A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae

In the post-genomic era, academic and biotechnological research is increasingly shifting its attention from single proteins to the analysis of complex protein networks. This change in experimental design requires the use of simple and experimentally tractable organisms, such as the unicellular eukaryote Saccharomyces cerevisiae, and a range of new high-throughput techniques. The Gateway system has emerged as a powerful high-throughput cloning method that allows for the in vitro recombination of DNA with high speed, accuracy and reliability. Two Gateway-based libraries of overexpression plasmids containing the entire complement of yeast open reading frames (ORFs) have recently been completed. In order to make use of these powerful resources, we adapted the widely used pRS series of yeast shuttle vectors for use in Gateway-based cloning. The resulting suite of 288 yeast Gateway vectors is based upon the two commonly used GPD and GAL1 promoter expression systems that enable expression of ORFs, either constitutively or under galactose-inducible conditions. In addition, proteins of interest can be fused to a choice of frequently used N- or C-terminal tags, such as EGFP, ECFP, EYFP, Cerulean, monomeric DsRed, HA or TAP. We have made this yeast Gateway vector kit available to the research community via the non-profit Addgene Plasmid Repository (http://www.addgene.org/yeast_gateway).     

New tools for phenotypic analysis in Candida albicans: the WAR1 gene confers resistance to sorbate

Availability of the complete sequence of the Candida albicans genome allows for global gene analysis. We designed a gene deletion method to facilitate such studies. First, we constructed C. albicans strains that are both Deltaura3 and Deltatrp1. Second, we designed a system that relies on in vitro recombination, using the Gateway((R)) technology, for efficient generation of deletion cassettes. They are generated in two steps: (a) upstream and downstream DNA fragments of the chromosomal region to be deleted are amplified by PCR and introduced into two separate entry vectors; (b) the second step involves a quadruple recombination event including the two entry vectors, a plasmid bearing a marker of interest and a destination vector, in order to generate a plasmid containing the deletion cassette. The deletion plasmid contains very rare restriction sites for convenient excision of the knockout cassette. Selection in C. albicans can be performed with one of the following markers: the C. albicans URA3 gene, a modified S. cerevisiae TRP1 gene or the mycophenolic acid resistance (MPA(R)) gene. Upon integration into the genome, these markers can be removed by the use of 5-fluoroorotic acid (URA3), 5-fluoroanthranilic acid (TRP1) or the FLP recombinase (MPA(R)). Using this approach, we show that removal of the C. albicans orf19.1035 gene results in sensitivity to the weak acid sorbate, while its overexpression increases resistance to this compound. We named it WAR1, in analogy to its S. cerevisiae orthologue.     

In vivo cloning by homologous recombination in yeast using a two-plasmid-based system

In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient ‘recombination in vivo’ technique. Two sets of vectors were developed. The first set, called ‘expression vectors’, contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a ‘transfer vector’ which is compatible with the second set of E. coli–yeast shuttle vectors. This set of ‘recombination vectors’ contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid-bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3-d as the selective marker together with an ARS/CEN and the 2 μm replicon. Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single-stranded DNA in E. coli for sequencing or site-directed mutagenesis. The sequence presented (Figure 1a) has been entered in the EMBL data library under Accession Number Z48747.     

A vector set for systematic metabolic engineering in Saccharomyces cerevisiae

A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 μ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering.     

Yeast vectors for the integration/expression of any sequence at the TYR1 locus

We have constructed new yeast vectors for targeted integration and conditional expression of any sequence at the Saccharomyces cerevisiae TYR1 locus which becomes disrupted. We show that vector integration is not neutral, causing prototrophy for tyrosine and auxotrophy for the vector's selectable marker (uracil or leucine, depending on the vector used). This feature allows a double screening of transformed yeast cells, improving the identification of colonies with the desired chromosomal structure. The GAL10 gene promoter has been added to drive conditional expression of cloned sequences. Using these vectors, chromosomal structure verification of recombinant clones is no longer necessary, since the noise of non-homologous recombination, as well as spontaneous reversion of the selected phenotype, can easily be identified. The ability of the vector to conditionally control gene expression has been confirmed using the gene for the green fluorescent protein (GFP) as a reporter.     

Development of series of gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation

We developed a new series of binary vectors useful for Gateway cloning to facilitate transgenic experiments in plant biotechnology. The new system, Gateway Binary Vectors (pGWBs) realized efficient cloning, constitutive expression using the cauliflower mosaic virus (CaMV) 35S promoter and the construction of fusion genes by simple clonase reaction with an entry clone. The reporters employable in this system are beta-glucuronidase (GUS), synthetic green fluorescent protein with S65T mutation (sGFP), luciferase (LUC), enhanced yellow fluorescent protein (EYFP), and enhanced cyan fluorescent protein (ECFP). The tags available are 6xHis, FLAG, 3xHA, 4xMyc, 10xMyc, GST, T7-epitope, and tandem affinity purification (TAP). In total, 13 kinds of reporter or tag were arranged and were almost applicable to both N- and C-fusions. The pGWBs could be used for many purposes, such as promoter::reporter analysis, observation of subcellular localization by the expression of proteins fused to a reporter or tag, and analysis of protein-protein interaction by copurification and immunodetection experiments. The pGWBs were constructed with modified pBI101 containing a CaMV35S promoter-driven hygromycin phosphotransferase (HPT) gene as the second selection marker. We also constructed pGWBs with the marker HPT driven by the nopaline synthase promoter. By using the pGWB system, the expression of tagged proteins, and the localization of GFP-fused proteins were easily analyzed. Moreover, tissue-specific and inducible gene expression using a promoter was also monitored with pGWBs. It is expected that, the pGWB system will serve as a powerful tool for plasmid construction in plant research.     

Development of episomal vectors carrying a nourseothricin‐resistance marker for use in minimal media for Schizosaccharomyces pombe

In the post‐genomic era, an immediate challenge is to assign biological functions to novel proteins encoded by the genome. This challenge requires the use of a simple organism as a genetic tool and a range of new high‐throughput techniques. Schizosacchromyces pombe is a powerful model organism used to investigate disease‐related genes and provides useful tools for the functional analysis of heterologous genes. To expand the current array of experimental tools, we constructed two series of Sz. pombe expression vectors, i.e. general and Gateway vectors, containing nourseothricin‐resistance markers. Vectors carrying nourseothricin‐resistance markers possess advantages in that they do not limit the parental strains with auxotrophic mutations with respect to availability for use in clone selection and can be used together with vectors carrying nutrient markers in minimal media. We modified the pSLF173, pSLF273 and pSLF373 vectors carrying a triple haemagglutinin epitope (3×HA) and an Ura4 marker. The vectors described here contain the nmt1 promoter with three different episomal expression strengths for proteins fused with 3×HA, EGFP or DsRed at the N‐terminus. These vectors represent an important contribution to the genome‐wide investigation of multiple heterologous genes and for functional and genetic analysis of novel human genes. Copyright © 2013 John Wiley & Sons, Ltd.     

pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast

A novel series of plasmid vectors named pDUAL have been developed. These vectors enable one to introduce not only multicopies of genes with episomal maintenance but also a single copy with chromosomal integration into the fission yeast, Schizosaccharomyces pombe. The multicopy plasmids can be easily converted to fragments for chromosomal integration by digestion of the plasmids with a certain restriction endonuclease before transformation of the yeast cells. The resultant fragments, lacking the autonomously replicating sequence, are designed for targeting into the chromosomal leu1 locus by homologous recombination. Whether the transformants are the results of episomal maintenance of the plasmid or homologous gene targeting can be readily checked by their requirement for uracil or leucine, or by the PCR diagnostic analysis. Furthermore, we propose the use of pDUAL derivatives for PCR-based chromosomal tagging of a gene to introduce several tags into 5'-terminus of a gene, employing a set of primers. Using these all-in-one vectors, a suitable mode of expression of a cloned gene can be selected for individual analysis without any complicated subcloning processes.     

烟草CHS基因RNA干扰载体的构建及其遗传转化

类黄酮化合物是烟草香气形成的重要前体物质,其形成需要多个调控基因的参与,查尔酮合成酶基因(CHS)是类黄酮物质合成途径中的关键基因,它的突变或沉默可能影响香气物质的形成。为了探讨CHS基因的下调表达对烟草香气的影响以及为选育优良的高香气品种提供技术,采用GATEWAY技术经BP反应和LR反应将CHS基因片段重组到表达载体pH7GWIWG2(I)上,成功构建了具有反向重复序列的CHS基因的RNAi表达载体,并通过农杆菌介导法转染烟草‘大白筋599’,以期为研究CHS基因在烟叶香气物质方面的作用奠定基础。经过鉴定,获得24株阳性转基因‘大白筋599’。     

New vectors for simple and streamlined CRISPR–Cas9 genome editing in Saccharomyces cerevisiae

Clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double‐strand breaks. Targeting of the DNA break is typically controlled by a single‐guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR–Cas9 technology can be used for efficient, marker‐free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time‐consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9–sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter‐selection using 5‐fluoro‐orotic acid (5‐FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR–Cas9 technology in yeast genome editing. Vectors pT040, pJH001, pML104 and pML107 have been deposited at Addgene ( www.addgene.org ). Copyright © 2015 John Wiley & Sons, Ltd.     

Integrating after CEN Excision (ICE) Plasmids: Combining the ease of yeast recombination cloning with the stability of genomic integration

Yeast recombination cloning is a straightforward and powerful method for recombining a plasmid backbone with a specific DNA fragment. However, the utility of yeast recombination cloning is limited by the requirement for the backbone to contain an CEN/ARS element, which allows for the recombined plasmids to propagate. Although yeast CEN/ARS plasmids are often suitable for further studies, we demonstrate here that they can vary considerably in copy number from cell to cell and from colony to colony. Variation in plasmid copy number can pose an unacceptable and often unacknowledged source of phenotypic variation. If expression levels are critical to experimentation, then constructs generated with yeast recombination cloning must be subcloned into integrating plasmids, a step that often abrogates the utility of recombination cloning. Accordingly, we have designed a vector that can be used for yeast recombination cloning but can be converted into the integrating version of the resulting vector without an additional subcloning. We call these “ICE” vectors, for “Integrating after CEN Excision.” The ICE series was created by introducing a “rare-cutter” NotI-flanked CEN/ARS element into the multiple cloning sites of the pRS series yeast integration plasmids. Upon recovery from yeast, the CEN/ARS is excised by NotI digest and subsequently religated without need for purification or transfer to new conditions. Excision by this approach takes ~3 hr, allowing this refinement in the same time frame as standard recombination cloning.     

Production of anti-CD2 chimeric antibody by recombinant animal cells

Expression vectors for chimeric anti-CD2 antibody were constructed in order to clarify the importance of the expression ratio of heavy (H-) and light (L-) chains of antibody to antibody production in animal cells. The antibody genes were introduced into cells using plasmid DNA vectors or replication-defective retroviral vectors. Productivity was maximal when the expression ratio of H-and L-chains was 1:1, and decreased when the ratio was not equal. We also examined the expression of antibody using one-packed vectors in which the bicistronic expression of H- and L-chain genes was mediated by an internal ribosomal entry site (IRES) sequence derived from encephalomyocarditis virus (EMCV). The translation efficiency was unbalanced between 5'Cap- and IRES-dependent genes. Using the retroviral vectors, it was estimated that the IRES-dependent translation efficiency was 5-fold lower than the 5'Cap-dependent translation efficiency. The cells exhibiting an unbalanced expression of H- and L-chains tended to accumulate H-chain protein.     

Application of the FLP/FRT system for conditional gene deletion in yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae has proved to be an excellent model organism to study the function of proteins. One of the many advantages of yeast is the many genetic tools available to manipulate gene expression, but there are still limitations. To complement the many methods used to control gene expression in yeast, we have established a conditional gene deletion system by using the FLP/FRT system on yeast vectors to conditionally delete specific yeast genes. Expression of Flp recombinase, which is under the control of the GAL1 promoter, was induced by galactose, which in turn excised FRT sites flanked genes. The efficacy of this system was examined using the FRT site-flanked genes HSP104, URA3 and GFP. The pre-excision frequency of this system, which might be caused by the basal activity of the GAL1 promoter or by spontaneous recombination between FRT sites, was detected ca. 2% under the non-selecting condition. After inducing expression of Flp recombinase, the deletion efficiency achieved ca. 96% of cells in a population within 9 h. After conditional deletion of the specific gene, protein degradation and cell division then diluted out protein that was expressed from this gene prior to its excision. Most importantly, the specific protein to be deleted could be expressed under its own promoter, so that endogenous levels of protein expression were maintained prior to excision by the Flp recombinase. Therefore, this system provides a useful tool for the conditional deletion of genes in yeast.     

The pYC plasmids, a series of cassette-based yeast plasmid vectors providing means of counter-selection

A series of 24 general-purpose yeast plasmid vectors has been constructed. The plasmid series is composed of inter-replaceable cassettes, allowing for easy interconversion of plasmid types. In addition to the usual replication origins, selectable markers and multiple cloning sites (MCS), cassettes dedicated to counter-selection have been constructed. A pair of unique 8 bp restriction enzyme recognition sites flank each type of cassette, FseI in the case of yeast replication origins, AscI in the case of selectable markers, PacI in the case of counter-selectable markers and NotI in the case of the MCS. Thus, any given cassette can be replaced by another cassette of the same type, facilitating interconversion of any given plasmid from one type to another, even after the insertion of DNA into the MCS. Hence, the plasmids have been named pYC for 'yeast cassettes'. The cassettes consist of either NONE, CEN4/ARS or 2micro as replication origin, either URA3, MET2-CA (Lg-MET2) or the G418 resistance gene (the apt1 gene from bacterial transposon Tn903, encoding aminoglycoside phosphotransferase) as selectable markers, either NONE, PMET25-PKA3 or PCHA1-PKA3 as counter-selectable marker, and the MCS, containing recognition sites for AflII, AvrII, BspEI, PmeI, SacII, SalI, SunI, BamHI, EcoRI, HindIII, KpnI, MluI, NarI and SacI (of which the seven first are unique in all plasmids). The counter-selectable markers consist of the PKA3 gene under control of the conditional MET25 or CHA1 promoters. At activating conditions these promoters express the PKA3 gene at toxic levels, facilitating easy selection for loss of plasmid or 'loop-out' of plasmid DNA sequence after genomic integration.     

The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae

The YDp plasmids (Yeast Disruption plasmids) are pUC9 vectors bearing a set of yeast gene disruption cassettes, all uniform in structure and differing only in the selectable marker used (HIS3, LEU2, LYS2, TRP1 or URA3). The markers, surrounded by translational termination codons, are embedded in the slightly modified sequence of the pUC9 multiple cloning sites.     

A family of low and high copy replicative, integrative and single-stranded S. cerevisiae/E. coli shuttle vectors.

We describe a set of replicative, integrative and single-stranded shuttle vectors constructed from the pUC19 plasmid that we use routinely in our experiments. They bear a yeast selectable marker: URA3, TRP1 or LEU2. Replicative vectors carrying different yeast replication origins have been constructed in order to have plasmids based on the same construction with a high or low copy number per cell and with different mitotic stabilities. All the vectors are small in size, provide a high yield in Escherichia coli and efficiently transform Saccharomyces cerevisiae. These plasmids have many of the unique sites of the pUC19 multicloning region and many of them allow for the screening of plasmids with an insert by alpha-complementation. The nucleotide sequence of each of them is completely known.     

A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences

Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 μm origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of ‘ZAP’-type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well-regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site-directed mutagenesis.     

藤黄微球菌过氧化氢酶基因在大肠杆菌中的重组与表达

目的应用基因重组技术在大肠杆菌中高效表达藤黄微球菌过氧化氢酶(catalase,CAT)。方法从藤黄微球菌DNA中获得CAT编码基因,并应用pProEx-HTa质粒构建融合表达载体并表达和检测活性。结果通过融合表达获得可溶性带6×His标签重组蛋白,该蛋白经过Ni-NTA纯化后可获得活性物质。结论从大肠杆菌表达体系中表达了具有生物活性的CAT。