Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair.

Recently, it was shown that Agrobacterium tumefaciens can transfer transferred DNA (T-DNA) to Saccharomyces cerevisiae and that this T-DNA, when used as a replacement vector, is integrated via homologous recombination into the yeast genome. To test whether T-DNA can be a suitable substrate for integration via the gap repair mechanism as well, a model system developed for detection of homologous recombination events in plants was transferred to S. cerevisiae. Analysis of the yeast transformants revealed that an insertion type T-DNA vector can indeed be integrated via gap repair. Interestingly, the transformation frequency and the type of recombination events turned out to depend strongly on the orientation of the insert between the borders in such an insertion type T-DNA vector.     

In vivo cloning of PCR products in E. coli.

This report describes an efficient method to clone PCR products exploiting endogenous Escherichia coli enzymatic activities. PCR products are engineered to contain terminal sequences identical to sequences at the two ends of a linearized vector. PCR products and vector DNA are then simply co-transfected into E. coli strain JC8679, obviating the requirement for enzymatic treatment of the PCR product or in vitro ligation. The high rate of homologous recombination in this strain results in efficient incorporation of the insert into the vector, a process we refer to as in vivo cloning (IVC).     

Retroviral vectors for homologous recombination provide efficient cloning and expression in mammalian cells

Homologous recombination technologies enable high-throughput cloning and the seamless insertion of any DNA fragment into expression vectors. Additionally, retroviral vectors offer a fast and efficient method for transducing and expressing genes in mammalian cells, including lymphocytes. However, homologous recombination cannot be used to insert DNA fragments into retroviral vectors; retroviral vectors contain two homologous regions, the 5′- and 3′-long terminal repeats, between which homologous recombination occurs preferentially. In this study, we have modified a retroviral vector to enable the cloning of DNA fragments through homologous recombination. To this end, we inserted a bacterial selection marker in a region adjacent to the gene insertion site. We used the modified retroviral vector and homologous recombination to clone T-cell receptors (TCRs) from single Epstein Barr virus-specific human T cells in a high-throughput and comprehensive manner and to efficiently evaluate their function by transducing the TCRs into a murine T-cell line through retroviral infection. In conclusion, the modified retroviral vectors, in combination with the homologous recombination method, are powerful tools for the high-throughput cloning of cDNAs and their efficient functional analysis.     

Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast.

We constructed representative large-insert bacterial artificial chromosome (BAC) libraries of two human pathogens (Trypanosoma brucei and Giardia lamblia) using a new hybrid vector, pTARBAC1, containing a yeast artificial chromosome (YAC) cassette (a yeast selectable marker and a centromere). The cassette allows transferring of BACs into yeast for their further modification. Furthermore, the new hybrid vector provides the opportunity to re-isolate each DNA insert without construction of a new library of random clones. Digestion of a BAC DNA by an endonuclease that has no recognition site in the vector, but which deletes most of the internal insert sequence and leaves the unique flanking sequences, converts a BAC into a TAR vector, thus allowing direct gene isolation. Cotransformation of a TAR vector and genomic DNA into yeast spheroplasts, and subsequent recombination between the TAR vector's flanking ends and a specific genomic fragment, allows rescue of the fragment as a circular YAC/BAC molecule. Here we prove a new cloning strategy by re-isolation of randomly chosen genomic fragments of different size from T. brucei cloned in BACs. We conclude that genomic regions of unicellular eukaryotes can be easily re-isolated using this technique, which provides an opportunity to study evolution of these genomes and the role of genome instability in pathogenicity.     

Yeast artificial chromosomes (YACs) and the analysis of complex genomes.

The development of yeast artificial chromosome (YAC) cloning vectors capable of carrying several hundred kilobase-pairs of DNA insert has greatly facilitated the study of complex genomes, and the cloning of large genes as single fragments. In addition, the ability to manipulate YAC sequences by homologous recombination makes this system extremely useful for the generation of disease models.     

Phenotypic expression of PCR-generated random mutations in a Pseudomonas putida gene after its introduction into an Acinetobacter chromosome by natural transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37 degrees C. Of recombinant clones that failed to express xylE at 37 degrees C, about 10% expressed the gene when grown at 22 degrees C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

Direct Cloning of Yeast Genes from an Ordered Set of Lambda Clones in Saccharomyces Cerevisiae by Recombination in Vivo

We describe a technique that facilitates the isolation of yeast genes that are difficult to clone. This technique utilizes a plasmid vector that rescues lambda clones as yeast centromere plasmids. The source of these lambda clones is a set of clones whose location in the yeast genome has been determined by L. Riles et al. in 1993. The Esherichia coli-yeast shuttle plasmid carries URA3, ARS4 and CEN6, and contains DNA fragments from the lambda vector that flank the cloned yeast insert. When yeast is cotransformed with linearized plasmid and lambda clone DNA, Ura(+) transformants are obtained by a recombination event between the lambda clone and the plasmid vector that generates an autonomously replicating plasmid containing the cloned yeast DNA sequences. Genes whose genetic map positions are known can easily be identified and recovered in this plasmid by testing only those lambda clones that map to the relevant region of the yeast genome for their ability to complement the mutant phenotype. This technique facilitates the isolation of yeast genes that resist cloning either because (1) they are underrepresented in yeast genomic libraries amplified in E. coli, (2) they provide phenotypes that are too marginal to allow selection of the gene by genetic complementation or (3) they provide phenotypes that are laborious to score. We demonstrate the utility of this technique by isolating three genes, GAL83, SSN2 and MAK7, each of which presents one of these problems for cloning.     

Selective isolation of genomic loci from complex genomes by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae

Here, we describe a protocol for the selective isolation of any genomic fragment or gene of interest up to 250 kb in size from complex genomes as a circular yeast artificial chromosome (YAC). The method is based on transformation-associated recombination (TAR) in the yeast Saccharomyces cerevisiae between genomic DNA and a linearized TAR cloning vector containing targeting sequences homologous to a region of interest. Recombination between the vector and homologous sequences in the co-transformed mammalian DNA results in the establishment of a YAC that is able to propagate, segregate and be selected for in yeast. Yield of gene-positive clones varies from 1% to 5%. The entire procedure takes 2 weeks to complete once the TAR vector is constructed and genomic DNA is prepared. The TAR cloning method has a broad application in functional and comparative genomics, long-range haplotyping and characterization of chromosomal rearrangements, including copy number variations.     

An inducible recA expression Bacillus subtilis genome vector for stable manipulation of large DNA fragments

Background

The Bacillus subtilis genome (BGM) vector is a novel cloning system based on the natural competence that enables B. subtilis to import extracellular DNA fragments into the cell and incorporate the recombinogenic DNA into the genome vector by homologous recombination. The BGM vector system has several attractive properties, such as a megabase cloning capacity, stable propagation of cloned DNA inserts, and various modification strategies using RecA-mediated homologous recombination. However, the endogenous RecA activity may cause undesirable recombination, as has been observed in yeast artificial chromosome systems. In this study, we developed a novel BGM vector system of an inducible recA expression BGM vector (iREX), in which the expression of recA can be controlled by xylose in the medium.

Results

We constructed the iREX system by introducing the xylose-inducible recA expression cassette followed by the targeted deletion of the endogenous recA. Western blot analysis showed that the expression of recA was strictly controlled by xylose in the medium. In the absence of xylose, recA was not expressed in the iREX, and the RecA-mediated recombination reactions were greatly suppressed. By contrast, the addition of xylose successfully induced RecA expression, which enabled the iREX to exploit the same capacities of transformation and gene modifications observed with the conventional BGM vector. In addition, an evaluation of the stability of the cloned DNA insert demonstrated that the DNA fragments containing homologous sequences were more stably maintained in the iREX by suppressing undesirable homologous recombination.

Conclusions

We developed a novel BGM vector with inducible recA expression system, iREX, which enables us to manipulate large DNA fragments more stably than the conventional BGM vector by suppressing undesirable recombination. In addition, we demonstrate that the iREX can be applied to handling the DNA, which has several homologous sequences, such as multiple-reporter expression cassettes. Thus, the iREX expands the utility of the BGM vector as a platform for engineering large DNA fragments.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1425-4) contains supplementary material, which is available to authorized users.     

Transfer of chimeric plasmids among Salmonella typhimurium strains by P22 transduction

Salmonella typhimurium bacteriophage P22 transduced plasmids having P22 sequences inserted in the vector pBR322 with high frequency. Analysis of the structure of the transducing particle DNA and the transduced plasmids indicates that this plasmid transduction involves two homologous recombination events. In the donor cell, a single recombination between the phage and the homologous sequences on the plasmid inserted the plasmid into the phage chromosome, which was then packaged by headfuls into P22 particles. The transducing particle DNA contained duplications of the region of homology flanking the integrated plasmid vector sequences and lacked some phage genes. When these defective phage genomes containing the inserted plasmid infected a recipient cell, recombination between the duplicated regions regenerated the plasmid. A useful consequence of this sequence of events was that genetic markers in the region of homology were readily transferred from phage to plasmid. Plasmid transduction required homology between the phage and the plasmid, but did not depend on the presence of any specific P22 sequence in the plasmid. When the infecting P22 carried a DNA sequence homologous to the ampicillin resistance region of pBR322, the vector plasmid having no P22 insert could be transduced. P22-mediated transduction is a useful way to transfer chimeric plasmids, since most S. typhimurium strains are poorly transformed by plasmid DNA.     

Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC

We describe a new cloning method, sequence and ligation-independent cloning (SLIC), which allows the assembly of multiple DNA fragments in a single reaction using in vitro homologous recombination and single-strand annealing. SLIC mimics in vivo homologous recombination by relying on exonuclease-generated ssDNA overhangs in insert and vector fragments, and the assembly of these fragments by recombination in vitro. SLIC inserts can also be prepared by incomplete PCR (iPCR) or mixed PCR. SLIC allows efficient and reproducible assembly of recombinant DNA with as many as 5 and 10 fragments simultaneously. SLIC circumvents the sequence requirements of traditional methods and functions much more efficiently at very low DNA concentrations when combined with RecA to catalyze homologous recombination. This flexibility allows much greater versatility in the generation of recombinant DNA for the purposes of synthetic biology.     

Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

PCR fragments and linear vectors containing overlapping ends are easily assembled into a propagative plasmid by homologous recombination in Escherichia coli. Although this gap-repair cloning approach is straightforward, its existence is virtually unknown to most molecular biologists. To popularize this method, we tested critical parameters influencing the efficiency of PCR fragments cloning into PCR-amplified vectors by homologous recombination in the widely used E. coli strain DH5α. We found that the number of positive colonies after transformation increases with the length of overlap between the PCR fragment and linear vector. For most practical purposes, a 20 bp identity already ensures high-cloning yields. With an insert to vector ratio of 2:1, higher colony forming numbers are obtained when the amount of vector is in the range of 100 to 250 ng. An undesirable cloning background of empty vectors can be minimized during vector PCR amplification by applying a reduced amount of plasmid template or by using primers in which the 5′ termini are separated by a large gap. DpnI digestion of the plasmid template after PCR is also effective to decrease the background of negative colonies. We tested these optimized cloning parameters during the assembly of five independent DNA constructs and obtained 94% positive clones out of 100 colonies probed. We further demonstrated the efficient and simultaneous cloning of two PCR fragments into a vector. These results support the idea that homologous recombination in E. coli might be one of the most effective methods for cloning one or two PCR fragments. For its simplicity and high efficiency, we believe that recombinational cloning in E. coli has a great potential to become a routine procedure in most molecular biology-oriented laboratories.     

Targeted alterations in yeast artificial chromosomes for inter-species gene transfer.

In order to facilitate alterations of large DNA molecules for their introduction into mammalian cells we have characterised the mechanism of site-specific modifications in yeast artificial chromosomes (YACs). Newly developed yeast integration vectors with dominant selectable marker genes allow targeted integration into left (centromeric) and right (non-centromeric) YAC arms as well as alterations to the human derived insert DNA. In transformation experiments, integration proceeds exclusively by homologous recombination although yeast prefers linear ends of homology for predefined insertions. Targeted regions can be rescued which expedite the cloning of internal human sequences and the identification of 5' and 3' YAC/insert borders. Integration of the neomycin resistance gene into various parts of the YAC allowed the transfer and stable integration of large DNA molecules into a variety of mammalian cells including embryonic stem cells.     

Transfer of P1 inserts into a yeast-bacteria shuttle vector by co-transformation mediated homologous recombination.

Manipulation of genomic inserts cloned into the bacteriophage P1 vector is hindered by the large size of the inserts. We have used co-transformation mediated recombination between the yeast-bacteria shuttle vector, pClasper, and various P1 clones to transfer the entire insert from the P1 into pClasper. This results in the insert being stably maintained in yeast, facilitating mutagenesis by homologous recombination. The recombinant plasmid can subsequently be transferred to and stably maintained in bacteria for efficient plasmid preparation. This method can also be applied to inserts from P1 artificial chromosome or bacterial artificial chromosome vectors.     

Defining the minimal length of sequence homology required for selective gene isolation by TAR cloning

The transformation-associated recombination (TAR) cloning technique allows selective and accurate isolation of chromosomal regions and genes from complex genomes. The technique is based on in vivo recombination between genomic DNA and a linearized vector containing homologous sequences, or hooks, to the gene of interest. The recombination occurs during transformation of yeast spheroplasts that results in the generation of a yeast artificial chromosome (YAC) containing the gene of interest. To further enhance and refine the TAR cloning technology, we determined the minimal size of a specific hook required for gene isolation utilizing the Tg.AC mouse transgene as a targeted region. For this purpose a set of vectors containing a B1 repeat hook and a Tg.AC-specific hook of variable sizes (from 20 to 800 bp) was constructed and checked for efficiency of transgene isolation by a radial TAR cloning. When vectors with a specific hook that was ≥60 bp were utilized, ~2% of transformants contained circular YACs with the Tg.AC transgene sequences. Efficiency of cloning dramatically decreased when the TAR vector contained a hook of 40 bp or less. Thus, the minimal length of a unique sequence required for gene isolation by TAR is ~60 bp. No transgene-positive YAC clones were detected when an ARS element was incorporated into a vector, demonstrating that the absence of a yeast origin of replication in a vector is a prerequisite for efficient gene isolation by TAR cloning.     

Phenotypic Expression of PCR-Generated Random Mutations in a Pseudomonas putida Gene after Its Introduction into an Acinetobacter Chromosome by Natural Transformation

Localized sets of random point mutations generated by PCR amplification can be transferred efficiently to the chromosome of Acinetobacter ADP1 (also known as strain BD413) by natural transformation. The technique does not require cloning of PCR fragments in plasmids: PCR-amplified DNA fragments are internalized by cells and directly incorporated into their genomes by homologous recombination. Previously such procedures for random mutagenesis could be applied only to Acinetobacter genes affording the selection of mutant phenotypes. Here we describe the construction of a vector and recipient that allow for mutagenesis, recovery, and expression of heterologous genes that may lack a positive selection. The plasmid carries an Acinetobacter chromosomal segment interrupted by a multiple cloning site next to a kanamycin resistance marker. The insertion of heterologous DNA into the multiple cloning site prepares the insert as a target for PCR mutagenesis. PCR amplifies the kanamycin resistance marker and a flanking region of Acinetobacter DNA along with the insert of heterologous DNA. Nucleotide sequence identity between the flanking regions and corresponding chromosomal segments in an engineered Acinetobacter recipient allows homologous recombination of the PCR-amplified DNA fragments into a specific chromosomal docking site from which they can be expressed. The recipient strain contains only a portion of the kanamycin resistance gene, so donor DNA containing both this gene and the mutagenized insert can be selected by demanding growth of recombinants in the presence of kanamycin. The effectiveness of the technique was demonstrated with the relatively GC-rich Pseudomonas putida xylE gene. After only one round of PCR amplification (35 cycles), donor DNA produced transformants of which up to 30% carried a defective xylE gene after growth at 37°C. Of recombinant clones that failed to express xylE at 37°C, about 10% expressed the gene when grown at 22°C. The techniques described here could be adapted to prepare colonies with an altered function in any gene for which either a selection or a suitable phenotypic screen exists.     

Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes

We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.     

Recovery and potential utility of YACs as circular YACs/BACs

A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.     

Recovery and potential utility of YACs as circular YACs/BACs

A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.     

Optimum conditions for selective isolation of genes from complex genomes by transformation-associated recombination cloning

Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library. The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5′ and 3′ gene-specific targeting hooks. Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases ~10-fold when the genomic DNA is sheared to 100–200 kb before being presented to the spheroplasts. Here we determine the most effective concentration of genomic DNA, and also show that the targeted sequences recombine much more efficiently with the vector’s targeting hooks when they are located at the ends of the genomic DNA fragment. We demonstrate that the yield of gene-positive clones increases ~20-fold after endonuclease digestion of genomic DNA, which caused double strand breaks near the targeted sequences. These findings have led to a greatly improved protocol.