SpinSmart Plasmid Purification Protocol: High-copy plasmid DNA from E. coli v1

protocols.io ◽  
2016 ◽  
Author(s):  
Denville Scientific
Keyword(s):  
Plasmid Dna ◽  
E Coli ◽  
Plasmid Purification ◽  
Purification Protocol ◽  
High Copy Plasmid

One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

2021 ◽  
Vol 2021 (11) ◽  
pp. pdb.prot101212 ◽  
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Escherichia Coli ◽  
Convenient Method ◽  
Plasmid Dna ◽  
Transformation Efficiency ◽  
Bacterial Cells ◽  
E Coli ◽  
One Step ◽  
And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

scholarly journals Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy

2019 ◽  
Vol 6 (2) ◽  
pp. 54
Author(s):  
Folarin ◽  
Nesbeth ◽  
Ward ◽  
Keshavarz-Moore
Keyword(s):  
Plasmid Dna ◽  
Downstream Processing ◽  
Point Of View ◽  
High Yield ◽  
Bacteriophage Mu ◽  
E Coli ◽  
Yield And Quality ◽  
Superhelical Density ◽  
High Level ◽  
Binding Sequence

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of −0.022, and pSVβ-Gal −0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.

scholarly journals Natural Transformation of Plasmid DNA in Escherichia coli in Sterilized Soil Column

1970 ◽  
Vol 25 (1) ◽  
pp. 49-52
Author(s):  
M Mahabub-Uz-Zaman ◽  
Zia Uddin Ahmed
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Natural Transformation ◽  
Soil Column ◽  
Limiting Factor ◽  
Broad Host Range ◽  
E Coli ◽  
Competent Cells ◽  
Broad Host Range Plasmid ◽  
Plasmid Puc18

The present study was carried out to assess transformability of natural and laboratory strains of Escherichia coli by plasmid DNA under different transformation conditions in sterilized soil column. Transformation experiments were carried out in laboratory conditions and in sterile soil columns with CaCl2-treated competent cells and non-competent cells at log phase and stationary phase of growth using the broad host range plasmid pUC18. In soil column experiments, transformants were obtained after CaCl2 induced competence in both E. coli K12 DH5α and strain BM09 in the frequency of 10-8 to 10-9. In natural transformation assays, transformants appeared only in log phase cells of strain DH5α at a lower frequency (5.0 x 10-9), and in CaCl2-competent BM09 cells, but not in fresh cells. Thus the major limiting factor for natural transformation in environmental E. coli in soil column is probably the absence of a competent state. The significance of this finding has been discussed with respect to generally observed lower antibiotic resistance in environmental E. coli isolates from aquatic sources. Keywords: Natural transformation; Plasmid DNA; Escherichia coli; Competent stateDOI: http://dx.doi.org/10.3329/bjm.v25i1.4856 Bangladesh J Microbiol, Volume 25, Number 1, June 2008, pp 49-52

Core-Shell Structured Fe3O4/PPy Microspheres with High Magnetization for Purification of Plasmid DNA

2013 ◽  
Vol 716 ◽  
pp. 314-319 ◽  
Author(s):  
Hai Hui Jiang ◽  
Yan Zhou ◽  
Xiao Yun Han ◽  
Xin Cheng Chen ◽  
Yun Hua Hou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Drug Delivery ◽  
Plasmid Dna ◽  
Magnetic Particles ◽  
Interfacial Polymerization ◽  
Core Shell ◽  
Magnetic Composite ◽  
Amino Group ◽  
High Quality ◽  
E Coli

Amino group-functionalized magnetic particles have wide applications in enzyme immobilization, DNA extraction, drug delivery, water purification, catalysis, and sensor. In this paper, Fe3O4/PPy microspheres with a well-defined coreshell structure have been prepared through an interfacial polymerization approach without surfactant. The magnetic composite spheres were characterized with XRD, FTIR, SEM, TEM, and magnetometry techniques, and further tested as the adsorbent to isolate plasmid DNA from Escherichia coli (E. coli) DH5α cells. The magnetic separation yields high-quality plasmid DNA in satisfying productivity as compared to the conventional phenolchloroform extraction.

Prevalence of Colonization with Antibiotic Resistant Gram-Negative Bacilli in a Nursing Home Care Unit: The Importance of Cross-Colonization as Documented by Plasmid Analysis

1986 ◽  
Vol 7 (11) ◽  
pp. 538-545 ◽  
Author(s):  
David M. Shlaes ◽  
Mary-Helen Lehman ◽  
Charlotte A. Currie-McCumber ◽  
C.H. Kim ◽  
Rachel Floyd
Keyword(s):  
Nursing Home ◽  
Plasmid Dna ◽  
Nursing Home Care ◽  
Restriction Endonuclease Digestion ◽  
Gram Negative ◽  
E Coli ◽  
Plasmid Analysis ◽  
Gentamicin Resistance ◽  
R Plasmids ◽  
Endonuclease Digestion

AbstractA prevalence study was carried out on a 100-bed Veterans Administration nursing home care unit to determine the extent of colonization with gentamicin-resistant gram-negative bacilli (GRGNB). Hand cultures of 12 employees and 17 environmental cultures were negative. Twenty-six of 86 (30%) patients were colonized with 49 GRGNB. Sixteen patients (19%) had urinary colonization. Multivariate analysis revealed significant associations between rectal or perineal colonization (P<0.01), and the presence of a urinary device (82% condom catheters) (P<0.05), with urinary colonization. The most common isolates were Providencia stuartii (20), Escherichia coli (nine) and Klebsiella pneumoniae (nine). Twenty-six of 49 isolates carried plasmids. Restriction endonuclease digestion of plasmid DNA was performed for 21. Cross-colonization, as defined by the presence of the identical species with the identical restriction endonuclease digestion profile of purified plasmid DNA found in different patients, was observed for eight of 21 (38%) strains. All were geographically clustered. No strains could transfer gentamicin-resistance by conjugation and only two plasmids could transform our E coli recipient to gentamicin resistance. One E coli plasmid was identical to two Citrobacter freundii plasmids and a P stuartii plasmid isolated from three different patients. This 105 kb plasmid is conjugative and encodes resistance to ampicillin, carbenicillin, tetracycline, and sulfonamides. Thus, 57% of strains were cross-colonizing or contained identical R-plasmids. Southern hybridization using a 1 kb TEM-1 gene probe demonstrated sequences homologous to this probe in five of five nursing home plasmids examined. These data demonstrate the utility of plasmid analysis in epidemiologic typing of multiple species of Enterobacteriaceae, and suggest wide dissemination of R-plasmids bearing the TEM-1β-lactamase gene among gram-negative bacilli colonizing patients residing in our nursing home.

scholarly journals De novo creation of MG1655-derived E. coli strains specifically designed for plasmid DNA production

2012 ◽  
Vol 97 (2) ◽  
pp. 611-620 ◽  
Author(s):  
Geisa A. L. Gonçalves ◽  
Duarte M. F. Prazeres ◽  
Gabriel A. Monteiro ◽  
Kristala L. J. Prather
Keyword(s):  
Plasmid Dna ◽  
De Novo ◽  
E Coli ◽  
Dna Production

scholarly journals Recombinant expression of Barnase in Escherichia coli and its application in plasmid purification

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Ram Shankar ◽  
Nina Schäffer ◽  
Marco Schmeer ◽  
Joe Max Risse ◽  
Karl Friehs ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cation Exchange ◽  
Plasmid Dna ◽  
Recombinant Expression ◽  
Exchange Chromatography ◽  
Dna Purification ◽  
Specific Productivity ◽  
Scale Production ◽  
Cation Exchange Chromatography ◽  
Plasmid Purification

Abstract Background The use of bovine-origin ribonucleases has been part of the standard protocol for plasmid DNA purification. As the field of gene therapy now enters the clinical stage, such enzymes need to be phased out or alternative purification protocols need to be developed to ensure product safety and regulatory compliance. The recombinant expression of bacterial RNase is fraught with toxicity problems making it a challenging enzyme to express. The current study describes a plasmid construct that allowed expression of barnase in Escherichia coli under co-expression of its native inhibitor barstar. Results The pure enzyme without the inhibitor barstar was exported to the extracellular space through the periplasm and then purified from the cell-free supernatant. Cation exchange chromatography was employed as a primary purification step. This was followed by hydrophobic interaction chromatography which resulted in a concentrated fraction of active enzyme. Although current levels of volumetric activity achieved are quite meagre (4 Kunitz units mL− 1), in principle its application to plasmid DNA purification could be proved. Currently, this is capable of processing small amounts (13 g) of bacterial biomass for plasmid production. Conclusions The current work focusses on the downstream purification strategies for a recombinant RNase and sets a framework for higher scale production if specific productivity is increased by optimal hosts and/or re-engineered plasmids. Also important is to curtail the massive enzyme loss during purification by cation exchange chromatography. Application of even a relatively small amount of recombinant RNase would contribute to greatly reducing the initial RNA levels in alkaline lysates thereby augmenting further downstream plasmid purification steps.

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