‘ATG vectors’ for regulated high-level expression of cloned genes in Escherichia coli

A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage A or a large portion of the coding region of the Herpes simplex virus type l glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.     

An Escherichia coli system for assay of Flp site-specific recombination on substrate plasmids

We have developed an Escherichia coli system for testing the behaviour of plasmids carrying target sites for the Flp site-specific recombinase. The E. coli strain BL-FLP is described, which carries a chromosomally integrated bacteriophage T7 RNA polymerase gene expressed from a lac promoter, and harbours the plasmid pMS40. pMS40 has the features: (i) it carries the FLP recombinase gene under the control of a bacteriophage T7 promoter, (ii) it confers kanamycin resistance, and (iii) it uses an R6K origin of replication; these two latter features make it compatible with most conventional cloning vectors. Substrate plasmids carrying Flp-recognition targets (FRT) are transformed into BL-FLP, and the consequences of Flp-mediated recombination can be analysed after subsequent extraction of plasmid DNA. We show that this system is capable of base-perfect Flp-mediated recombination on plasmid substrates. We also present a corrected sequence of the commonly used Flp substrate plasmid, pNEOβGAL (O'Gorman et al. (1991) Science 251, 1351–1355).     

Development of cloning vehicles from the Streptomyces plasmid pFJ103

A 20-kb plasmid, pFJ103, was isolated from a strain of Streptomyces granuloruber. A restriction endonuclease map of the plasmid was constructed. A Streptomyces gene that specifies resistance to the antibiotic thiostrepton was subcloned into Escherichia coli plasmid pBR322, inserted into pFJ103 and transformed into Streptomyces ambofaciens protoplasts. Two classes of transformants were obtained. One carries the pFJ104 plasmid consisting of the entire pFJ103 with the 1.8-kb thiostrepton resistance gene insert. The other carries the pFJ105 plasmid consisting of the 2.9-kb replicon segment of pFJ103 with the same thiostrepton resistance insert. A gene for neomycin resistance together with the entire E. coli pBR322 plasmid were cloned into pFJ105. The resulting E. coli-Streptomyces bifunctional vector, pFJ123, transformed both E. coli and Streptomyces. The small size of pFJ105, its ease of isolation, and efficient transformation of Streptomyces protoplasts establishes it, and its derivatives, as useful plasmid cloning vehicles for fundamental and applied studies     

Use of transmissible plasmids as cloning vectors in Caulobacter crescentus

Cloning vectors for studies of Caulobacter crescentus genes should be transferable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (Su^R-Sm^R) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.     

A versatile plasmid system for the study of prokaryotic transcription signals in Escherichia coli

For comparing the relative efficiencies of Escherichia coli promoters, a modified plasmid system, pKO-2 and pKM-2, has been constructed using short synthetic DNA fragments. The new vectors were derived from the plasmids pKO-1 and pKM-1. The plasmids contain seven clustered unique restriction sites which can be used for promoter insertions. Also, three adjacent stop codons were introduced to abort any undesired translational initiation from various upstream origins. The DNA sequence of any insert in pKO-2 and pKM-2 can be determined rapidly by the supercoiled plasmid DNA sequencing method using a single oligonucleotide primer. The plasmid pKM-2 is especially suitable for the cloning and sequence determination of strong promoters.     

A bacteriophage lambda vector for cloning with BamHI and Sau3A

A phage lambda cloning vector has been constructed which contains a single site for the restriction endonuclease BamHI. Since Sau3A and BglII produce the same cohesive ends as BamHI, this vector can also be used to clone DNA fragments generated with either of these enzymes. We have used this vector to construct an Escherichia coli library using partial digestion with Sau3A. This vector will be most useful for applications requiring genetic analysis of cloned E. coli genes.     

A binary-BAC system for plant transformation with high-molecular-weight DNA

A binary-BAC (BIBAC) vector suitable for Agrobacterium-mediated plant transformation with high-molecular-weight DNA was constructed. A BIBAC vector is based on the bacterial artificial chromosome (BAC) library vector and is also a binary vector for Agrobacterium-mediated plant transformation. The BIBAC vector has the minimal origin region of the Escherichia coli F plasmid and the minimal origin of replication of the Agrobacterium rhizogenes Ri plasmid, and thus replicates as a single-copy plasmid in both E. coli and in A. tumefaciens. The T-DNA of the BIBAC vector can be transferred into the plant nuclear genome. As examples, a 30-kb yeast genomic DNA fragment and a 150-kb human genomic DNA fragment were inserted into the BIBAC vector; these constructs were maintained in both E. coli and A. tumefaciens. In order to increase the efficiency of transfer of unusually large BIBAC T-DNAs, helper plasmids that carry additional copies of A. tumefaciens virulence genes virG and virE were constructed. These helper plasmids are compatible with, and can be present in addition to, the BIBAC vector in the A. tumefaciens host. This report details the components of the BIBAC system, providing information essential to the general understanding and the application of this new technology.     

A novel DNA microarray design for accurate and straightforward identification of Escherichia coli safety and laboratory strains

Escherichia coli K-12, B, C and W strains and their derivates are declared in biological safety guidelines as risk group 1 organisms as they are unable to colonise the human gut.

Differentiation and identification of these safety strains is mainly based on pulsed-field gel electrophoresis (PFGE), phage sensitivity tests or PCR-based methods. However, these methods are either tedious and time consuming (phage sensitivity, PFGE) or based on single specific fragments (PCR) or patterns (PFGE) lacking additional information for further differentiation of the strains.

In the current study, subtractive hybridisation techniques were applied to detect specific DNA fragments which were used to design a microarray (chip) for accurate and simple identification of these organisms, and to differentiate them from other E. coli strains. The chip can be used to identify E. coli safety strains and monitor them during ongoing experiments for changes in their genome and culture purity. The hybridisation layout of the microarray was arranged in such a way that the respective lineages of safety strains could be easily identified as distinct letters (K, B, C or W). Differentiation of single strains or subtyping was possible with further probes. In addition, a set of probes targeting genes coding for common virulence factors has been included, both to differentiate safety strains from pathogenic variants and to make sure that no transfer of these genes happens during handling or storage. The reliability of the approach has been tested on a comprehensive selection of E. coli laboratory strains and pathogenic representatives.     

Inhibition of soil-borne plant pathogens by the treatment of sinigrin and myrosinases released from reconstructed Escherichia coli and Pichia pastoris

Myrosinases (thioglucoside glucohydrolase, EC 3.2.3.1) are able to hydrolyse glucosinolates in natural plant products. In Arabidopsis thaliana three different genes with different tissue-specific expressions and distribution patterns encode myrosinases. cDNAs of myrosinase genes (TGG1 and TGG2) were isolated from A. thaliana and expressed in Escherichia coli and Pichia pastoris. The enzyme activities of myrosinase TGG1 and TGG2 genes expressed in P. pastoris were higher than those expressed in E. coli. Among six glucosinolates tested for specificity to myrosinases TGG1 and TGG2, the suitable substrates for these two genes expressed in P. pastoris and E. coli were sinigrin, gluconapin, glucobrassicanapin and glucoraphanin. Treatment of sinigrin with myrosinases excreted from reconstructed E. coli and P. pastoris with TGG1 and TGG2 genes showed strong fungicidal effects on mycelial growth of Rhizoctonia solani AG-4, Sclerotium rolfsii, and Pythium aphanidermatum. This study suggests that the combination of glucosinolate with myrosinases excreted from the reconstructed microbes may be of potential for control of soil-borne diseases.     

A novel host-vector system for direct selection of recombinant baculoviruses (bacmids) in Escherichia coli

Site-specific transposition in Escherichia coli was used to introduce foreign genes into the Autographica californica nuclear polyhedrosis baculovirus genome. Using a temperature-sensitive donor plasmid and an E. coli host strain with an occupied Tn7 attachment site it was possible to select directly for ‘bacmid’ recombinants at 44°C. A blue to white color screen provided further confirmation of insertion at the correct site in the baculovirus genome. After cloning the gene of interest into a donor plasmid, a single transformation and plating on selective medium resulted in homogeneous baculovirus DNA which could immediately be transfected into insect cells. The utility of the host-vector system for expression in insect cells was illustrated using three heterologous genes encoding β-glucuronidase, human N-myristoyl transferase and murine preproguanylin. Using this approach, bacmid recombinants could be produced at a frequency of 10⁵ per pg input DNA. This system should not only greatly enhance the ability to obtain recombinant viruses for heterologous protein production, but should also be useful for protein engineering applications and expression cloning in insect cells.     

The cyclization of linear DNA in Escherichia coli by site-specific recombination

The efficiency with which linearized plasmid DNA can transform competent Escherichia coli can be significantly increased by use of the Cre-lox site-specific recombination system of phage P1. Linear plasmid molecules containing directly repeated loxP sites (lox² plasmids) are cyclized in Cre⁺ E. coli strains after introduction either by transformation or by mini-Mu transduction, Exonuclease V activity of the RecBC enzyme inhibits efficient cyclization of linearized lox² plasmids after transformation. By use of E. coli mutants which lack exonuclease V activity, Cre-mediated cyclization results in transformation efficiencies for linearized lox² plasmids identical to those obtained with covalently closed circular plasmid DNA. Moreover, Cre⁺ E. coli recBC strains allow the efficient recovery of lox² plasmids integrated within large linear DNA molecules such as the 150-kb genome of pseudorabies virus.     

Plasmid vector pBR322 and its special-purpose derivatives — a review

The plasmid pBR322 was one of the first EK2 multipurpose cloning vectors to be designed and constructed (ten years ago) for the efficient cloning and selection of recombinant DNA molecules in Escherichia coli. This 4363-bp DNA molecule has been extensively used as a cloning vehicle because of its simplicity and the availability of its nucleotide sequence. The widespread use of pBR322 has prompted numerous studies into its molecular structure and function. These studies revealed two features that detract from the plasmid's effectiveness as a cloning vector: (a) plasmid instability in the absence of selection and, (b) the lack of a direct selection scheme for recombinant DNA molecules. Several vectors based on pBR322 have been constructed to overcome these limitations and to extend the vector's versatility to accomodate special cloning purposes. The objective of this review is to provide a survey of these derivative vectors and to summarize information currently available on pBR322.     

Isolation and characterization of the Neisseria meningitidis lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis

The lpxD-fabZ-lpxA gene cluster involved in lipid A biosynthesis in Neisseria meningitidis has been cloned and sequenced. By complementation of a temperature-sensitive E. coli lpxD mutant, we first cloned a meningococcal chromosomal fragment that carries the lpxD homologue. Cloning and sequence analysis of chromosomal DNA downstream of lpxD revealed the presence of the fabZ and lpxA genes. This gene cluster shows high homology to the corresponding genes from several other bacterial species. The LpxA and LpxD proteins catalyze early steps in the lipid A biosynthetic pathway, adding the O- and N-linked 3-OH fatty acyl chains, respectively. In E. coli and N. meningitidis, LpxD has the same specificity, in both cases adding 3-OH myristoyl chains; in contrast to E. coli, the meningococcal LpxA protein is presumed to add 3-OH lauroyl chains instead. The established sequence points the way to further experiments to define the basis for this difference in specificity, and should allow modification of meningococcal lipid A biosynthesis through gene exchange.     

A selective λ phage cloning vector with automatic excision of the insert in a plasmid

A bacteriophage λ cloning vehicle has been constructed for the generation of cDNA libraries. The vector has the following properties. (1) It has a unique BamHI site engineered into the λ gam gene. Segments of DNA can be cloned into this site and clones with an insert can be selected by their ability to grow on an Escherichia coli host lysogenic for phage P2 (Spi⁻ phenotype). (2) When the recombinant phage infects a Cre-producing E. coli strain, a site-specific recombination event results in the excision of a plasmid replicon with the cloned insert. (3) Single-stranded DNAs can be recovered by growing helper M13 phages on bacteria harboring such plasmids. The vector, λMGU2, has been used to construct a nematode (Caenorhabditis elegans) cDNA library.     

Verification of elicitor efficacy of lipopolysaccharides and peptidoglycans on antibacterial peptide gene expression in Bombyx mori

We verified the efficacy of lipopolysaccharide (LPS) in activating the cecropin B gene (CecB) in an immune-competent Bombyx mori cell line. Strong activation of CecB by the LPSs from Escherichia coli, Pseudomonas aeruginosa, and Salmonella minnesota were completely eliminated after digestion of the LPSs with muramidase. The results clearly indicate that a polymer form of PGN in the LPSs elicited CecB. An oligonucleotide microarray screen revealed that none of the 16,000 genes on the array were activated by LPS in the cells. In contrast, E. coli PGN strongly elicited five antibacterial peptide genes and numerous other genes, and PGN from Micrococcus luteus activated only several genes. Semi-quantitative RT-PCR revealed that all antibacterial genes activated by both PGNs, but the extents were 10–100 times higher with E. coli PGN. Similarly, higher elicitor activity of E. coli than M. luteus was indicated using peptidoglycan recognition protein gene, which is involved in pro-phenol oxidase cascade.     

Surface display of a glucose binding protein

Glucose binding protein (GBP) from Escherichia coli has been widely used to develop minimally invasive glucose biosensors for diabetics. To develop a cell-based glucose biosensor, it is essential to functionally display GBP on the cell surface. In this study, we designed a molecular structure to display GBP on the outer membrane of E. coli. We fused GBP with the first nine N-terminal residues of Lpp (major E. coli lipoprotein) and the 46–150 residues of OmpA (an outer membrane protein of E. coli). With this molecular design, we have successfully displayed GBP on the surface of E. coli. Using FITC-conjugated Dextran, we demonstrated that glucose’s binding sites of surface-displayed GBP were accessible to glucose. Furthermore, we showed that glucose transport in a GBP-deficient E. coli NM303 could be restored by displaying GBP on the surface of NM303. 0.51 h⁻¹ of specific growth rate was attained for NM303/pESDG grown in M9 minimal medium supplemented with 2 g/l glucose, whereas no growth was observed for NM303 in the same medium. Both NM303 and NM303/pESDG grew in M9 medium supplemented with 1 mM of fucose. Because cell surface is an interface between intracellular and extracellular molecular events, this technique paves a way to develop cell-based glucose biosensors.     

枯草芽孢杆菌Mn-SOD基因的克隆及原核表达

为了实现Mn-SOD基因在大肠杆菌(E.coli)中的可溶性表达,根据枯草芽孢杆菌(Bacillus subtilis)168sodA核酸序列设计引物,以枯草芽孢杆菌ATCC 9372基因组为模板,PCR扩增获得了Mn-SOD基因.将此基因重组至原核表达载体pET-28a,构建含Mn-SOD基因的重组表达质粒,并转化至大肠杆菌BL21(DE3).异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达获得Mn-SOD,蛋白分子量约为26kD,占全菌蛋白的5.6%.改良的连苯三酚自氧化法测定SOD活力,菌体可溶性总蛋白SOD比活为51.09U·mg^-1,是对照组的.8倍.枯草芽孢杆菌ATCC 9372 Mn-SOD基因在大肠杆菌BL21(DE3)中首次成功表达,产物具有较高的可溶性和活性,为大量制备Mn-SOD奠定了基础.