Plasmid Extraction Research Articles

High-throughput plasmid extraction using microtiter plates.

Plasmid extraction is typically performed to produce template DNA for a desired molecular biological reaction, or set of reactions, such as restriction endonuclease digestion (see Chapter 20), DNA sequencing (see Chapter 22), in vitro mutagenesis (see Chapters 23– 26), transformation (see Chapters 4 and 5), transfection, or probe generation. The principal methodologies of extraction, involving either alkaline lysis or boiling are well established () and are described in detail in Chapters 8 and 9. These methods are relatively crude but are both inexpensive and rapid, and they yield DNA that is suitable for many molecular biology protocols. DNA of higher purity can be prepared using silica resin-based anion exchange as described in Chapter 10 or by separation on cesium chloride gradients (for details, see ref. , Chapter 1, Protocol 10). Plasmid minipreps are a ubiquitous part of molecular biology. They are simple and easy to perform but can be very tedious when large numbers of samples are involved. In recent years, endeavors such as the human genome project have led to the development of methods for the manageable high-throughput extraction of plasmids (, , , , , , , , , , , , , ).

Encapsulation of plasmid DNA in PLGA-stearylamine microspheres: A comparison of solvent evaporation and spray-drying methods

Stearylamine, a positively charged hydrophobic molecule, was tested as a formulation agent for the encapsulation of a model plasmid in PLGA microspheres. The primary objective was to compare the spray-drying and double emulsion solvent evaporation methods and evaluate their suitability for fabricating PLGA-stearylamine plasmid-entrapped microspheres. A luciferase reporter gene plasmid (pGL3-Con) was formulated into microspheres using a 64 kDa PLGA 50:50 polymer blended with stearylamine (SA) at a range of concentrations up to 15%m/m, by the solvent evaporation and spray-drying methods. The microspheres were characterized regarding their size distributions, zeta potentials and morphology by laser diffraction, electrophoretic mobility and scanning electron microscopy (SEM), respectively. Formulated plasmid extracts were assessed for physical damage by agarose gel electrophoresis, and the in vitro biological activity was determined by tranfection of a human embryo kidney epithelial (293) cell line. Size distribution analysis showed that SA reduced the median diameters of spray-dried particles from 8.32 to 3.64 microns, with a corresponding reduction in the spread of the distribution, but solvent evaporation microspheres showed an increased median diameter on addition of SA. Concentrations of SA above 10%m/m resulted in disruption of the smooth morphology of the solvent evaporation particles. There was a SA concentration-dependent tendency in the increase of surface positive charge and resistance to serum nuclease assault and in vitro expression of luciferase protein. These results show that SA and possibly other charged hydrophobic molecules may be useful agents in the formulation of particulate DNA vaccines by both methods.

Construction of multivalent DNA vaccines forMycobacterium tuberculosis and its immunogenicity

The coding regions of Ag85B MPT-64, and ESAT-6 secreted proteins were cloned initially into the eukaryotic expression vector pJW4303, then transformed toE coli Top 10 strain for plasmid DNA extraction and further analysis. Plasmids containing the right insertion were sequenced to confirm their identity. COS7 cells were transfected with a mixture containing serially diluted plasmid DNA encoding three secreted proteins and Lipofectin (Gibco). The supernatants and pellets prepared from various cell lines were run on SDS-PAGE gel and the expression of these proteins in COS7 cells were demonstrated by immunoblot using polyclonal or monoclonal antiserum of M.TBH37Rv. 21 days after first vaccination of C57BL-6 mice by all three recombinant eukaryotic expressing vectors, antibody titer for Ag85B reached 1: 3200. 21 days after second vaccination, the antibody titer reached 1: 102400. The highest antibody levels induced by multivalent vaccines after the second injection were equal to or even greater than the highest antibody levels of single DNA vaccine reported in literature after third injections. Antibody titer of MPT-64 was 1: 50 after the first injection and it reached 1: 200 after the second injection. No antigen-specific antibody against ESAT-6 was detected in sera harvested from immunized mice 21 days after both injections. Antigen-specific IFN-γ level of Ag85B was 110 pg/mL while no antigen-specific IFN-γ level of ESAT-6 and MPT-64 was detected even after third injections. To our knowledge, it is the first time that studies of polyvalent recombinant DNA vaccines against TB were carried out in C57BL-6 mice. Our results indicated that multiple DNA vaccines could be used to enhance protective responses against MTB.

A rapid and easy method for production and selection of recombinant adenovirus genomes

Adenoviruses are used widely as vectors for gene therapy. Due to the large size of their genome there is a low frequency of unique restriction sites and many techniques have been described to construct recombinant viruses. Whatever the considered technique, the Escherichia coli strain BJ5183 is used to obtain recombinant adenovirus genomes in a plasmid, or to construct defective viral backbones which will be used to produce infectious viral particles by homologous recombination in HEK293 cells. Unfortunately BJ5183 bacteria do not produce a sufficient amount of plasmid DNA to allow for restriction analysis. Plasmids have to be transferred into another strain to detect the expected construction. It is reported now that the common E. coli strain, Top10F′ can be used for the construction of recombinant adenovirus genomes. A plasmid carrying a kanamycin resistance gene and containing the two ends of the adenovirus genome was used. It permits modification by classical molecular biology techniques or homologous recombination at both ends of the genome. The remainder of the genome is introduced by homologous recombination in Top10F′. Several homologous recombination steps were successfully performed without the steps of extraction and introduction of plasmid DNA in another strain to check the plasmids obtained.

The answer is

The objective of this study was to investigate the molecular mechanisms explaining the multidrug-resistant (MDR) phenotype found in a novel clinical Shewanella sp. strain (Shew256) recovered from a diabetic patient. Whole-genome shotgun sequencing was performed using Illumina MiSeq-I and Nextera XT DNA library. De novo assembly was performed with SPAdes. RAST Server was used to predict the open-reading frames and the predictions were confirmed using BLAST. Further genomic analysis was carried out using average nucleotide identity (ANI), ACT (Artemis), OrthoMCL, ARG-ANNOT, ISfinder, PHAST, tRNAscan-SE, plasmidSPAdes, PlasmidFinder and MAUVE. PCR and plasmid extraction were also performed. Genomic analysis revealed a total of 456 predicted genes unique to Shew256 compared with other Shewanella genomes. Moreover, the presence of different resistance genes, including blaPER-2, was found. A complex class 1 integron containing the ISCR1 gene, disrupted by two putative transposase genes, was identified. Furthermore, other resistance genes, a transposon containing aph(3′), insertion sequences, phages and non-coding RNAs were also found. In conclusion, evidence of acquisition of resistance genes and mobile elements that could explain the MDR phenotype were observed. This Shewanella sp. represents a prime example of how antibiotic resistance determinants can be acquired by uncommon pathogens.

Control of chromosomal DNA replication in the early Xenopus embryo.

In order for the genome to be faithfully maintained, chromosomal DNA must be precisely replicated and segregated in each cell cycle. Over the last decade an enormous amount has been learned about how this is achieved. Much of the progress has come from genetic analysis in yeast. However, biochemical analysis of cell cycle events using extracts prepared from eggs of the South African clawed toad Xenopus laevis has also played an important role. Although there are differences in the detailed regulation of the yeast and frog cell cycles, the basic cell cycle machinery appears to have been conserved throughout evolution. The results obtained in these model organisms, therefore, seem likely to be generally applicable to the cell cycles of all eukaryotes. ### Xenopus eggs and egg extracts The fertilized Xenopus egg undergoes 12 synchronous rounds of cell division in ∼8 h. These cell divisions take place in the absence of growth, subdividing the large (∼1 mm diameter) single‐celled egg into ∼4000 smaller cells. Transcription does not occur during these early embryonic divisions, although translation of pre‐existing maternal mRNA continues. Most of the proteins required for cell cycle progression are pre‐formed in the egg, and the continuing translation of a single protein (cyclin B) can support passage through the whole cell cycle (Murray and Kirschner, 1989). The stockpile of cell cycle proteins present in the Xenopus egg became exploitable by biochemical means following the development of cell‐free extracts that support all the nuclear events of the early embryonic cell division cycle (Lohka and Masui, 1983). Gentle lysis associated with minimal dilution of the cytoplasm yields a ‘low speed supernatant’ that maintains all the cell cycle activities present in the intact egg. These ‘low speed supernatants’ support precise rounds of DNA replication on exogenously added DNA templates, and like the intact egg, only re‐replicate DNA after passage through …

Construction and identification of the eukaryotic expression vector of vaccinia virus expressing human interleukin‐2

OBJECTIVE: Cancer gene therapy using interleukin‐2 (IL‐2) has generated much interest because of the potent antitumor effect of this cytokine. There is ongoing research into one promising new gene therapy approach for cancer using the vaccinia virus vector. The purpose of this study was to construct a vaccinia eukaryotic expression vector, pMJ601, which contains human IL‐2 (hIL‐2; pMJ601hIL‐2) and can be used for the treatment of gastric carcinomas. METHODS: Genetic engineering techniques such as plasmid extraction, agarose gel electrophoresis, restriction analysis, ligation, preparation of competent cells, transformation and DNA sequence analysis were used to clone the hIL‐2 gene into pBluescript II SK+/– and pMJ601 and identify these vectors. RESULTS: Fragments of hIL‐2 DNA from pLXSN from an EcoR1–BamHI restriction enzyme digest were successfully cloned into pBluescript II SK+/–. In addition, hIL‐2 DNA from pBluescript II SK+/– with a SalI‐BamHI restriction enzyme digest was also successfully cloned into pMJ601. CONCLUSION: Constructing a pMJ601 vector that expresses the hIL‐2 gene is an important step toward being able to treat gastric carcinoma using gene therapy.

Use of a ‘Miniprep’ for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium

Conventional methods of plasmid extraction are largely unsuited to diagnostic laboratories. The 'Miniprep' is a rapid method that utilises a centrifugable column to separate plasmid DNA from chromosomal DNA. We have modified this technique to extract plasmid DNA from seven strains of vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF): 1% mannitol was added to the growth medium and cell lysis was achieved by incubation in 10 mg of lysozyme/ml in 10 mM Tris, 1 mM EDTA and 25% sucrose at pH 8.0. RNase A was added to plasmid eluate rather than at the lytic step. In comparison to a standard phenol/chloroform method, Miniprep completely eliminated chromosomal interference in gel electrophoresis but otherwise produced identical plasmid profiles. Plasmids obtained from the VGREF ranged from 42 to 1.3 Md. Band densities on a single elution from the Miniprep varied from 8.3 to 106.3 relative units. Double elution increased band densities from the same preparation from 30.4 to 196 relative units; mean percentage increases per track between 7.0 and 34.6%. This method is suitable to achieve plasmid DNA extraction from VGREF within 1 h, making the process more suitable for diagnostic laboratories.

A coelectroporation method for the isolation of cryptic plasmids from Lactococcus lactis.

A coelectroporation method using a marker plasmid for indirect selection of lactococcal plasmids with unassigned functions was evaluated. Cryptic plasmids were mixed with an erythromycin resistance (Eryr) marker plasmid and introduced into a recipient strain by electroporation, followed by plasmid extraction of erythromycin-resistant transformants. By optimizing the ratio between the marker plasmid and the cryptic plasmids, an average of 20% cotransformants was obtained, including combinations of more than one cryptic plasmid. The marker plasmid pSA3 was easily eliminated from the cotransformed cells by subculture without selective pressure. This cotransformation approach reduces the number of colonies that must be screened to find transformants harbouring cryptic plasmids. The method facilitates the isolation of cryptic plasmids, helps in assigning functions to unknown plasmids and allows construction of food-grade lactococcal strains with new combinations of wild-type plasmids.

Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction

There is a clear association between heavy antimicrobial consumption within a population and the frequent recovery of resistant bacteria, but whether a reduction in antimicrobial use can reverse this process is less clear. We investigated the effect of a national restriction of sulphonamide prescribing in the UK on the prevalence of sulphonamide resistance in Escherichia coli. Consecutive clinical isolates of E coli were collected at the Royal London Hospital in 1991 and 1999. These collections, each of more than 350 isolates, were compared. Minimum inhibitory concentrations of sulphamethoxazole and eight other antimicrobials were determined. The presence and locations of sulphonamide-resistance genes were examined by PCR, plasmid extraction, Southern hybridisation, and transconjugation. Despite a huge decrease in sulphonamide prescriptions (from 3,208,000 [corrected] prescriptions per year in 1991 to 77,000 [corrected] in 1999), the frequency of resistance remained high in 1999 (165/359 [46.0%] vs 143/360 [39.7%] in 1991; difference 6.2% [95% CI 20.9 to 13.3]). Integron-borne sulI was present in 16.4% of isolates in 1991 and 17.5% in 1999. The prevalence of sulII increased from 26.7% in 1991 to 36.5% in 1999 (difference 9.8% [3.1 to 16.5] p=0.0046). SulII was located on large plasmids, at least some of which were conjugative multiresistance determinants. These results show that a huge decrease in antibiotic prescribing does not necessarily reduce resistance within a useful time. The main reason seems to be the genetic linkage of the index resistance to other resistance determinants.

Purification of essentially RNA free plasmid DNA using a modified Escherichia coli host strain expressing ribonuclease A

Regulatory agencies have stringent requirements for the large-scale production of biotherapeutics. One of the difficulties associated with the manufacture of plasmid DNA for gene therapy is the removal of the host cell-related impurity RNA following cell lysis. We have constructed a modified Escherichia coli JM107 plasmid host (JMRNaseA), containing a bovine pancreatic ribonuclease (RNaseA) expression cassette, integrated into the host chromosome at the dif locus. The expressed RNaseA is translocated to the periplasm of the cell, and is released during primary plasmid extraction by alkaline lysis. The RNaseA protein is stable throughout incubation at high pH (∼12–12.5), and subsequently acts to hydrolyse host cell RNA present in the neutralised solution following alkaline lysis. Results with this strain harbouring pUC18, and a 2.4 kb pUC18Δ lacO, show that sufficient levels of ribonuclease (RNase) activity are produced to hydrolyse the bulk of the host RNA. This provides a suitable methodology for the removal of RNA, whilst avoiding the addition of exogenous animal sourced RNase and its associated regulatory requirements.

Extraction of plasmid DNA using reactor scale alkaline lysis and selective precipitation for scalable transient transfection.

DNA extracted and purified for vaccination, gene therapy or transfection of cultured cells has to meet different criteria. We describe herein, a scalable process for the primary extraction of plasmid DNA suitable for transient expression of recombinant protein. We focus on the scale up of alkaline lysis for the extraction of plasmid DNA from Escherichia coli, and use a simple stirred tank reactor system to achieve this. By adding a series of three precipitations (including a selective precipitation step with ammonium acetate) we enrich very quickly the plasmid DNA content in the extract. The process has been thus far used to extract up to 100 mg of plasmid from 1.5 l of clarified lysate, corresponding to an E.coli bioreactor fermentation of 3 l.

The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR), DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

Plasmid vectors for the analysis of protein-induced DNA bending.

Bending of DNA by proteins plays an important role in transcription initiation, DNA replication, and recombination. The degree of protein-induced DNA-bending can be simply and conveniently determined by combining gel electrophoresis of DNA-protein complexes with the use of special plasmid vectors carrying the bendable DNA sequence (,). The vectors contain duplicate sets of restriction sites in a direct repeat order and cloning sites for insertion of protein-binding sequences between the two sets. Restriction enzyme digestion readily generates DNA fragments, which are identical in size but differ in the location of the protein-binding site Fig. 1). Open image in new window Fig. 1. Schematic representation of the pBend3 insert located between the EcoRI and HindIII sites. pBend3 was constructed by cloning of the 236-base-pair EcoRI-HindIII fragment of pBend2 (4) into pBluescript SK (Stratagene). pBluescript is a high-copy-number plasmid and generates a large amount of DNA upon plasmid extraction. The EcoRI-HindIII fragment contains 17 duplicated restriction sites. The duplicated sites can be used to generate DNA fragments of identical length, but in which the protein-binding sequence (gray rectangle) is shifted. The sites XbaI and SalI (in boxes) are unique and suitable for cloning of the protein binding sequence. Restriction sites are not drawn to scale. The sequence of the insert is shown in the lower part of the figure.

Purification of plasmid DNA by tangential flow filtration.

A simple, scalable method for purification of plasmid DNA is described. The method includes modification of the classical alkaline-lysis-based plasmid extraction method by extending the solubilization step from less than 30 min to 24 h. The extraction is followed by the novel use of tangential flow filtration (TFF) for purification of the remaining contaminants. The method does not include the use of any organic solvents, RNase, high-speed centrifugation, or column chromatography steps. The method typically yields 15 to 20 mg of plasmid DNA per liter of bacterial culture and results in removal of >99% of RNA and >95% of the protein that remains after the modified alkaline lysis procedure. The procedure has been demonstrated to be effective in the isolation of seven different plasmids. Plasmids isolated using this method had comparable transfection capability relative to plasmid isolated using a classical, cesium chloride gradient-based method.

Expression of the Naegleria intron endonuclease is dependent on a functional group I self-cleaving ribozyme.

NaSSU1 is a complex nuclear group I intron found in several species of Naegleria, consisting of a large self-splicing group I ribozyme (NaGIR2), which itself is interrupted by a small, group I-like ribozyme (NaGIR1) and an open reading frame (ORF) coding for a homing endonuclease. The GIR1 ribozyme cleaves in vitro transcripts of NaSSU1 at two internal processing sites about 400 nt downstream of the 5' end of the intron, proximal to the endonuclease ORF. Here we demonstrate that self-cleavage of the excised intron also occurs in vivo in Naegleria gruberi, generating an ORF-containing RNA that possesses a short leader with a sequence element likely to be involved in gene expression. To assess the functional significance of self-cleavage, we constructed a genetic system in Saccharomyces cerevisiae. First, a mutant yeast strain was selected with a mutation in all the rRNA genes, rendering the rDNA resistant to cleavage by the Naegleria endonuclease. Active endonuclease, which is otherwise lethal, could be expressed readily in these cells. Endonuclease activity also could be detected in extracts of yeast harboring plasmids in which the endonuclease ORF was embedded in its native context in the intron. Analysis of the RNA from these yeast cells showed that the excised intron RNA was processed as in N. gruberi. A mutant intron constructed to prevent self-cleavage of the RNA failed to express endonuclease activity. These results support the hypothesis that the NaGIR1-catalyzed self-cleavage of the intron RNA is a key event in expression of the endonuclease.

Physiological aspects of hydrocarbon emulsification, metal resistance and DNA profile of biodegrading bacteria isolated from oil polluted sites

The production of biosurfactants was evaluated for seven bacterial strains isolated from different oil contaminated sites by the Emulsification Index using diesel oil as the hydrocarbon source. Minimum Inhibitory Concentrations of Mg2+, Cr3+ and Cu2+ were determined to identify the less sensitive bacteria in order to select the best strains for bioremediation. Plasmid extraction was also performed in order to search for gene sequences involved with biosurfactant synthesis. All strains were able to emulsify diesel oil. Rhodococcus ruber AC239 presented the best index (58%), followed by other Rhodococcus strains. Pseudomonas aeruginosa, R. ruber AC239, AC87 and R. erytropolis AC272 presented smallest sensitivities to heavy metals used, being suitable for use in sites contaminated with high concentrations of them. No plasmid DNA was detected showing that biosurfactant coding genes should be in the chromosomal DNA.

Effects of growth medium selection on plasmid DNA production and initial processing steps

Cultures of recombinant Escherichia coli containing the plasmid pSVβ were grown in three medium formulations to assess their effects on the characteristics of supercoiled plasmid DNA production for plasmid-based gene therapy. A semi-defined medium containing casamino acids (SDCAS) was found to support higher cell densities and higher plasmid stability than a similar medium containing soya amino acids (SDSOY) or Luria–Bertani medium (LB). Differences were observed in the cell harvest characteristics, plasmid DNA primary recovery, plasmid DNA yield and quality between cells grown on LB and on SDCAS medium. Cells grown on SDCAS medium were more difficult to resuspend after harvest than those grown in LB medium and were less susceptible to alkaline lysis. The plasmid DNA content from SDCAS was predominantly supercoiled and was less contaminated by chromosomal DNA than plasmid DNA extracts derived from cells grown on LB medium. It was hypothesised that the different carbon:nitrogen ratio (C:N) of the medium may have been responsible for changing the cell wall polysaccharide composition resulting in the change in cell harvest and lysis characteristics. Results indicated that changing the C:N ratio of SDCAS medium between 1.21:1 and 12.08:1 resulted in no alteration in cell wall polysaccharide composition or in cell susceptibility to chemical lysis or physical breakage. Plasmid DNA yields increased ten-fold with ten-fold increase in the C:N ratio of SDCAS medium.

Extraction of Superior-Quality Plasmid DNA by a Combination of Modified Alkaline Lysis and Silica Matrix

Extraction of Superior-Quality Plasmid DNA by a Combination of Modified Alkaline Lysis and Silica Matrix

Lipid mediated gene delivery in the adult rat brain: quantitative analysis of expression

Gene transfer into the adult brain is potentially an attractive alternative to commonly employed transgenic approaches. DNA-lipid complexes have been used to obtain brain gene transfer, but data are sparse to indicate to what extent this results in significant expression of functional protein. Here, an expression construct encoding the functional reporter, chloramphenicol-acetyl-transferase (CAT), was complexed to a novel biodegradable lipid, and delivered into the rat brain. CAT-activity was assayed in tissue extracts to allow a precise quantitation of functional enzyme protein. Following bilateral intraventricular (i.c.v.) injection, robust enzyme activity was found in all brain regions studied, peaking at 4 weeks. Other routes of administration, e.g. intra-parenchymal injection or chronic infusion of complexes, resulted in marginal or no activity. Presence of CAT mRNA and plasmid DNA in tissue extracts was confirmed at 4 weeks post i.c.v. administration. In agreement with previous studies, labelled lipid-DNA complexes were mainly found in the ventricular ependyma. Present data support the feasibility of lipid mediated brain gene transfer, and outline some of its anatomical and temporal limitations.