Plasmid DNA Synthesis Research Articles

A plasmid system with tunable copy number

Plasmids are one of the most commonly used platforms for genetic engineering and recombinant gene expression in bacteria. The range of available copy numbers for cloning vectors is largely restricted to the handful of Origins of Replication (ORIs) that have been isolated from plasmids found in nature. Here, we introduce two systems that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell: a plasmid with an anhydrotetracycline-controlled copy number, and a parallelized assay that is used to generate a continuous spectrum of 1194 ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our systems enable the precise control of gene expression, and our results highlight the importance of tuning plasmid copy number as a powerful tool for the optimization of synthetic biological systems.

In situ bone tissue engineering using gene delivery nanocomplexes

Gene delivery offers promising outcomes for functional recovery or regeneration of lost tissues at cellular and tissue levels. However, more efficient carriers are needed to safely and locally delivery of genetic materials. Herein, we demonstrate microfluidic-assisted synthesis of plasmid DNA (pDNA)-based nanocomplexe (NC) platforms for bone tissue regeneration. pDNA encoding human bone morphogenesis protein-2 (BMP-2) was used as a gene of interest. Formation and fine-tuning of nanocomplexes (NCs) between pDNA and chitosan (CS) as carriers were performed using a micromixer platform. Flow characteristics were adjusted to tune mixing time and consequently size, zeta potential, and compactness of assembled NCs. Subsequently, NCs were immobilized on a nanofibrous Poly(ε-caprolactone) (PCL) scaffold functionalized with metalloprotease-sensitive peptide (MMP-sensitive). This construct can provide an environmental-sensitive and localized gene delivery platform. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells (MSCs) was studied using chemical and biological assays. The presented results converge to indicate a great potential of the developed methodology for in situ bone tissue engineering using immobilized microfluidic-synthesized gene delivery nanocomplexes, which is readily expandable in the field of regenerative nanomedicine. Statement of significanceIn this study, we demonstrate microfluidic-assisted synthesis of plasmid DNA (pDNA)-based nanocomplexes (NCs) platforms for bone tissue regeneration. We used pDNA encoding human bone morphogenesis protein-2 (BMP-2) as the gene of interest. Using micromixer platform nanocomplexes (NCs) between pDNA and chitosan (CS) were fabricated and optimized. NCs were immobilized on a nanofibrous polycaprolactone scaffold functionalized with metalloprotease-sensitive peptide. In vitro and in vivo assays confirmed the osteogenic differentiation of mesenchymal stem cells (MSCs). The obtained data indicated great potential of the developed methodology for in situ bone tissue engineering using immobilized microfluidic-synthesized gene delivery nanocomplexes, which is readily expandable in the field of regenerative nanomedicine.

Hfq protein deficiency in Escherichia coli affects ColE1-like but not λ plasmid DNA replication

Hfq is a nucleic acid-binding protein involved in controlling several aspects of RNA metabolism. It achieves this regulatory function by modulating the translational activity and stability of different mRNAs, generally via interactions with stress-related small regulatory sRNAs. However, besides its role in the coordination of translation of bacterial mRNA, Hfq is also a nucleoid-associated DNA-binding protein. Motivated by the above property of Hfq, we investigated if hfq gene mutation has implications for the regulation of DNA replication. Efficiency of ColE1-like (pMB1- and p15A replicons) and bacteriophage λ-derived plasmids’ replication has been investigated in wild-type strain and otherwise isogenic hfq mutant of Escherichia coli. Significant differences in plasmid amount and kinetics of plasmid DNA synthesis were observed between the two tested bacterial hosts for ColE1-like replicons, but not for λ plasmid. Furthermore, ColE1-like plasmids replicated more efficiently in wild-type cells than in the hfq mutant in the early exponential phase of growth, but less efficiently in late exponential and early stationary phases. Hfq levels in the wild-type host, estimated by Western-blotting, were increased at the latter phases relative to the former one. Moreover, effects of the hfq mutation on ColE1-like plasmid replication were impaired in the absence of the rom gene, coding for a protein enhancing RNA I–RNA II interactions during the control of the replication initiation. These results are discussed in the light of a potential mechanism by which Hfq protein may influence replication of some, but not all, replicons in E. coli.

RK2 plasmid dynamics in Caulobacter crescentus cells – two modes of DNA replication initiation

Undisturbed plasmid dynamics is required for the stable maintenance of plasmid DNA in bacterial cells. In this work, we analysed subcellular localization, DNA synthesis and nucleoprotein complex formation of plasmid RK2 during the cell cycle of Caulobacter crescentus. Our microscopic observations showed asymmetrical distribution of plasmid RK2 foci between the two compartments of Caulobacter predivisional cells, resulting in asymmetrical allocation of plasmids to progeny cells. Moreover, using a quantitative PCR (qPCR) method, we estimated that multiple plasmid particles form a single fluorescent focus and that the number of plasmids per focus is approximately equal in both swarmer and predivisional Caulobacter cells. Analysis of the dynamics of TrfA-oriV complex formation during the Caulobacter cell cycle revealed that TrfA binds oriV primarily during the G1 phase, however, plasmid DNA synthesis occurs during the S and G2 phases of the Caulobacter cell cycle. Both in vitro and in vivo analysis of RK2 replication initiation in C. crescentus cells demonstrated that it is independent of the Caulobacter DnaA protein in the presence of the longer version of TrfA protein, TrfA-44. However, in vivo stability tests of plasmid RK2 derivatives suggested that a DnaA-dependent mode of plasmid replication initiation is also possible.

Comparative proteomic and genetic analyses reveal unidentified mutations in Escherichia coli XL1-Blue and DH5α

Escherichia coli has been used widely in laboratory and the biotech industry. However, the genetic and metabolic characteristics remain inadequately studied, particularly for those strains with extensive genetic manipulations that might have resulted in unknown mutations. Here, we demonstrate a comparative proteomics and genetics approach to identify unknown mutations in E. coli K-12 derivatives. The comparative proteomic and genetic analyses revealed an IS5 disruption of the kdgR gene in two commonly used derivative strains of E. coli K-12, XL1-Blue and DH5α, compared with K-12 wild-type strain W3110. In addition, a controversial deoR mutation was clarified as a wild type in E. coli DH5α using the same approach. This approach should be useful in characterizing the unknown mutations in various mutant strains developed. At the same time, comparative proteomic analysis also revealed the distinct metabolic characteristic of the two derivatives: higher biosynthetic flux to purine nucleotides. This is potentially beneficial for the synthesis of plasmid DNA.

Pyruvate Kinase-Deficient Escherichia coli Exhibits Increased Plasmid Copy Number and Cyclic AMP Levels

Previously established consequences of abolishing pyruvate kinase (Pyk) activity in Escherichia coli during aerobic growth on glucose include reduced acetate production, elevated hexose monophosphate (HMP) pathway flux, elevated phosphoenolpyruvate carboxylase (Ppc) flux, and an increased ratio of phosphoenolpyruvate (PEP) to pyruvate. These traits inspired two hypotheses. First, the mutant (PB25) may maintain more plasmid than the wild type (JM101) by combining traits reported to facilitate plasmid DNA synthesis (i.e., decreased Pyk flux and increased HMP pathway and Ppc fluxes). Second, PB25 likely possesses a higher level of cyclic AMP (cAMP) than JM101. This is based on reports that connect elevated PEP/pyruvate ratios to phosphotransferase system signaling and adenylate cyclase activation. To test the first hypothesis, the strains were transformed with a pUC-based, high-copy-number plasmid (pGFPuv), and copy numbers were measured. PB25 exhibited a fourfold-higher copy number than JM101 when grown at 37 degrees C. At 42 degrees C, its plasmid content was ninefold higher than JM101 at 37 degrees C. To test the second hypothesis, cAMP was measured, and the results confirmed it to be higher in PB25 than JM101. This elevation was not enough to elicit a strong regulatory effect, however, as indicated by the comparative expression of the pGFPuv-based reporter gene, gfp(uv), under the control of the cAMP-responsive lac promoter. The elevated cAMP in PB25 suggests that Pyk may participate in glucose catabolite repression by serving among all of the factors that tighten gene expression.

Essential Elements of a Licensed, Mammalian Plasmid Origin of DNA Synthesis

We developed a mammalian plasmid replicon with a formerly uncharacterized origin of DNA synthesis, 8xRep*. 8xRep* functions efficiently to support once-per-cell-cycle synthesis of plasmid DNA which initiates within Rep*. By characterizing Rep*'s requirements for acting as an origin, we have uncovered several striking properties it shares with DS, the only other well-characterized, licensed, mammalian plasmid origin of DNA synthesis. Rep* contains a pair of previously unrecognized Epstein-Barr nuclear antigen 1 (EBNA1)-binding sites that are both necessary and sufficient in cis for its origin activity. These sites have an essential 21-bp center-to-center spacing, are bent by EBNA1, and recruit the origin recognition complex. The properties shared between DS and Rep* define cis and trans characteristics of a mammalian, extrachromosomal replicon. The role of EBNA1 likely reflects its evolution from cellular factors involved in the assembly of the initiation machinery.

Gyrase inhibitors and thymine starvation disrupt the normal pattern of plasmid RK2 localization in Escherichia coli.

Multicopy plasmids in Escherichia coli are not randomly distributed throughout the cell but exist as defined clusters that are localized at the mid-cell, or at the 1/4 and 3/4 cell length positions. To explore the factors that contribute to plasmid clustering and localization, E. coli cells carrying a plasmid RK2 derivative that can be tagged with a green fluorescent protein-LacI fusion protein were subjected to various conditions that interfere with plasmid superhelicity and/or DNA replication. The various treatments included thymine starvation and the addition of the gyrase inhibitors nalidixic acid and novobiocin. In each case, localization of plasmid clusters at the preferred positions was disrupted but the plasmids remained in clusters, suggesting that normal plasmid superhelicity and DNA synthesis in elongating cells are not required for the clustering of individual plasmid molecules. It was also observed that the inhibition of DNA replication by these treatments produced filaments in which the plasmid clusters were confined to one or two nucleoid bodies, which were located near the midline of the filament and were not evenly spaced throughout the filament, as is found in cells treated with cephalexin. Finally, the enhanced yellow fluorescent protein-RarA fusion protein was used to localize the replication complex in individual E. coli cells. Novobiocin and nalidixic acid treatment both resulted in rapid loss of RarA foci. Under these conditions the RK2 plasmid clusters were not disassembled, suggesting that a completely intact replication complex is not required for plasmid clustering.

Modulation of DNA Synthesis in Saccharomyces cerevisiae Nuclear Extract by DNA Polymerases and the Origin Recognition Complex

We have analyzed the modulation of DNA synthesis on a supercoiled plasmid DNA template by DNA polymerases (pol), minichromosome maintenance protein complex (Mcm), topoisomerases, and the origin recognition complex (ORC) using an in vitro assay system. Antisera specific against the four-subunit pol alpha, the catalytic subunit of pol delta, and the Mcm467 complex each inhibited DNA synthesis. However, DNA synthesis in this system appeared to be independent of polepsilon. Consequently, DNA synthesis in the in vitro system appeared to depend only on two polymerases, alpha and delta, as well as the Mcm467 DNA helicase. This system requires supercoiled plasmid DNA template and DNA synthesis absolutely required DNA topoisomerase I. In addition, we also report here a novel finding that purified recombinant six subunit ORC significantly stimulated the DNA synthesis on a supercoiled plasmid DNA template containing an autonomously replicating sequence, ARS1.

Development of a green fluorescent protein‐based laboratory curriculum

A laboratory curriculum has been designed for an undergraduate biochemistry course that focuses on the investigation of the green fluorescent protein (GFP). The sequence of procedures extends from analysis of the DNA sequence through PCR amplification, recombinant plasmid DNA synthesis, bacterial transformation, expression, isolation, and characterization of the protein by SDS-PAGE. A survey of participants found that the majority of them were performing most of these procedures for the first time and that participants found the exercises enjoyable and considered them a significant aid to their understanding of biochemistry, cell biology, and molecular genetics.

Template directed DNA photoligation via substituted 2'-deoxyuridine.

An efficient and reversible template-directed photoligation of oligodeoxynucleotides (ODNs) using 5-carboxyvinyldeoxyuridine (CVU) was described. We have also demonstrated about photochemical synthesis of circularized DNA and padlocking plasmid DNA via CVU mediated photoreaction.

An SSB encoded by and operating on linear killer plasmids from Kluyveromyces lactis.

The Kluyveromyces lactis linear plasmids k1 and k2 belong to the family of protein-primed linear DNA genomes, which includes adenoviruses. Here we identify the 18 kDa gene product of k2ORF5 as a novel putative single-stranded DNA binding protein, SSB. As judged from Western analysis using an epitope-tagged fusion protein and ssDNA-agarose affinity chromatography, the Orf5 protein preferentially binds to ssDNA in vitro. Consistently, electrophoretic mobility shift assays demonstrate that ssDNA plasmid probes from k1 and k2 are retarded by this Orf5-associated SSB activity. ORF5 gene shuffle-mediated mutagenesis in vivo results in k1/k2 plasmid instability, pointing towards a role for the Orf5 protein in plasmid replication. Consistently, the Orf5 protein protects ssDNA from exonuclease digestion and stimulates Klenow enzyme. Our findings suggest a functional role for the Orf5 protein as a putative SSB probably required during k1/k2 plasmid DNA synthesis.

Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis.

To develop a simple, speedy, economical and widely applicable method for multiple-site mutagenesis, we have substantially modified the Quik-Change Site-Directed Mutagenesis Kit protocol (Stratagene, La Jolla, CA). Our new protocol consists of (i) a PCR reaction using an in vitro technique, LDA (ligation-during-amplification), (ii) a DPN:I treatment to digest parental DNA and to make megaprimers and (iii) a synthesis of double-stranded plasmid DNA for bacterial transformation. While the Quik Change Kit protocol introduces mutations at a single site, requiring two complementary mutagenic oligonucleotides, our new protocol requires only one mutagenic oligonucleotide for a mutation site, and can introduce mutations in a plasmid at multiple sites simultaneously. A targeting efficiency >70% was consistently achieved for multiple-site mutagenesis. Furthermore, the new protocol allows random mutagenesis with degenerative primers, because it does not use two complementary primers. Our mutagenesis strategy was successfully used to alter the fluorescence properties of green fluorescent protein (GFP), creating a new-color GFP mutant, cyan-green fluorescent protein (CGFP). An eminent feature of CGFP is its remarkable stability in a wide pH range (pH 4-12). The use of CGFP would allow us to monitor protein localization quantitatively in acidic organelles in secretory pathways.

Separate roles of Escherichia coli replication proteins in synthesis and partitioning of pSC101 plasmid DNA.

We report here that the Escherichia coli replication proteins DnaA, which is required to initiate replication of both the chromosome and plasmid pSC101, and DnaB, the helicase that unwinds strands during DNA replication, have effects on plasmid partitioning that are distinct from their functions in promoting plasmid DNA replication. Temperature-sensitive dnaB mutants cultured under conditions permissive for DNA replication failed to partition plasmids normally, and when cultured under conditions that prevent replication, they showed loss of the entire multicopy pool of plasmid replicons from half of the bacterial population during a single cell division. As was observed previously for DnaA, overexpression of the wild-type DnaB protein conversely stabilized the inheritance of partition-defective plasmids while not increasing plasmid copy number. The identification of dnaA mutations that selectively affected either replication or partitioning further demonstrated the separate roles of DnaA in these functions. The partition-related actions of DnaA were localized to a domain (the cell membrane binding domain) that is physically separate from the DnaA domain that interacts with other host replication proteins. Our results identify bacterial replication proteins that participate in partitioning of the pSC101 plasmid and provide evidence that these proteins mediate plasmid partitioning independently of their role in DNA synthesis.

Rep*: a viral element that can partially replace the origin of plasmid DNA synthesis of Epstein-Barr virus.

Replication of the Epstein-Barr viral (EBV) genome occurs once per cell cycle during latent infection. Similarly, plasmids containing EBV's plasmid origin of replication, oriP, are replicated once per cell cycle. Replication from oriP requires EBV nuclear antigen 1 (EBNA-1) in trans; however, its contributions to this replication are unknown. oriP contains 24 EBNA-1 binding sites; 20 are located within the family of repeats, and 4 are found within the dyad symmetry element. The site of initiation of DNA replication within oriP is at or near the dyad symmetry element. We have identified a plasmid that contains the family of repeats but lacks the dyad symmetry element whose replication can be detected for a limited number of cell cycles. The detection of short-term replication of this plasmid requires EBNA-1 and can be inhibited by a dominant-negative inhibitor of EBNA-1. We have identified two regions within this plasmid which can independently contribute to this replication in the absence of the dyad symmetry element of oriP. One region contains native EBV sequences within the BamHI C fragment of the B95-8 genome of EBV; the other contains sequences within the simian virus 40 genome. We have mapped the region contributing to replication within the EBV sequences to a 298-bp fragment, Rep*. Plasmids which contain three copies of Rep* plus the family of repeats support replication more efficiently than those with one copy, consistent with a stochastic model for the initiation of DNA synthesis. Plasmids with three copies of Rep* also support long-term replication in the presence of EBNA-1. These observations together indicate that the latent origin of replication of EBV is more complex than formerly appreciated; it is a multicomponent origin of which the dyad symmetry element is one efficient component. The experimental approach described here could be used to identify eukaryotic sequences which mediate DNA synthesis, albeit inefficiently.

Regulation of cellular response to cisplatin-induced DNA damage and DNA repair in cells overexpressing p185(erbB-2) is dependent on the ras signaling pathway.

We have examined the role of erbB-2 expression in the modulation of cellular toxicity to cisplatin. We have demonstrated that treatment of NIH3T3-erbB-2 cells, which overexpress the p185(erbB-2) product of the human erbB-2 gene, with a monoclonal antibody directed against the extracellular domain (TAb-250), results in enhanced cisplatin cytotoxicity. A similar enhancement was obtained when cells were exposed to herbimycin A and its analogue CP127 374, both of which inhibit tyrosine kinase activity. Using the host cell reactivation (HCR) of reporter gene expression from cisplatin-damaged plasmid and unscheduled DNA synthesis (UDS) following cisplatin treatment of cells, we have found that modulation of erbB-2 by TAb-250 was associated with inhibition of DNA repair. TAb-250 alone, under conditions which modulate DNA repair, slightly reduces the S-phase of the cell cycle, while cisplatin induced arrest at S and G2 phases. Combination of TAb-250 and cisplatin only slightly prevented cisplatin-induced S and G2 blocks. Since the ras pathway is one of the major signaling components coupled to erbB-2, we have examined the role of ras in DNA repair regulation. Transient expression of a ras dominant negative mutant, Asn-17-ras(H), prevents DNA repair modulation by TAb-250, suggesting that the erbB-2 receptor regulates DNA repair mechanism(s), at least in part, through ras-coupled pathway(s).

Replication of plasmids derived from P1, F, R1, R6K and RK2 replicons in amino acid-starved Escherichia coli stringent and relaxed strains.

Replication of mini-plasmids derived from bacteriophage P1 and naturally existing plasmids F, R1, R6K and RK2 in otherwise isogenic relA+ and relA- Escherichia coli strains during amino acid starvation and limitation was investigated. Since it was previously demonstrated that inhibition of DNA synthesis or amplification of plasmid DNA may depend on the nature of deprived amino acid, we starved bacteria for five different amino acids. We found differential replication of all these plasmids but RK2 (which did not replicate at all in amino acid-starved bacteria) during the stringent and relaxed response. While in almost all cases plasmid DNA replication was inhibited during the stringent response irrespective of the nature of deprived amino acid, wild-type or copy-up mini-P1, mini-F and mini-R1 plasmids replicated in relA- bacteria depending on the kind of starvation. R6K-derived plasmids harbouring ori beta and gamma (but not those containing ori alpha, beta and gamma or only ori gamma) were able to replicate in relA- bacteria starved for all tested amino acids. Possible explanations for the mechanisms of regulation of replication of plasmids derived from P1, F, R1, R6K and RK2 during amino acid starvation are discussed. Our results also indicate that, like in the case of some other replicons, appropriate amino acid starvation or limitation may be used as a method for efficient amplification of plasmids derived from P1, F, R1 and R6K.

Differential Replication of Plasmids during Stringent and Relaxed Response of Escherichia coli

Stringent control of DNA replication has been demonstrated for a few replicons like oriC, pBR322, and plasmids derived from coliphage λ. In this study we investigated the replication of other plasmids harboring a well defined origin (orip15A, oripSC101, and ori RK2 = oriV) in amino-acid-starved stringent and relaxed strains of Escherichia coli. We found differential replication of plasmids during stringent and relaxed response. Inhibition of DNA synthesis or amplification of plasmid DNA in amino acid-starved relA+ and rel A- cells depends on the kind of replicon and, surprisingly, on the nature of deprived amino acid. We conclude that there are no general rules for stringent control of DNA replication and each replicon must be considered separately. There are, however, possible explanations for the differences shown between replicons in their response to stringent and relaxed conditions.

Regulation of replication of plasmid pBR322 in amino acid-starved Escherichia coli strains.

The stringent response causes inhibition of replication of plasmid pBR322 in amino acid-starved Escherichia coli cells whereas in relaxed mutants the replication of this plasmid proceeds for several hours. On the basis of density shift experiments and pulse-labelling experiments we showed that most of the pBR322 molecules begin replication during the relaxed response and the rate of plasmid DNA synthesis in unstarved and isoleucine-starved relA- bacteria is similar. We found that the Rom function plays a key role in the stringent control of plasmid pBR322 replication, as insertional inactivation of the rom gene causes amplification of pBR322rom- in both relA- and relA+ strains during amino acid starvation. Moreover, pUC19, which is a pBR322-derived plasmid lacking the rom gene, behaves like pBR322rom-, whereas introduction of the rom gene into the pUC19 replicon drives it into the pBR322 mode of replication in amino acid-starved bacteria. A model for the regulation of pBR322 plasmid DNA replication by Rom protein in amino acid-starved Escherichia coli strains is proposed.

Control of replication of the Lactobacillus pentosus plasmid p353-2: evidence for a mechanism involving transcriptional attenuation of the gene coding for the replication protein.

The synthesis of plasmid DNA and of RNA encoded by the replication protein gene (rep) of plasmid p353-2 of Lactobacillus pentosus was studied for the wild-type plasmid and for a mutant plasmid with a deletion in the 5' untranslated region of the rep gene. Plasmid p353-2 codes for two countertranscript RNAs (CT-RNA) of approximately 75 and 250 nucleotides transcribed from the 5' untranslated region of the rep gene, in opposite directions. In a mutant plasmid with a deletion of the promoter and part of the CT-RNA-encoding sequence which shows a 5- to 10-fold increase in copy number compared to the wild-type plasmid, no CT-RNA could be detected. In the wild-type plasmid more than 90% of transcription initiated at a promoter upstream of the rep gene is prematurely terminated to form a 190 nucleotide truncated RNA, whereas in the mutant plasmid nearly all transcripts reach a size (1100 nucleotides) corresponding to that of the rep gene. A model is presented for the role of CT-RNA in control of plasmid replication, similar to that previously presented for the staphylococcal plasmid pT181, involving a mechanism of transcriptional attenuation of rep RNA at a site just upstream of the rep gene.