pYEMF,a pUC18-Derived <Emphasis Type="Italic">Xcm</Emphasis>I T-Vector for Efficient Cloning of PCR Products

A 1330-bp DNA sequence with two XcmI cassettes was inserted into pUC18 to construct an efficient XcmI T-vector parent plasmid, pYEMF. The large size of the inserted DNA fragment improved T-vector cleavage efficiency, and guaranteed good separation of the molecular components after restriction digestion. The pYEMF-T-vector generated from parent plasmid pYEMF permits blue/white colony screening; cloning efficiency analysis showed that most white colonies (>75%) were putative transformants which carried the cloning product. The sequence analysis and design approach presented here will facilitate applications in the fields of molecular biology and genetic engineering.     

An efficient plasmid vector for expression cloning of large numbers of PCR fragments in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The described plasmid pEamTA was designed for parallel polymerase chain reaction (PCR) cloning of open reading frames (ORFs) in Escherichia coli. It relies on the well-known TA-cloning principle, and the “T-vector” can be generated from a plasmid preparation by digestion with the restriction enzyme Eam1105I. The single 3′-T-overhangs of the vector fragment are positioned in a way that A-tailed PCR-products beginning with the start-ATG of an ORF end up in optimal position for expression from a strong tac-promoter when ligated in correct orientation. The orientation of the insert can be checked via a reconstituted NdeI site (catATG) present in correct clones. The protocol works regardless of internal restriction sites of the PCR fragment, a major advantage when cloning a number of fragments in parallel. It also does not require 5′-primer extensions and finally delivers an expression clone for the preparation of untagged protein in less than a week.     

Positive-selection and ligation-independent cloning vectors for large scale <Emphasis Type="Italic">in Planta</Emphasis> expression for plant functional genomics

Transient expression is an easy, rapid and powerful technique for producing proteins of interest in plants. Recombinational cloning is highly efficient but has disadvantages, including complicated, time consuming cloning procedures and expensive enzymes for large-scale gene cloning. To overcome these limitations, we developed new ligationindependent cloning (LIC) vectors derived from binary vectors including tobacco mosaic virus (pJL-TRBO), potato virus X (pGR106) and the pBI121 vector-based pMBP1. LIC vectors were modified to enable directional cloning of PCR products without restriction enzyme digestion or ligation reactions. In addition, the ccdB gene, which encodes a potent cell-killing protein, was introduced between the two LIC adapter sites in the pJL-LIC, pGR-LIC, and pMBP-LIC vectors for the efficient selection of recombinant clones. This new vector does not require restriction enzymes, alkaline phosphatase, or DNA ligase for cloning. To clone, the three LIC vectors are digested with SnaBI and treated with T4 DNA polymerase, which includes 3′ to 5′ exonuclease activity in the presence of only one dNTP (dGTP for the inserts and dCTP for the vector). To make recombinants, the vector plasmid and the insert PCR fragment were annealed at room temperature for 20 min prior to transformation into the host. Bacterial transformation was accomplished with 100% efficiency. To validate the new LIC vector systems, we were used to coexpressed the Phytophthora AVR and potato resistance (R) genes in N. benthamiana by infiltration of Agrobacterium. Coexpressed AVR and R genes in N. benthamiana induced the typical hypersensitive cell death resulting from in vivo interaction of the two proteins. These LIC vectors could be efficiently used for high-throughput cloning and laboratory-scale in planta expression. These vectors could provide a powerful tool for high-throughput transient expression assays for functional genomic studies in plants.     

Next generation synthetic vectors for transformation of the plastid genome of higher plants

Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Simple Method for Markerless Gene Deletion in Multidrug-Resistant Acinetobacter baumannii

The traditional markerless gene deletion technique based on overlap extension PCR has been used for generating gene deletions in multidrug-resistant Acinetobacter baumannii. However, the method is time-consuming because it requires restriction digestion of the PCR products in DNA cloning and the construction of new vectors containing a suitable antibiotic resistance cassette for the selection of A. baumannii merodiploids. Moreover, the availability of restriction sites and the selection of recombinant bacteria harboring the desired chimeric plasmid are limited, making the construction of a chimeric plasmid more difficult. We describe a rapid and easy cloning method for markerless gene deletion in A. baumannii, which has no limitation in the availability of restriction sites and allows for easy selection of the clones carrying the desired chimeric plasmid. Notably, it is not necessary to construct new vectors in our method. This method utilizes direct cloning of blunt-end DNA fragments, in which upstream and downstream regions of the target gene are fused with an antibiotic resistance cassette via overlap extension PCR and are inserted into a blunt-end suicide vector developed for blunt-end cloning. Importantly, the antibiotic resistance cassette is placed outside the downstream region in order to enable easy selection of the recombinants carrying the desired plasmid, to eliminate the antibiotic resistance cassette via homologous recombination, and to avoid the necessity of constructing new vectors. This strategy was successfully applied to functional analysis of the genes associated with iron acquisition by A. baumannii ATCC 19606 and to ompA gene deletion in other A. baumannii strains. Consequently, the proposed method is invaluable for markerless gene deletion in multidrug-resistant A. baumannii.     

Vectors for ligation-independent construction of lacZ gene fusions and cloning of PCR products using a nicking endonuclease

Several ligation-independent cloning methods have been developed that offer advantages for construction of recombinant plasmids at high efficiency while minimizing cloning artifacts. Here we report new plasmid vectors that use the nicking endonuclease Nt.BspQI to generate extended single stranded tails for direct cloning of PCR products. The vectors include pLacCOs1, a ColE1-derivative plasmid imparting resistance to ampicillin, which allows facile construction of lacZ translational fusions and pKanCOs1, a pSC101-derivative cloning vector that imparts resistance to kanamycin, for cloning of PCR amplicons from genomic DNA as well as from ampicillin-based plasmids. We have successfully used these plasmids to directionally clone and characterize bacterial promoters that exhibit temperature regulated expression, as well as for cloning a variety of PCR products. In all cases, constructs with the correct configurations were generated at high efficiency and with a minimal number of manipulations. The cloning vectors can also be easily modified to incorporate additional reporter genes or to express epitope-tagged gene products.     

Construction ofClostridium butyricum hybrid plasmids and transfer toBacillus subtilis

Summary Small plasmids were isolated from type strains ofClostridium butyricum. Strain NCIB 7423 carries one plasmid (pCBU1) of 6.4 kb, whereas strain NCTC 7423 carries two unrelated plasmids of 6.3 kb (pCBU2) and 8.4 kb (pCBU3). Cleavage sites for 18 restriction endonucleases have been mapped on these plasmids and detailed physical maps are presented. For the purpose of developing vector plasmids for gene cloning in saccharolytic clostridia these crypticC. butyricum plasmids were joined to a selectable marker that will likely be expressed in clostridia. This was achieved by cloning the clostridial plasmids into theE. coli vector pBR322 carrying the chloramphenicol acetyltransferase (CAT) gene from theStaphylococcus aureus plasmid pC194. The recombinant plasmids were tested for their ability to confer chloramphenicol resistance toBacillus subtilis. Hybrid plasmids (pHL105, pHL1051) derived from pCBU2 were identified, which are capable of replication and expression of theS. aureus drug resistance marker in bothE. coli andB. subtilis. No structural instability was detected upon retransformation of pHL105 fromB. subtilis intoE. coli. The recombinant plasmids might thus be useful as shuttle vectors for the gene transfer betweenE. coli and a wide range of bacilli and clostridia.     

Electroporation of Tobacco Protoplasts with Homologous and Nonhomologous Transformation Vectors

A modified protoplast selection/plating technique was used to quantitate and compare the transformation efficiencies of tobacco NTl protoplasts electroporated with plasmid molecules harboring a chimeric nos-neo gene (pMON213) or both this chimeric gene and a region of homology with the host chromosome (pCPI). The latter plasmid was constructed by cloning the pMON213 EcoR I cassette carrying the nos-neo gene into pNtSS233, a pBR322 derivative harboring a 1.2-kbp piece of Nicotiana tabacum DNA comprising the 5'-end of a gene coding for 1,5-ribulose bisphosphate carboxylase-oxygenase (Rubisco). These plasmids were linearized by restriction endonuclease digestion and electroporated into tobacco protoplasts which were subsequently selected on kanamycin-containing medium. Both plasmids displayed the same transformation efficiency, indicating that the presence of a homologous region in the selectable vector had no influence on the rates of transformation.     

Construction and characterization of three yeast-Escherichia coli shuttle vectors designed for rapid subcloning of yeast genes on small DNA fragments

We have constructed three new subcloning plasmid vectors, pRC1, pRC2, and pRC3, derived from pKC7, which allow the rapid, single-step subcloning of yeast genes. Subcloning with these vectors utilizes a partial digestion with Sau3A to generate a quasi-random set of DNA fragments from the original plasmid. All three vectors contain a kanamycin resistance gene. Therefore, if the original cloned yeast DNA fragment is present in a vector that does not specify kanamycin resistance, the subclone pool can be propagated in Escherichia coli in the presence of kanamycin to select against parent plasmids that escaped restriction by Sau3A. Selection by complementation in yeast yields a collection of plasmids with smaller yeast DNA inserts containing the gene of interest. In the vectors pRC2 and pRC3, constructed from pRC1, the unique BamHI site is located within an intact tetracycline resistance gene, thus making it possible to screen bacterial transformants for those containing recombinant plasmid molecules. Vectors pRC2 and pRC3 also contain the yeast 2 micrometers DNA replication origin, and thus are more stable than plasmids carrying only the TRP1-associated replicator (ars1).     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

An Alternative Method to Facilitate cDNA Cloning for Expression Studies in Mammalian Cells by Introducing Positive Blue White Selection in Vaccinia Topoisomerase I-Mediated Recombination

One of the most basic techniques in biomedical research is cDNA cloning for expression studies in mammalian cells. Vaccinia topoisomerase I-mediated cloning (TOPO cloning by Invitrogen) allows fast and efficient recombination of PCR-amplified DNAs. Among TOPO vectors, a pcDNA3.1 directional cloning vector is particularly convenient, since it can be used for expression analysis immediately after cloning. However, I found that the cloning efficiency was reduced when RT-PCR products were used as inserts (about one-quarter). Since TOPO vectors accept any PCR products, contaminating fragments in the insert DNA create negative clones. Therefore, I designed a new mammalian expression vector enabling positive blue white selection in Vaccinia topoisomerase I–mediated cloning. The method utilized a short nontoxic LacZα peptide as a linker for GFP fusion. When cDNAs were properly inserted into the vector, minimal expression of the fusion proteins in E. coli (harboring lacZΔM15) resulted in formation of blue colonies on X-gal plates. This method improved both cloning efficiency (75%) and directional cloning (99%) by distinguishing some of the negative clones having non-cording sequences, since these inserts often disturbed translation of lacZα. Recombinant plasmids were directly applied to expression studies using GFP as a reporter. Utilization of the P2A peptide allowed for separate expression of GFP. In addition, the preparation of Vaccinia topoisomerase I-linked vectors was streamlined, which consisted of successive enzymatic reactions with a single precipitation step, completing in 3 hr. The arrangement of unique restriction sites enabled further modification of vector components for specific applications. This system provides an alternative method for cDNA cloning and expression in mammalian cells.     

A hybrid promoter-containing vector for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells

An efficient vector, designated as pCAGX, was designed for direct cloning and enhanced expression of PCR-amplified ORFs in mammalian cells. It relied on the well-known TA-cloning principle, and utilized the CMV enhancer/chicken β-actin/rabbit β-globin (CAG) hybrid promoter instead of the classical CMV promoter to drive more efficient transgene expression in wider host cells. The specially designed cassette under CAG hybrid promoter contained two tandemly arrayed XcmI sites which were spaced by an additional EcoRV site. For direct cloning and expressing PCR-amplified ORFs, the T-vector was prepared by further digesting the EcoRV-linearized pCAGX with XcmI to produce T tails on both 3′-ends, which could efficiently minimize the non-recombinant background of T-vector and eliminate the necessity of selective marker genes such as LacZ that allowed blue/white screening. Various PCR fragments in length were prepared to verify the cloning efficiency by ligation with this vector, and GFP gene expression under control of the CAG hybrid promoter in different host cells was assayed by flow cytometry. The results indicated that this vector was higher efficient, especially suitable for cloning and expressing a number of interesting ORFs in parallel, and higher-level transgene expression in different mammalian cells was obtained than the reported vectors using the CMV promoter.     

中国葡萄属野生种抗白粉病抗逆基因植物表达载体的构建

将质粒pSB166中包含ED35s启动子、Omega元件及TNOS终止子的一段核苷酸序列定向克隆到质粒pCAMBIA1303,构建了中间表达载体pWR306;以中国野生葡萄华东葡萄白河35-1 cDNA为模板,通过PCR扩增出葡萄芪合成酶基因（STS）、醛脱氢酸基因（ALDH）,与pGEM-T Easy克隆载体连接,获得重组质粒pGEM-T Easy-STS和pGEM-TEasy-ALDH;双酶切重组质粒及表达载体pWR306,将STS、ALDH基因片段与线性表达载体pWR306进行定向连接,构建了葡萄芪合成酶基因及醛脱氢酶基因的植物表达载体pWR-STS、pWR-ALDH,并用改进冻融法导入农杆菌GV3101。     

Construction of cloning vectors using the Vibrio harveyi luminescence genes luxA and luxB as markers

A synthetic oligodeoxyribonucleotide harboring four new restriction sites was inserted into the luxB gene of Vibrio harveyi. This insertion did not disrupt the reading frame. An active beta-subunit was synthesized since a plasmid with both the luxA and mutated luxB genes conferred upon Escherichia coli the bacterial luciferase (Lux) phenotype in the presence of an aldehyde. Ligation of a piece of foreign DNA at these new cloning sites in the vector extinguish the Lux phenotype of the transformed bacteria. Therefore, the plasmid was used as a cloning vector, and recombinant DNA-containing bacteria were detected by the loss of bioluminescence. To create more versatile plasmids, the intergenic region of phage f1 was inserted outside of the lux genes. The selection by loss of bioluminescence presents several advantages over the white/blue selection of the lacZ gene on indicator plates.     

Development of a suicidal vector-cloning system based on butanal susceptibility due to an expression of YqhD aldehyde reductase

Previously, we observed butanal/propanal sensitivity of Escherichia coli K-12 when cells overexpress YqhD protein, a NADPH dependent aldehyde reductase, possibly due to an accumulation of butanol/propanol in vivo as the reaction products. Based on this finding, we developed a suicidal vector-cloning system derived from pUC19, in which lacZ was substituted with the yqhD gene. As a result, when foreign DNA was inserted into its multiple cloning sites by disrupting an expression of YqhD, the recombinants survived on butanal/propanal containing plate, whereas cells containing the YqhD vector died because of the alcohol production by YqhD. The cloning efficiency, estimated based on colony PCR and enzyme digestion, was achieved more than 90% when the suicidal vector system was used. Moreover, the plasmid vector itself was stably maintained in the cell, presumably due to its ability to remove toxic aldehydes being accumulated in E. coli cell by metabolic stress.     

Using a modified TA cloning method to create entry clones

We describe a noncommercial alternative method to create entry clones compatible with all kinds of destination vectors based on an improved TA cloning approach. To generate Gateway T vectors, we first constructed gentamicin- and chloramphenicol-resistant entry vectors designated pGWG and pGWC, respectively. Each entry vector contains an AhdI cassette flanked by attL sites, with each AhdI cassette containing two AhdI restriction enzyme sites spaced by the ccdB killer gene, which is lethal to most Escherichia coli strains. Gateway T vectors can be prepared by simple digestion of these entry vectors with the AhdI enzyme or its isoschizomers. The use of the ccdB gene as a negative selection marker is an important improvement over conventional TA cloning in that it eliminates the necessity of blue/white color screening based on alpha-complementation. Another important improvement that we have implemented is to retail the T vectors using Taq polymerase and dTTP so as to improve the cloning efficiency. Together, these improvements allow TA cloning to realize its full potential. Using Gateway T vectors prepared by this improved method, entry clones for PCR products or restriction enzyme fragments can be created simply, efficiently, and inexpensively while at the same time introducing greater compatibility.     

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211–213]. A family of related plasmids containing different antibiotic-resistance markers (Cm^R or Gm^R or Km^R and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.     

Construction of shuttle vectors for cloning inEscherichia coli andAcetobacter pasteurianus

New cloning vectors were prepared with the aid of a large plasmid isolated fromAcetobacter pasteurianus and from plasmids pBR322 and pUC4-KAPA. Of the prepared cloning vectors, pACK5 contains a gene coding for kanamycin resistance, pACT7 and pACT71 contain a gene coding for tetracycline resistance and vector pACG3 with a gene coding for both kanamycin and tetracycline resistance. The vectors prepared only contained the beginning of replication from the pAC1 plasmid and possessed the ability to replicate withinE. coli andA. pasteurianus. The vectors are highly stable in both strains and during the 5-d cultivation under nonselective conditions are not eliminated.