Overcoming cloning problems by staining agarose gels with crystal violet instead of ethidium bromide in lactate dehydrogenase gene from<i>Plasmodium vivax</i>and<i>Plasmodium falciparum</i>

Turgut-Balık, Dilek; Çelik, Venhar; Moreton, Kathleen M.; Brady, R.L.

doi:10.1556/abiol.56.2005.3-4.20

Cited by 4 publications

(3 citation statements)

References 7 publications

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“…Furthermore, exposing DNA or plasmid to UV light has the potential to damage the DNA, resulting in false or empty recombinants. However, the latter challenge can be overcome by staining agarose gels with crystal violet instead of ethidium bromide to visualize DNA and improve cloning efficiency ( Rand, 1996 ; Turgut-Balik et al, 2005 ).…”

Section: Limiting Factors That Contribute To Insufficient Sequence-fu...mentioning

confidence: 99%

Insufficiency in functional genomics studies, data, and applications: A case study of bio-prospecting research in ruminant microbiome

2022

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Over the last two decades, biotechnology has advanced at a rapid pace, propelled by the incorporation of bio-products into various aspects of pharmaceuticals, industry, and the environment. These developments have sparked interest in the bioprospecting of microorganisms and their products in a variety of niche environments. Furthermore, the use of omics technologies has greatly aided our analyses of environmental samples by elucidating the microbial ecological framework, biochemical pathways, and bio-products. However, the more often overemphasis on taxonomic identification in most research publications, as well as the data associated with such studies, is detrimental to immediate industrial and commercial applications. This review identifies several factors that contribute to the complexity of sequence data analysis as potential barriers to the pragmatic application of functional genomics, utilizing recent research on ruminants to demonstrate these limitations in the hopes of broadening our horizons and drawing attention to this gap in bioprospecting studies for other niche environments as well. The review also aims to emphasize the importance of routinely incorporating functional genomics into environmental metagenomics analyses in order to improve solutions that drive rapid industrial biocatalysis developments from derived outputs with the aim of achieving potential benefits in energy-use reduction and environmental considerations for current and future applications.

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Section: Limiting Factors That Contribute To Insufficient Sequence-fu...mentioning

confidence: 99%

Insufficiency in functional genomics studies, data, and applications: A case study of bio-prospecting research in ruminant microbiome

2022

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“…PvD GAG TAA ATC ATC TCG GCT CTT TCC TGG 3 0 . After that, mutant double-stranded PvLDH genes were purified from a 1% crystal violet stained agarose gel [Turgut-Balik et al, 2005]. They were digested using Eco RI and Pst I, and were ligated into similarly digested pKK223-3 and transformed into calcium chloride competent E. coli cells for sequencing, expression, and Western blotting.…”

Section: Mutagenesis To Construct Mutant Pvldh Genesmentioning

confidence: 99%

Analysis of active site loop amino acids of lactate dehydrogenase from Plasmodium vivax by site‐directed mutagenesis studies

Turgut-Balık

Sadak

Çelik

2006

Drug Development Research

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The spread of resistance of malaria parasites to conventional chemotherapies necessitates the development of new drugs. The present study analyzes the active site loop of the enzyme lactate dehydrogenase from Plasmodium vivax (PvLDH). A five-amino acids insertion was observed in the active site loop of PvLDH, as in other plasmodial LDHs, which is absent in human LDH and is therefore a potential novel target site for new antimalarial drugs. This study describes the mutagenic testing of overproduced Plasmodium vivax lactate dehydrogenase to evaluate the importance of the inserted amino acids in the active site loop, which were previously identified as important residues by crystal structure studies. Deletion of these 5 extra amino acids in the active site loop resulted in an inactive form of the protein, supporting the notion of using this site as a novel target for antimalarial drug design. Drug Dev.

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“…for 30 cycles. Double-strand amplicons run on a 1% crystal violet stained agarose gel for preparative purposes and a parallel PCR product obtained using the same reaction was run on the 1% ethidium bromide (EtBr) stained agarose gel for visualisation of the amplicons (18). The DNA was recovered by QIA quick Gel Extraction Kit (QIAGEN, Germany) following the electrophoresis.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Analysis of the Amino Terminal End of Active Site Loop of Lactate Deyhdrogenase from Plasmodium Vivax

Mutlu¹,

Balik²

2012

Balkan Med J

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Objective: In this study, kinetic analysis was performed to understand the functional importance of the amino terminal of the active site of previously mutated Plasmodium vivax Lactate Dehydrogenase enzyme by mimicking Toxoplasma gondii I, II, Eimeria acervulina and Eimeria tenella LDH's.Material and Methods: Mutant LDH genes were amplified by PCR and 6xHistag was added to the C-terminal of the enzymes. Then LDH enzymes are overproduced as recombinant in E. coli cells, purified by Ni-NTA agarose matrix and kinetic properties were analysed.Results: Observing increase of K m values of mutant enzymes showed that mutations in this place caused decreasing affinity of enzyme for its substrate. However k cat values were about the same throughout all mutant proteins.Conclusion: Sensitivity of the studied region emphasizes the significance of this site for drug design studies for both Plasmodium and some other Apicomplexans.

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Overcoming cloning problems by staining agarose gels with crystal violet instead of ethidium bromide in lactate dehydrogenase gene fromPlasmodium vivaxandPlasmodium falciparum

Cited by 4 publications

References 7 publications

Insufficiency in functional genomics studies, data, and applications: A case study of bio-prospecting research in ruminant microbiome

Insufficiency in functional genomics studies, data, and applications: A case study of bio-prospecting research in ruminant microbiome

Analysis of active site loop amino acids of lactate dehydrogenase from Plasmodium vivax by site‐directed mutagenesis studies

Kinetic Analysis of the Amino Terminal End of Active Site Loop of Lactate Deyhdrogenase from Plasmodium Vivax

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