Coupled analysis of bacterial transformants and ligation mixture by duplex PCR enables detection of fatal instability of a nascent recombinant plasmid

When a DNA cloning experiment fails, it is often difficult to distinguish between an inadequate cloning protocol and instability of the new recombinant plasmid. The identification of plasmid instability is particularly challenging when the instability is fatal and no DNA of the expected construct can be isolated. We have effectively addressed this problem by employment of duplex PCR (insert-insert, vector-insert) to analyse both the ligation mixture and the resultant bacterial transformants. Using this approach we found a fatal maintenance instability of one of the plasmids generated during subcloning of the cDNA for human LDLR in Escherichia coli STBL2. The described duplex PCR screening method allows monitoring of the fate of nascent recombinant plasmid from ligation, through the initial bacterial colony and the subsequent overnight culture.     

Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.     

Self-assembling supramolecular complexes by single-stranded extension from plasmid DNA

Self-assembling supramolecular complexes are of great interest for bottom-up research like nanotechnology. DNA is an inexpensive building block with sequence-specific self-assembling capabilities through Watson-Crick and/or Hoogsteen base pairing and could be used for applications in surface chemistry, material science, nanomechanics, nanoelectronics, nanorobotics, and of course in biology. The starting point is usually single-stranded DNA, which is rather easily accessible for base pairing and duplex formation. When long stretches of double-stranded DNA are desirable, serving either as genetic codes or electrical wires, bacterial expansion of plasmids is an inexpensive approach with scale-up properties. Here, we present a method for using double-stranded DNA of any sequence for generating simple structures, such as junctions and DNA lattices. It is known that supercoiled plasmids are strand-invaded by certain DNA analogs. Here we add to the complexity by using "Self-assembling UNiversal (SUN) anchors" formed by DNA analog oligonucleotides, synthesized with an extension, a "sticky-end" that can be used for further base pairing with single-stranded DNA. We show here how the same set of SUN anchors can be utilized for gene therapy, plasmid purification, junction for lattices, and plasmid dimerization through Watson-Crick base pairing. Using atomic force microscopy, it has been possible to characterize and quantify individual components of such supra-molecular complexes.     

High frequency of yeast transformation by plasmids carrying part or entire 2-micron yeast plasmid.

By using two chimeric plasmids containing yeast ura3 gene and 2-micron yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-micron DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-micron-ura3 sequence can be considered as a "transposable" block. In contrast, the second one bears the entire 2-micron plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-micron DNA of the recipient strain. The specific activity of orotidine 5'-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.     

A Model for the Evolution of Biological Specificity: a Cross-Reacting DNA-Binding Protein Causes Plasmid Incompatibility

Few biological systems permit rigorous testing of how changes in DNA sequence give rise to adaptive phenotypes. In this study, we sought a simplified experimental system with a detailed understanding of the genotype-to-phenotype relationship that could be altered by environmental perturbations. We focused on plasmid fitness, i.e., the ability of plasmids to be stably maintained in a bacterial population, which is dictated by the plasmid''s replication and segregation machinery. Although plasmid replication depends on host proteins, the type II plasmid partitioning (Par) machinery is entirely plasmid encoded and relies solely on three components: parC, a centromere-like DNA sequence, ParR, a DNA-binding protein that interacts with parC, and ParM, which forms actin-like filaments that push two plasmids away from each other at cell division. Interactions between the Par operons of two related plasmids can cause incompatibility and the reduced transmission of one or both plasmids. We have identified segregation-dependent plasmid incompatibility between the highly divergent Par operons of plasmids pB171 and pCP301. Genetic and biochemical studies revealed that the incompatibility is due to the functional promiscuity of the DNA-binding protein ParR_pB171, which interacts with both parC DNA sequences to direct plasmid segregation, indicating that the lack of DNA binding specificity is detrimental to plasmid fitness in this environment. This study therefore successfully utilized plasmid segregation to dissect the molecular interactions between genotype, phenotype, and fitness.     

Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and two-site plasmids but has little effect on the three-site plasmid. Taken together the data indicate that EcoRII requires simultaneous binding of three rather than two recognition sites in cis to achieve concerted DNA cleavage at a single site. We show that the orthodox type IIP enzyme PspGI which is an isoschisomer of EcoRII, cleaves different plasmid substrates with equal rates. Data provided here indicate that type IIE restriction enzymes EcoRII and NaeI follow different mechanisms. We propose that other type IIE restriction enzymes may employ the mechanism suggested here for EcoRII.     

Infectious plasmid resistance and efflux pump mediated resistance

Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.     

pcD-awte候选疟疾DNA疫苗制备及其质量相关性分析

本实验室构建的疟疾DNA疫苗经动物试验表明具有很好的免疫原性,为申请临床试验,进行了制备工艺的研究。本研究将含pcD-awte质粒的大肠杆菌DH5α在发酵罐中发酵培养,碱裂解法粗提质粒,再依次通过Sepharose 6FF分子筛层析、Plasmidselect 亲硫吸附层析和Source 30Q离子交换层析精制获得质粒纯品,并对纯品进行质量分析。结果每升培养液可获得质粒纯品43.9mg,质量符合Ferreira等推荐的药用标准。     

大肠杆菌菌蜕装载质粒DNA实验方法的优化

目的:优化大肠杆菌菌蜕装载质粒的效率,并将装载质粒的菌蜕转染抗原提呈细胞,以提高核酸疫苗的递送水平。方法:将质粒pHH43转化大肠杆菌DH5α,制备大肠杆菌菌蜕;优化菌蜕装载质粒时菌蜕、质粒和膜囊的比例,获得更高的装载效率,通过扫描及透射电镜、流式细胞术观察其形态变化及装载效率;将装载质粒的菌蜕与抗原提呈细胞——巨噬细胞RAW264.7和树突状细胞DC2.4共孵育,观察吞噬效果。结果:优化了大肠杆菌菌蜕装载质粒的效率,当菌蜕、质粒、膜囊的比例为7∶10∶4时效率达到最佳,装载DNA效率达98%以上;抗原提呈细胞吞噬装载了质粒的菌蜕,效率达100%。结论:大肠杆菌菌蜕可高效装载核酸疫苗,且高效被抗原提呈细胞捕获,有助于提高核酸疫苗的递送和免疫效果的提高。     

The Selection of Recombinant Binary Plasmids Generated by Gateway® LR Cloning in the Escherichia coli Strain C2110

Gateway^® cloning is widely used in molecular biology laboratories. Various binary vectors used for Agrobacterium-mediated plant transformation have been modified as destination vectors that are convenient for the sub-cloning of targeted genes from Entry plasmids. However, when the destination and Entry plasmids have the same antibiotic resistance genes for bacterial selection, the non-recombinant Entry plasmid in the LR reaction mixture can compete with the recombinant destination plasmid during bacterial transformation and selection. Methods for the effective selection of recombinant destination plasmids are highly desirable. In this study, we demonstrated that Escherichia coli strain C2110, which is defective in DNA polymerase I (pAL1), could be used to select a recombinant binary destination plasmid with a RK2 replication origin, while the replication of the Entry plasmid with a ColE1 replication origin was inhibited. Plasmid DNA isolated from C2110 by a traditional mini-prep kit was used for restriction enzyme digestion, DNA sequencing, and Arabidopsis protoplast transfection. The binary plasmid in C2110 was also efficiently mobilized into Agrobacterium tumefaciens via the tri-parental conjugation method.     

Simple and rapid method for isolating large plasmid DNA from lactic streptococci.

A procedure for the rapid isolation of plasmid DNA larger than 30 megadaltons from lactic streptococci is described. This protocol can be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments. A scaled-down protocol is very useful for rapidly screening the plasmid content of streptococcal strains. With this methodology, previously undetected large plasmids were observed.     

Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: experiments with a model system

We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context.     

PLASMIDS AND EVOLUTIONARY RESCUE BY DRUG RESISTANCE

Antibiotic resistance provides evolutionary rescue for bacterial populations under the threat of extinction through antibiotics. It can arise de novo through mutation in the population, or be obtained from other bacterial populations via the transfer of a resistance‐conferring plasmid. We use stochastic modeling methods to establish whether the most likely source of rescue is via a plasmid or via the chromosome, and show that contrary to what is assumed plasmids are not necessarily beneficial locations for resistance genes. Competition at the plasmid level of selection is of great importance—the spread of a resistant plasmid in the population can be slowed or entirely stopped by a nonresistant version of the same plasmid. We suggest that future studies on antibiotic‐resistant plasmids should explicitly consider competition at this level of selection.     

Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier

The efficacious delivery of eukaryotic expression plasmids to inductive cells of the immune system constitutes a key prerequisite for the generation of effective DNA vaccines. Here, we have explored the use of bacteria as vehicles to orally deliver expression plasmids. Attenuated Salmonella typhimurium aroA harbouring eukaryotic expression plasmids that encoded virulence factors of Listeria monocytogenes were administered orally to BALB/c mice. Strong cytotoxic and helper T cell responses as well as antibody production were elicited even after a single administration. Mice immunised four times with Salmonella that carried a eukaryotic expression plasmid encoding the secretory listerial protein listeriolysin were protected against a subsequent lethal challenge with this pathogen. A single dose was already partially protective. The efficiency of this vaccination procedure was due to transfer of the expression plasmid from the bacterial carrier to the mammalian host. Evidence for such an event could be obtained in vivo and in vitro. Expression of the desired antigen in various lymphoid tissues was already detectable 1 day after administration of the DNA vaccine and persisted for at least 1 month in spleen and mesenteric lymph nodes. Induction of cytotoxic and helper T cell responses was observed in all mouse strains tested including outbred strains whereas antibodies were mainly detected in BALB/c. Furthermore, we could show that immunogenicity could be improved by increasing the invasiveness of the bacterial carrier.     

A novel antibiotic free plasmid selection system: advances in safe and efficient DNA therapy

The presence of antibiotic resistance genes in the delivered plasmids is one of the drawbacks of modern gene therapy and DNA vaccine applications. Here, we describe a strategy that allows for plasmid selection in bacterial hosts, without the requirement of any selection marker. Several bacterial strains were modified, so that the plasmid's replicational inhibitor RNA I could suppress the translation of a growth essential gene by RNA-RNA antisense reaction. An essential gene (murA) was modified such that a repressor protein (tetR) would hamper its expression. Only in the presence of plasmid and, hence, RNA I, was tetR turned down and murA expressed. Different commercially available plasmids could be selected by various modified Escherichia coli strains. We further designed a minimalistic plasmid devoid of any selection marker. All of the clones (n=6) examined, when the modified strain JM109-murselect was used for selection, contained plasmids. Thus, we have designed bacterial host strains that for the first time serve to select and maintain plasmids without the use of any selection marker or other additional sequence on the plasmid. Consequently, such plasmids may not only be safer, but due to their decreased size, advantages for the manufacturer and higher transfection efficiencies are anticipated.     

Hfr-mediated conjugative transfer of pBR322 vector carrying the chromosomal DNA of Escherichia coli

Hybrid plasmids consisting of a non-mobilized plasmid, pBR322, and a segment of chromosomal DNA of Escherichia coli could be transferred from an Hfr donor to recipient cells by a bacterial mating. When the chromosomal DNA in the plasmid corresponded to the early transfer region of the Hfr, the frequency of the transfer was high. The recA function of both donor and recipient cells was required in the transfer. The physical association of the hybrid plasmid with the transferring Hfr chromosome through the homologous sequences may mediate the transfer of the non-mobilized plasmid. This phenomenon is useful for the determination of the chromosomal location of an unidentified fragment cloned in a non-mobilized plasmid.     

A method for sequencing polymerase chain reaction products can be used to sequence Bacillus subtilis "miniprep" plasmid DNA

Several methods for sequencing double-stranded plasmid DNA isolated from E. coli have been described. These methods are usually not effective when used to sequence plasmid DNA isolated from Bacillus subtilis. In the course of developing a simplified version of a previously published protocol for polymerase chain reaction product sequencing, it was found that this protocol could be used for sequencing plasmid DNA isolated from Bacillus subtilis.     

Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants.

The partition system of the low-copy-number plasmid/prophage of bacteriophage P1 encodes two proteins, ParA and ParB, and contains a DNA site called parS. ParB and the Escherichia coli protein IHF bind to parS to form the partition complex, in which parS is wrapped around ParB and IHF in a precise three-dimensional conformation. Partition can be thought of as a positioning reaction; the plasmid-encoded components ensure that at least one copy of the plasmid is positioned within each new daughter cell. We have used an E. coli chromosomal partition mutant to test whether this positioning is mediated by direct plasmid-chromosomal attachment, for example, by pairing of the partition complex that forms at parS with a bacterial attachment site. The E. coli MukB protein is required for proper chromosomal positioning, so that mukB mutants generate some cells without chromosomes (anucleate cells) at each cell division. We analyzed the plasmid distribution in nucleate and anucleate mukB cells. We found that P1 plasmids are stable in mukB mutants and that they partition into both nucleate and anucleate cells. This indicates that the P1 partition complex is not used to pair plasmids with the host chromosome and that P1 plasmids must be responsible for their own proper cellular localization, presumably through host-plasmid protein-protein interactions.