Methods enabling mutational analysis of distinct chromosomal locations, like site-directed mutagenesis, insertion of foreign sequences or in-frame deletions, have become of fast growing interest since complete bacterial genome sequences became available. Various approaches have been described to modify any nucleotide(s) in almost any manner. Some genetic engineering technologies do not rely on the
Over several decades, researchers developed and refined various strategies for genetic engineering that make use of the homologous recombination system. Its natural main functions are restoring collapsed replication forks, repairing damage-induced double-strand breaks and maintaining the integrity of the chromosome (Poteete, 2001).
We want to focus on a technique for recombination-mediated genetic engineering ("recombineering", Copeland et al., 2001). Recombineering requires only minimal
In the early 1990s, the DNA double-strand break and repair recombination pathway proved to be very efficient for recombining incoming linear DNA with homologous DNA in the yeast
In contrast to
One highly applicable RecET-mediated recombination reaction, termed ‘ET-cloning’, combines a homologous recombination reaction between linear DNA fragments and circular target molecules, like BAC episomes (Zhang et al., 1998). After co-transformation of linear and circular DNA molecules, only
1.1. The bacteriophage λ Red recombination system
Besides the mutagenesis pathway described above, Red recombination is one of the most commonly exploited techniques to foster recombination between the bacterial chromosome and linear dsDNA introduced into the cell (Murphy, 1998). The Red recombination system of the bacteriophage λ leads to a precise and rapid approach with greatly enhanced rates of recombination, compared to those found in
Which components make up the λ Red system? The genes of the Red system,
1.2. Use of λ Red recombination for manipulation of bacterial genomes
The basic strategy of the λ Red system is the replacement of a chromosomal sequence with a (e.g., PCR-amplified) selectable antibiotic resistance gene flanked by homology extensions of distinct lengths. For genetic engineering in the
The second very efficient λ Red-mediated recombination approach, involved a low-copy plasmid with λ
One example of a possible refinement of the λ Red procedure promotes high-frequency recombination using ssDNA substrates. It has been discovered that only λ Redβ is absolutely required for ssDNA recombination (Ellis et al., 2001). Neither
These methods offer a technology for studying bacterial gene functions or even for introducing mutations or markers in the chromosomes of eukaryotic cells, e.g., to provide special ‘‘tags’’ in the DNA of living cells (Ellis et al., 2001).
1.2.1. Gene deletion
λ Red recombination has been successfully used for convenient generation of gene deletions in
The first step in generating gene deletions is creating a linear targeting construct which consists usually of a resistance gene (“
In the next step, the PCR product is used to transform bacteria expressing λ Red proteins. Homologous recombination results in insertion of the cassette at the precise position determined by the homology extensions (Fig. 1). Transformants can be selected using their acquired antibiotic resistance. Target regions for site-specific recombinases (Fig. 1, yellow triangles) provide the option for subsequent removal of the resistance cassette (see also 1.2.3).
1.2.2. DNA insertion
In addition to removing DNA from bacterial genomes (1.2.1), λ Red recombination can also be applied to precisely insert any DNA within a genome. This approach has been widely used for analyzing bacterial gene expression via the generation of reporter gene fusions (Gerlach et al., 2007a, Lee et al., 2009, Yamamoto et al., 2009) or epitope tagging (Cho et al., 2006, Lee et al., 2009, Uzzau et al., 2001). In a similar approach, promoter sequences can be inserted or exchanged within the genome (Alper et al., 2005, Wang et al., 2009).
In these cases, the targeting construct includes besides a selectable marker the DNA to be inserted. Using primers with homology extensions, these targeting constructs can be amplified by PCR from sets of template vectors available for different reporter genes (e.g., ß-galactosidase, luciferase, green fluorescent protein (
Depending on the scientific question to be answered, different integration strategies for reporter genes are available. For transcriptional fusions, a promoterless reporter gene is inserted downstream of a promoter of interest. The reporter gene may have optimized translational signals, including an optimized ribosome binding site (RBS) at the optimal distance from the start codon. If such a construct is inserted within an operon, hybrid operons are generated (Gerlach et al., 2007a). We have introduced so-called “start codon fusions,” in which the reporter gene is inserted behind the native RBS and start codon of the gene under study, so that expression is assessed in the native genomic context (Gerlach et al., 2007a, Wille et al., 2012). This gene fusion strategy is closely related to translational fusions. The classical Red recombination protocol enables the easy generation of C-terminal fusion proteins, in which the reporter gene or epitope tag is inserted in-frame at any position in an open-reading-frame (ORF). For the generation of N-terminal fusions, a “scarless“ recombination protocol (see 2.) has to be applied.
1.2.3. Site-specific recombination for removal of antibiotic resistance genes
Several methods, involving various site-specific recombination systems, have been developed for the removal of unwanted marker sequences from the chromosome. The most frequently used site-specific recombinases for subsequent excision of antibiotic resistance genes are Flp and Cre. Flp and Cre recombinases recognize 34-bp long sequences with palindromic elements (
Although there is limited homology between the scar sequences themselves, they might serve as hotspots for recombination in successive recombination steps, representing a risk for unwanted deletions or chromosomal rearrangements (Datsenko et al., 2000). In addition, these scars might have influence on gene functions when operon structures or intragenic regions were modified (Blank et al., 2011). Mutations within the inverted repeats of
2. Site-directed mutagenesis using oligonucleotides
Precise insertion of chromosomal mutations has been established as the “gold standard” for analysis of bacterial gene function. Generation of point mutations, seamless deletions and in-frame gene fusions without leaving selectable markers or a recombination target site (e.g.,
2.1. Counterselection with SacB
Linear targeting constructs harboring
The latter two methods were used to generate gene deletions within the chromosomes of
In the homologous recombination step, clones were selected for the respective antibiotic resistance. Recombinants were selected on medium plates containing 5-7% sucrose to select for loss of the cassette. Exact timing of counterselection is a critical issue when working with SacB or SBn, since
2.2. Dual selection of recombinants with GalK or ThyA
Besides the fusion protein SBn,
2.3. Counterselection using streptomycin resistance
Several mutants of the ribosomal protein S12 (RpsL) were shown to confer streptomycin resistance (SmR, Springer et al., 2001). Strains harboring such an
2.4. Selection with the fusaric acid sensitivity system
A counterselection technique developed by Bochner et al. (1980) enables direct selection of tetracycline sensitive (TcS) clones from a predominantly tetracycline resistant (TcR) bacterial population. The method is based on the hypersensitivity of lipophilic TcR cells to chelating agents, like fusaric acid or quinaldic acids. The precise mechanism of tetracycline exclusion is so far unknown and the subject of much speculation. The hypersensitivity seems to be caused by alterations of the host cell membrane, which are evoked from the expression of the tetracycline resistance gene. These alterations interfere, on one hand, with tetracycline permeation to confer tetracycline resistance, but, on the other hand, also to increase susceptibility to other toxic compounds (Bochner et al., 1980). This effect was exploited by using a medium that was effective for the selection of TcS revertants. The counterselection was successful in
The counterselection of TcR clones on Bochner-Maloy plates was sometimes used as the final step in recombineering protocols. Point mutations were inserted in BACs using a combination of λ
A PCR product carrying the desired mutation was used in the second recombination step to exchange
The selection efficiency of Bochner-Maloy plates was reported not to exceed 50% (Podolsky et al., 1996). Therefore, the selection procedure was not very stringent. Exact timing of all incubation steps was necessary; but still high background might be observed, making purification of positive clones difficult. Highly increased selection efficiencies were obtained with plates containing 5-7 mM NiCl2, which led to 80-100% positive TcS revertants (Podolsky et al., 1996).
2.5. Double-strand breaks introduced by I-
SceI can be used to select recombinants
The endonuclease I-
Several methods for site-directed mutagenesis of BACs and/or bacterial genomes utilizing I-
For mutagenesis of the genomes of
Because no specific mechanisms were implemented in pGETrec3.1 and pBC-I-SceI to promote convenient plasmid curing, it might be difficult to get plasmid-free host strains after site-directed mutagenesis. We solved that problem by integrating a tetracycline-inducible I-
The Red recombination system can anneal single-stranded DNA derived from dsDNA substrates into replicating homologous target sequences (1.1). Usually homologous sequences for recombination are supplied with the homology extensions flanking the targeting construct. In contrast, homologous regions flanking a DSB generated by I-
One major problem of the I-
2.6. Screening methods for recombinants
An underestimated problem is the screening effort needed to identify correct recombinants when using seamless recombination techniques. Although PCR fragment length polymorphism can be used in case of deletions and insert-specific PCRs in case of DNA insertion, successful single nucleotide exchanges are hard to detect. Direct phenotypical screening or the parallel introduction of novel restriction sites together with the nucleotide exchange are solutions of the problem.
2.6.1. Introduction of silent mutations to generate novel restriction sites
A screening problem arises if mutations introduced via recombineering have no direct or indirect impact on the phenotype or if the phenotypic test required is very time-consuming. Introduction of a novel restriction site adjacent to the mutation was proven to be very useful for colony screening. Designing the oligonucleotides for λ Red recombination offers the prospect of introducing silent mutations in the target region. Identification of silent mutations generating novel restriction sites can be done
2.6.2. Phenotypical screening
If available, phenotypical screening is the fastest way for selecting recombinants with the desired mutation. The screening is based on phenotypic differences between the mutant and the wt. In the simplest case, activity of an integrated reporter gene like
Two successive recombination steps catalyzed by the phage λ Red or phage Rac RecE/T recombination systems in combination with a negative selection procedure provide a venue for scarless mutagenesis within bacterial genomes and BACs. The outstanding ability of these enzymes to use homologous sequences as short as 35 bp as substrates for recombination allows the use of linear DNA derived from synthetic oligonucleotides as targeting constructs. The limiting step of this rationale is the availability of a reliable counterselection method. Here we gave an overview about recombination and the counterselection techniques successfully applied to the manipulation of bacterial genomes, as well as BACs.
Alper H Fischer C Nevoigt E Stephanopoulos G 2005 Tuning genetic control through promoter engineering
Alper M. D Ames B. N 1975 Positive selection of mutants with deletions of the gal-chl region of the Salmonella chromosome as a screening procedure for mutagens that cause deletions.
and efficient method for direct gene deletion in Baudin A Ozier-kalogeropoulos O Denouel A Lacroute F Cullin C 1993 A Simple
Blank K Hensel M Gerlach R. G 2011 Rapid and Highly Efficient Method for Scarless Mutagenesis within the Salmonella enterica Chromosome
Blomfield I. C Vaughn V Rest R. F Eisenstein B. I 1991 Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon.
Bochner B. R Huang H. C Schieven G. L Ames B. N 1980 Positive selection for loss of tetracycline resistance.
Cho B. K Knight E. M Palsson B. O 2006 PCR-based tandem epitope tagging system for Escherichia coli genome engineering.
Clark A. J Satin L Chu C. C 1994 Transcription of the Escherichia coli recE gene from a promoter in Tn5 and IS50.
Copeland N. G Jenkins N. A Court D. L 2001 Recombineering: a powerful new tool for mouse functional genomics
Cox M. M Layton S. L Jiang T Cole K Hargis B. M Berghman L. R Bottje W. G Kwon Y. M 2007 Scarless and site-directed mutagenesis in Salmonella enteritidis chromosome.
Datsenko K. A Wanner B. L 2000 One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products
and simple method for inactivating chromosomal genes in Derbise A Lesic B Dacheux D Ghigo J. M Carniel E 2003 A Rapid
DiTizio, T. & Court, D.L. ( Ellis H. M Yu D 2001 High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides
Fouts K. E Wasie-gilbert T Willis D. K Clark A. J Barbour S. D 1983 Genetic analysis of transposon-induced mutations of the Rac prophage in Escherichia coli K-12 which affect expression and function of recE.
Le Coq, D., Steinmetz, M., Berkelman, T. & Kado, C.I. ( Gay P 1985 Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria.
Gerlach R. G Hölzer S. U Jäckel D Hensel M 2007a Rapid engineering of bacterial reporter gene fusions by using red recombination.
Gerlach R. G Jäckel D Hölzer S. U Hensel M 2009 Rapid oligonucleotide-based recombineering of the chromosome of Salmonella enterica.
Gerlach R. G Jäckel D Stecher B Wagner C Lupas A Hardt W. D Hensel M 2007b Salmonella Pathogenicity Island 4 encodes a giant non-fimbrial adhesin and the cognate type 1 secretion system.
genetic manipulation of antibiotic-producing Streptomyces. Gust B Chandra G Jakimowicz D Yuqing T Bruton C. J Chater K. F 2004 L Red-mediated
Hansen-wester I Hensel M 2002 Genome-based identification of chromosomal regions specific for Salmonella spp.
Heermann R Zeppenfeld T Jung K 2008 Simple generation of site-directed point mutations in the Escherichia coli chromosome using Red®/ET® Recombination.
Jamsai D Orford M Nefedov M Fucharoen S Williamson R Ioannou P. A 2003Targeted modification of a human β-globin locus BAC clone using
Jasin M Schimmel P 1984 Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments.
Kang Y Durfee T Glasner J. D Qiu Y Frisch D Winterberg K. M Blattner F. R 2004 Systematic mutagenesis of the Escherichia coli genome.
range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA Kaniga K Delor I Cornelis G. R 1991 A Wide-host
Karakousis G Ye N Li Z Chiu S. K Reddy G Radding C. M 1998The beta protein of phage L binds preferentially to an intermediate in DNA renaturation.
Karlinsey J. E 2007L-Red genetic engineering in
0036-8075 5333 277 1824 1827 Kovall, R. & Matthews, B.W. (1997). Toroidal structure of λ-exonuclease. Science, Vol.277, No.5333, pp. 1824-1827, ISSN 0036-8075
Lambert J. M Bongers R. S Kleerebezem M 2007 Cre-lox-based system for multiple gene deletions and selectable-marker removal in Lactobacillus plantarum.
Lederberg J 1951 Streptomycin resistance: a genetically recessive mutation
Lee D. J Bingle L. E Heurlier K Pallen M. J Penn C. W Busby S. J Hobman J. L 2009 Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains
Martinez de Velasco, J., Tessarollo, L., Swing, D. Lee E. C Yu D A., Court, D.L., Jenkins, N.A. & Copeland, N.G. (for recombinogenic targeting and subcloning of BAC DNA. 2001A highly efficient Escherichia coli-based chromosome engineering system adapted
Li Z Karakousis G Chiu S. K Reddy G Radding C. M 1998The beta protein of phage L promotes strand exchange.
Liang R Liu J 2010 Scarless and sequential gene modification in Pseudomonas using PCR product flanked by short homology regions
Little J. W 1967An exonuclease induced by bacteriophage λ. II. Nature of the enzymatic reaction.
Lorenz M. G Wackernagel W 1994 Bacterial gene transfer by natural genetic transformation in the environment.
Maloy S. R Nunn W. D 1981 Selection for loss of tetracycline resistance by Escherichia coli.
Maresca M Erler A Fu J Friedrich A Zhang Y Stewart A. F 2010Single-stranded heteroduplex intermediates in λ Red homologous recombination.
Monteilhet C Perrin A Thierry A Colleaux L Dujon B 1990 Purification and characterization of the in vitro activity of I-Sce I, a novel and highly specific endonuclease encoded by a group I intron.
Murphy K. C 1991Gam protein inhibits the helicase and χ-stimulated recombination activities of
Murphy K. C 1998Use of bacteriophage λ recombination functions to promote gene replacement in
Muyrers J. P Zhang Y Benes V Testa G Ansorge W Stewart A. F 2000a Point mutation of bacterial artificial chromosomes by ET recombination
α/Redβ initiate double-stranded break repair by specifically interacting with their respective partners. Genes Dev, Vol.14, No.15, pp. 1971-1982, ISSN 0890-9369 ., Muyrers, J.P ., Zhang, Y . & Buchholz, F . (2000 Stewart, A.F E/RecT and Red b). Rec
Nefedov M Williamson R Ioannou P. A 2000 Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli.
Passy S. I Yu X Li Z Radding C. M Egelman E. H 1999Rings and filaments of β protein from bacteriophage L suggest a superfamily of recombination proteins.
Pelicic V Reyrat J. M Gicquel B 1996 Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria.
Podolsky T Fong S. T Lee B. T 1996 Direct selection of tetracycline-sensitive Escherichia coli cells using nickel salts.
Pósfai G Kolisnychenko V Bereczki Z Blattner F. R 1999 Markerless gene replacement in Escherichia coli stimulated by a double-strand break in the chromosome.
Poteete A. R 2001What makes the bacteriophage λ Red system useful for genetic engineering: molecular mechanism and biological function.
Quénée L Lamotte D Polack B 2005 Combined sacB-based negative selection and cre-lox antibiotic marker recycling for efficient gene deletion in Pseudomonas aeruginosa.
Ranallo R. T Barnoy S Thakkar S Urick T Venkatesan M. M 2006Developing live
Reyrat J. M Pelicic V Gicquel B Rappuoli R 1998 Counterselectable markers: untapped tools for bacterial genetics and pathogenesis
Sawitzke J Austin S 2001 An analysis of the factory model for chromosome replication and segregation in bacteria.
Schweizer H. P 2003Applications of the
Semsey S Krishna S Sneppen K Adhya S 2007Signal integration in the galactose network of
Springer B Kidan Y. G Prammananan T Ellrott K Böttger E. C Sander P 2001Mechanisms of streptomycin resistance: selection of mutations in the 16S rRNA gene conferring resistance.
rd ( Sun W Wang S Curtiss R 2008Highly efficient method for introducing successive multiple scarless gene deletions and markerless gene insertions into the
Swaminathan S Ellis H. M Waters L. S Yu D Lee E. C Court D. L Sharan S. K 2001Rapid engineering of bacterial artificial chromosomes using oligonucleotides.
Tischer B. K Smith G. A Osterrieder N 2010
Tischer B. K Von Einem J Kaufer B Osterrieder N 2006Two-step red-mediated recombination for versatile high-efficiency markerless DNA manipulation in
Uzzau S Figueroa-bossi N Rubino S Bossi L 2001Epitope tagging of chromosomal genes in
Wang H. H Isaacs F. J Carr P. A Sun Z. Z Xu G Forest C. R Church G. M 2009Programming cells by multiplex genome engineering and accelerated evolution.
Wang J Chen R Julin D. A 2000A single nuclease active site of the
Warming S Costantino N Court D. L Jenkins N. A Copeland N. G 2005Simple and highly efficient BAC recombineering using
Wille T Blank K Schmidt C Vogt V Gerlach R. G 2012
Wong Q. N Ng V. C Lin M. C Kung H. F Chan D Huang J. D 2005Efficient and seamless DNA recombineering using a thymidylate synthase A selection system in
Yamamoto S Izumiya H Morita M Arakawa E Watanabe H 2009Application of λ Red recombination system to
Yu B. J Kang K. H Lee J. H Sung B. H Kim M. S Kim S. C 2008Rapid and efficient construction of markerless deletions in the
Yu D Ellis H. M Lee E. C Jenkins N. A Copeland N. G Court D. L 2000An efficient recombination system for chromosome engineering in
Zhang Y Buchholz F Muyrers J. P Stewart A. F 1998A new logic for DNA engineering using recombination in
Zhang Y Muyrers J. P Testa G Stewart A. F 2000DNA cloning by homologous recombination in
Zhang Z Lutz B 2002Cre recombinase-mediated inversion using lox66 and lox71: method to introduce conditional point mutations into the CREB-binding protein.