1. Introduction
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
The
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,
The
A
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-Sce I 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
3. Conclusion
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.
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