Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells

Wood, Whitney N.; Smith, Kyle D.; Ream, Jennifer A.; Lewis, L. Kevin

doi:10.1016/j.mimet.2016.12.016

Cited by 16 publications

(10 citation statements)

References 17 publications

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“…The main advantage of TB over LB and 2xHK-SII is its buffering capacity, maintaining a controlled pH during growth, and the presence of glycerol as an additional carbon source. TB had previously demonstrated improved performance over other culture media for the production of recombinant proteins (Osadska et al 2014;Zamani et al 2015) and plasmid DNA (Wood et al 2017). The auto-induction medium described by Studier (2005) was also evaluated, but the growth and solubility of ΔNS1 were not better than that observed with TB (data not shown).…”

Section: Discussionmentioning

confidence: 97%

Optimization and scale-up production of Zika virus ΔNS1 in Escherichia coli: application of Response Surface Methodology

et al. 2019

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Diagnosing Zika virus (ZIKV) infections has been challenging due to the cross-reactivity of induced antibodies with other flavivirus. The concomitant occurrence of ZIKV and Dengue virus (DENV) in endemic regions requires diagnostic tools with the ability to distinguish these two viral infections. Recent studies demonstrated that immunoassays using the C-terminal fragment of ZIKV NS1 antigen (ΔNS1) can be used to discriminate ZIKV from DENV infections. In order to be used in serological tests, the expression/solubility of ΔNS1 and growth of recombinant E. coli strain were optimized by Response Surface Methodology. Temperature, time and IPTG concentration were evaluated. According to the model, the best condition determined in small scale cultures was 21 °C for 20 h with 0.7 mM of IPTG, which predicted 7.5 g/L of biomass and 962 mg/L of ΔNS1. These conditions were validated and used in a 6-L batch in the bioreactor, which produced 6.4 g/L of biomass and 500 mg/L of ΔNS1 in 12 h of induction. The serological ELISA test performed with purified ΔNS1 showed low cross-reactivity with antibodies from DENV-infected human subjects. Denaturation of ΔNS1 decreased the detection of anti-ZIKV antibodies, thus indicating the contribution of conformational epitopes and confirming the importance of properly folded ΔNS1 for the specificity of the serological analyses. Obtaining high yields of soluble ΔNS1 supports the viability of an effective serologic diagnostic test capable of differentiating ZIKV from other flavivirus infections.

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Section: Discussionmentioning

confidence: 97%

Optimization and scale-up production of Zika virus ΔNS1 in Escherichia coli: application of Response Surface Methodology

et al. 2019

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“…An on-colony PCR of patched E. coli colonies using HH-F 5′-CCGTGAGGACGAAACGAGT-3′ and HDV-R 5′-CCGAAGCATGTTGCCCAGC-3′ primers was performed to confirm the integrity of the sgRNA array prior to inoculating a culture for plasmid purification. All liquid cultures for E. coli (plasmid isolation and conjugation) were inoculated in LB24 medium ( Wood et al, 2017 ) and grown at 30°C with appropriate antibiotics and shaken at 225 rpm.…”

Section: Methodsmentioning

confidence: 99%

Multiplexed Genome Editing via an RNA Polymerase II Promoter-Driven sgRNA Array in the Diatom Phaeodactylum tricornutum: Insights Into the Role of StLDP

et al. 2022

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CRISPR/Cas9-mediated genome editing has been demonstrated in the model diatom P. tricornutum, yet the currently available genetic tools do not combine the various advantageous features into a single, easy-to-assemble, modular construct that would allow the multiplexed targeting and creation of marker-free genome-edited lines. In this report, we describe the construction of the first modular two-component transcriptional unit system expressing SpCas9 from a diatom episome, assembled using the Universal Loop plasmid kit for Golden Gate assembly. We compared the editing efficiency of two constructs with orthogonal promoter-terminator combinations targeting the StLDP gene, encoding the major lipid droplet protein of P. tricornutum. Multiplexed targeting of the StLDP gene was confirmed via PCR screening, and lines with homozygous deletions were isolated from primary exconjugants. An editing efficiency ranging from 6.7 to 13.8% was observed in the better performing construct. Selected gene-edited lines displayed growth impairment, altered morphology, and the formation of lipid droplets during nutrient-replete growth. Under nitrogen deprivation, oversized lipid droplets were observed; the recovery of cell proliferation and degradation of lipid droplets were impaired after nitrogen replenishment. The results are consistent with the key role played by StLDP in the regulation of lipid droplet size and lipid homeostasis.

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“…Standard procedures were used for DNA manipulation, and genomic DNA from Streptomyces was extracted according to Sambrook Molecular Cloning: A Laboratory Manual (Sambrook 2001). Plasmid DNA was purified using commercial chromatography kits from Thermo Fisher Scientific (Waltham, MA, USA) and QIAGEN (Hilden, Germany); silica columns from these kits were regenerated and reused following the procedure described by Siddappa (Siddappa et al, 2007), while standard protocols were modified to obtain greater yields of DNA (Pronobis, Deuitch, and Peifer 2016;Wood et al, 2017). Restriction enzymes and thermostable polymerases were used according to the manufacturers' instructions.…”

Section: Dna Manipulation and Ss-ired_s Cloningmentioning

confidence: 99%

Identification, Characterization, and In Silico Analysis of New Imine Reductases From Native Streptomyces Genomes

et al. 2021

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The development of biocatalytic tools for the synthesis of optically pure amines has been the focus of abundant research in recent years. Among other enzymes, imine reductases have attracted much attention associated with the possibility of attaining chiral secondary amines. Furthermore, the reductive aminase activity associated with some of these enzymes has facilitated the production of optically pure amines from a prochiral ketone, a transformation that opens doors to an incredible array of products. In this work, the genomes from native Streptomyces strains isolated in our lab have been explored on the search for novel imine reductases. Application of different structural criteria and sequence motif filters allowed the identification of two novel enzymes, Ss-IRED_S and Ss-IRED_R. While the former presented outstanding activity towards bulky cyclic imine substrates, the latter presented reductive aminase activity with the assayed ketones. A bioinformatic analysis based on modeling and docking studies was performed in order to explain the differences in enzyme activity, searching for additional criteria that could be used to analyze enzyme candidates in silico, providing additional tools for enzyme selection for a particular application. Our findings suggest that imine reductase activity could be predicted by this analysis, overall accounting for the number of docking positions that meet the catalytic requirements.

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Enhancing yields of low and single copy number plasmid DNAs from Escherichia coli cells

Cited by 16 publications

References 17 publications

Optimization and scale-up production of Zika virus ΔNS1 in Escherichia coli: application of Response Surface Methodology

Optimization and scale-up production of Zika virus ΔNS1 in Escherichia coli: application of Response Surface Methodology

Multiplexed Genome Editing via an RNA Polymerase II Promoter-Driven sgRNA Array in the Diatom Phaeodactylum tricornutum: Insights Into the Role of StLDP

Identification, Characterization, and In Silico Analysis of New Imine Reductases From Native Streptomyces Genomes

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