Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

Kostylev, Maxim; Otwell, Anne E.; Richardson, Ruth; Suzuki, Yo

doi:10.1371/journal.pone.0137466

Cited by 123 publications

(148 citation statements)

References 40 publications

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“…After purification using NucleoSpin Gel and the PCR Clean-up kit (Macherey-Nagel, Bethlehem, PA), the linearized plasmid was introduced into High Efficiency NEB 5-alpha chemically competent cells (New England Biolabs, Ipswich, MA), per the manufacturer’s protocol5455. Transformed colonies were selected on LB-ampicillin agar plates.…”

Section: Methodsmentioning

confidence: 99%

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

Goldgof

Durrant

Ottilie

et al. 2016

Sci Rep

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The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.

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Section: Methodsmentioning

confidence: 99%

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

Goldgof

Durrant

Ottilie

et al. 2016

Sci Rep

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“…Vectors were constructed using standard molecular biology techniques including one-pot cloning method [63], Escherichia coli DH5α-mediated DNA assembly method [64], and Body Double cloning method [65]. For detailed cloning and sequence information see Additional file 3. sgRNA target sites and mismatching sgRNAs are available in Additional file 2.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

et al. 2017

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Background: The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side by side to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects. Results: These three increased-fidelity nucleases can routinely be used only with perfectly matching 20-nucleotide-long spacers, a matching 5′ G extension being more detrimental to their activities than a mismatching one. HeFSpCas9 exhibit substantially improved specificity for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. The targets can also be ranked by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s.

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“…There was a positive response to pDNA production and students were fascinated by the use of bacteria to produce pVAX1‐ LacZ . The host used was E. coli DH5 α , a GRAS microorganism with low endonuclease activity, that promotes high yields of pVAX1‐ LacZ . The growth curve of cell culture was determined by students by measuring the optical densities at 600 nm (OD 600 ).…”

Section: Resultsmentioning

confidence: 99%

Plasmid production and purification: An integrated experiment‐based biochemistry and biotechnology laboratory course

Santos

Pereira

Queiroz

et al. 2019

Biochem Molecular Bio Educ

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This laboratory experiment describes the production and purification of plasmid DNA for undergraduate biochemistry and biotechnology courses. This experiment performed in a one‐week period includes the protocols for plasmid pVAX1‐LacZ production in Escherichia coli DH5α cells and subsequent purification of supercoiled pVAX1‐LacZ. Firstly, the students use a growth medium that favors the replication of the plasmid resulting in a higher plasmid production during exponential growth. Afterwards, alkaline lysis is done to disrupt the bacterial cells and recover pVAX1‐LacZ plasmid. Lastly, they perform the purification of pVAX1‐LacZ supercoiled isoform by L‐histidine chromatography, followed by agarose gel electrophoresis to characterize the separation of supercoiled isoform from contaminants. The proposed experiment provides an opportunity for students to acquire these skills that are routinely used in biochemistry and biotechnology laboratories. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):638–643, 2019.

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Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

Cited by 123 publications

References 40 publications

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

Comparative chemical genomics reveal that the spiroindolone antimalarial KAE609 (Cipargamin) is a P-type ATPase inhibitor

Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

Plasmid production and purification: An integrated experiment‐based biochemistry and biotechnology laboratory course

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