and vertically shaded circles, respectively.

Fig. 4. Cloning of the isc operon from E. coli with the adjacent “excisable” marker and their integration into the desired locus of the E. coli chromosome. In the case of the insertion of the pre-selected isc-harboring CRIM plasmid, the step involving the removal of the vector portion of the plasmid is omitted for simplicity.

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by the lox66/lox71 sites could be removed from the chromosome via the expressed Cre recombinase (Albert et al., 1995; Lambert et al., 2007), which would finalize the construction of the marker-less recombinant strain. Therefore, using the example of isc operon integration, we demonstrated the usefulness of the developed method for the insertion of a long specific DNA fragment into a desired locus without pre-amplification by PCR. 4. Conclusion In this work, with the help of widely used methods based on application of bacteriophage recombination systems, a new strategy of E. coli chromosome editing was developed. This approach enables the cloning of long DNA fragments (more than 5 kb) without its PCR amplification, and the fragment of interest may be inserted into any chromosomal locus. The expression of genes in the inserted fragment can be further modified using recombineering procedures (Court et al., 2002; Datsenko and Wanner, 2000; Katashkina et al., 2005; Gulevich et al., 2009). The developed method allows the duplication of any DNA fragment in the E. coli chromosome with the assistance of phage integration/excision systems (Haldimann and Wanner, 2001; Minaeva et al., 2008). This approach may be applied to the ORF deletion collection of E. coli strains (Baba et al., 2006) to enable the cloning and targeted integration of long DNA fragments bracketed by any two genes using universal oligonucleotide primers. Using repeated integrations into different loci followed by integrative vector excision, and the combination of these chromosomal modifications by P1-transduction followed by the elimination of the selective marker, the insert can be multiplied in a step-wise fashion. This may be of particular interest for the construction of industrial strains. In addition, this approach provides an opportunity for the systematic investigation of the structural instability of the bacterial genome, initiated by direct or inverted repeats of different lengths. Certainly, in this case, insertions should be relatively neutral for cell growth and viability. Beyond this, the insertion of I-SceI meganuclease recognition sites using the “empty” CRIM vector, pAH162-λattL-tetA-tetR-λattR-2Ter-I-SceI, allows the generation of controlled double-strand DNA breaks with the help of the meganuclease-encoding gene. Therefore, this tool may be useful for studies of repair mechanisms and for a variety of gene engineering applications, as described elsewhere (Friedberg et al., 2006; Simmons et al., 2009). Additionally, in a bacterial recipient containing genomic insertions of the I-SceI recognition sites, the meganuclease-encoding gene could be used as a counter-selective marker. Thus, the approach developed here is particularly convenient for the design of plasmid-less and marker-less strains for the biotechnology industry, and it could also be used for fundamental studies of E. coli chromosome rearrangements and DNA repair mechanisms. Acknowledgments We thank Dr. N. Minaeva for providing the CRIM vector pAH162λattL-tetA-tetR-λattR-2Ter and the MG1655 trs5.9::φ80-attB strain. We also would like to thank Prof. B.L. Wanner (Dept. of Biological Sciences, Purdue Univ., West Lafayette, Indiana 47907, USA) who kindly provided plasmids pKD46 and pAH123. References Akhverdyan, V.Z., Gak, E.R., Tokmakova, I.L., Stoynova, N.V., Yomantas, Y.A.V., Mashko, S.V., 2011. Application of the bacteriophage Mu-driven system for the integration/ amplification of target genes in the chromosomes of engineered Gram-negative bacteria. Appl. Microbiol. Biotechnol. 91, 857–871. Albert, H., Dale, E.C., Lee, E., Ow, D.W., 1995. Site-specific integration of DNA into wildtype and mutant lox sites placed in the plant genome. Plant J. 7 (4), 649–659.

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