High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme

Posted on by Nicholas under Antibodies, Assay Kits, cDNA, Clia Kits, Culture Cells, Devices, DNA, Elisa Kits, Enzymes, Exosomes, Gels, Isotypes, NATtrol, Particles, PCR, Pcr Kits, Peptides, Recombinant Proteins, Test Kits, Vector & Virus

Hyperthermophilic archaea with distinctive biochemical and physiological traits are necessary organisms for elementary analysis of life science and have nice potential for biotechnological purposes. Nonetheless, low transformation effectivity of international DNA molecules impedes developments in genetic modification instruments and industrial purposes. On this research, we utilized extended overlap extension PCR (POE-PCR) to generate multimeric DNA molecules after which remodeled them into two hyperthermophilic archaea, Thermococcus kodakarensis KOD1 and Pyrococcus yayanosii A1.

This research was the primary instance to exhibit the improved transformation efficiencies of POE-PCR merchandise by an element of roughly 100 for T. kodakarensis KOD1 and eight for P. yayanosii A1, respectively, relative to round shuttle plasmids. Moreover, directed evolution of a modestly thermophilic enzyme, Methanothermococcus okinawensis 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), was carried out to acquire extra steady ones attributable to excessive transformation effectivity of T. kodakarensis. T. kodakarensis harbouring probably the most thermostable MoHMGR mutant can develop within the presence of a thermostable antibiotic simvastatin at 85°C and even greater temperatures. This excessive transformation effectivity approach couldn’t solely assist develop extra hyperthermophilic enzyme mutants through directed evolution but additionally simplify genetical modification of archaea, which may very well be novel hosts for industrial biotechnology.

Turmeric, or Curcuma longa, is usually consumed within the South East Asian nations as a medical product and as meals attributable to its therapeutic properties. Nonetheless, with growing demand for turmeric powder, adulterated turmeric powders combined with different low-cost starch powders, comparable to from corn or cassava, are being distributed by meals suppliers for financial profit. Right here, we developed molecular markers utilizing quantitative real-time PCR to determine adulteration in business turmeric powder merchandise. Chloroplast genes, comparable to matK, atpF, and ycf2, had been used to design species-specific primers for C. longa and Zea mays.

 

Screening of economic meat merchandise from grocery store chains for feline derivatives utilizing SP-PCR-RLFP and lab-on-a-chip

Dedication of feline meat in meals merchandise is a vital difficulty for social, well being, financial and spiritual concern. Therefore this paper documented the applying of species particular polymerase chain reaction-restriction fragment size polymorphism (SP-PCR-RFLP) assay focusing on a short-fragments (69 bp) of mitochondrial cytochrome b (cytb) gene to display screen feline meat in business meat merchandise utilizing lab-on-a-chip. The SP-PCR assay proved its specificity theoretically and experimentally whereas testing with totally different frequent animal, aquatic and plant species of DNA. The feline particular (69 bp, 43- and 26-bp) attribute molecular DNA sample was noticed by SP-PCR and RFLP evaluation.
For assay efficiency, it was examined in three several types of business dummy meat merchandise comparable to frankfurters, nuggets and meatballs and digested with AluI-restriction enzyme. The best sensitivity of the assay utilizing lab-on-a-chip was as little as 0.1 pg or 0.01 % (w/w) in business dummy meat merchandise. We’ve got additionally utilized this assay to display screen three necessary business meat merchandise of six totally different model from six grocery store chains situated at three totally different states of Malaysia. Thus complete 378 samples had been examined to validate the specificity, sensitivity, stability of the assay and utilization of it for business meat product screening. Amongst others, the households Opisthorchiidae and Heterophyidae consists of a number of genera inflicting fish-borne zoonoses and distributed additionally in European International locations and which can be included within the ParaFishControl (Superior Instruments and Analysis Methods for Parasite Management in European farmed fish) H2020 EU challenge. As a result of small measurement of the metacercariae, the infective stage for human, these parasites can’t be detected visually in fish and monitoring requires skilled utility of time-consuming strategies.
The goal of this was to develop a speedy and inexpensive molecular technique primarily based on multiplex PCR for simultaneous identification of metacercariae of the most typical European Opisthorchiid and Heterophyid in fish or fish merchandise.

High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme

High-efficiency transformation of archaea by direct PCR products with its application to directed evolution of a thermostable enzyme

TaqMan probe primarily based multiplex quantitative PCR assay for dedication of bovine, porcine and fish DNA in gelatin admixture, meals merchandise and dietary dietary supplements.

Detection of animal supplies in gelatin-based merchandise is required to handle non secular and cultural considerations, as a result of porcine and bovine gelatins are prohibited in Halal, Kosher and Hindus shopper items. On this paper, multiplex quantitative polymerase chain response (qPCR) assay utilizing TaqMan probe was developed to discriminate bovine, porcine and fish gelatin species in a single assay platform. The assay was particular to cattle, pigs and fish, having been examined towards 14 non-target species. The restrict of detection, beneath gelatin admixed circumstances, was 0.005 ng/µL. Lastly, a pilot survey was undertaken testing 35 Halal branded processed meals and dietary gadgets. Out of 35 samples, solely two had been discovered to be optimistic for porcine species. The authenticity of those two qPCR merchandise was confirmed by DNA sequencing evaluation, which confirmed 99-100% similarity with Sus scrofa (Wild boar) species.
Dried squid merchandise are widespread in China as a snack meals, facet dishes, or refreshments, and the market attraction might be mirrored by the excessive worth that often reaches 497 RMB per kg. Nonetheless, the absence of harmonisation across the definition of squid, in addition to the issues with visible inspection for processed seafood merchandise, make various species substitution for dried squid merchandise a frequent incidence. The goal of the current research was to use a DNA barcoding method for species identification of 48 dried squid merchandise collected from the most important on-line procuring platform in China. Furthermore, we additionally developed a novel SYBR inexperienced real-time PCR assay (simplex and duplex adopted by a melting curve evaluation) particular for Illex argentinus and Todarodes pacificus primarily based on cytochrome C oxidase subunit I (COI) gene.

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Outcomes highlighted the profitable DNA extraction and PCR amplification of a 655 bp COI gene fragment from all merchandise. A most similarity worth within the vary of 98-100% was obtained for all readable sequences utilizing the BOLD and BLAST public databases and 4 species had been recognized.
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Nicholas

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