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Bakteriyel enfeksiyonlarda ilaç direnci frekansındaki artış, yeni mekanizmaları hedefleyen yeni antibakteriyel ajanların araştırılmasına yol açmıştır. NAD+-bağımlı DNA ligazın fonksiyonlarının bir çoğu, onu antibakteriyel ilaç keşfi için dikkate değer bir hedef haline getirmiştir. Escherichia coli (E. coli) NAD+ bağımlı DNA ligazı, ATP bağımlı insan DNA ligazı ile karşılaştrıldığında, benzersiz substrat özgüllüğü nedeniyle potansiyel bir hedef olarak görülmektedir. Bu çalışmada antibakteriyel ilaç keşiflerinde sıklıkla kullanılan E. coli NAD+ bağımlı DNA ligaz enziminin, yüksek miktarda ve saflıkta üretilmesi amaçlamıştır. E. coli DNA ligaz gen dizisi, pTOLT vektör sistemine klonlanmıştır. E. coli DNA ligaz enzimi, E. coli BL21 (DE3) pLysE hücrelerinde üretildikten sonra saflaştırılmıştır. Bu çalışmada üretilen DNA ligaz enziminin DNA fragmanlarının ligasyonunu sağlayabildiği, aktivite testi ile açıkça gösterilmiştir. Sonuç olarak, bu enzim üzerinde aday inhibitörlerin etkisinin basitçe incelenebileceği ortaya koyulmuştur., The increase in the frequency of drug resistance in bacterial infections has led to the research of new antibacterial agents targeting new mechanisms. Many of the functions of NAD+-dependent DNA ligase have made it a remarkable target for antibacterial drug discovery. Escherichia coli (E. coli) NAD+-dependent DNA ligase is presented as a potential target due to its unique substrate specificity compared to the ATP-dependent human DNA ligase. In this study, it was aimed to produce and purify the E. coli NAD + dependent DNA ligase enzyme, which is frequently used in antibacterial drug discovery. The E. coli DNA ligase gene sequence was cloned into pTOLT vector system. E. coli DNA ligase enzyme was purified after the production in E. coli BL21 (DE3) pLysE cells. It was clearly demonstrated by the activity test that the DNA ligase enzyme produced in this study can ligate the DNA fragments. As a result, it was revealed that the effect of candidate inhibitors can be studied simply on the enzyme.

Moloney murin lösemi virüs (MMLV) ters transkriptazı (RT), cDNA sentezi ve RNA amplifikasyonu için en yaygın kullanılan enzimdir. Bu çalışmada moleküler çalışmalardaki önemi nedeniyle MMLV RT enziminin rekombinant olarak üretilmesi amaçlanmıştır. Bu bağlamda, MMLV RT enzimini kodlayan DNA fragmanı pTOLT plazmidine klonlanmış ve E. coli BL21 (DE3) pLysE hücrelerinde eksprese edilmiştir. Proteinin yüksek düzeyde ekspresyonu, protein moleküllerinin inklüzyon cisimciklerinde toplanmasına neden olduğundan MMLV RT proteininin çözünür formda elde edilebilmesi için MMLV RT ve şaperon plazmidlerin (pG-KJE8, pGro7, pKJE7, pGTf2, pTf16) birlikte ekspresyonu gerçekleştirilmiştir. Beklentilerimizin aksine, protein çözünür formda elde edilemediği için, yeniden katlama prosesi kullanılarak inklüzyon cisimciklerinden geri kazanılmıştır. Son olarak protein, afinite kromatografisiyle saflaştırılmış ve proteinin aktivitesi RT-PCR tekniği kullanılarak kontrol edilmiştir., Reverse transcriptase (RT) of Moloney murine leukemia virus (MMLV) is the most widely used enzyme for cDNA synthesis and RNA amplification. In this study, we aimed to produce MMLV RT enzyme recombinantly due to its importance in molecular studies. In this context, the DNA fragment encoding the MMLV RT enzyme was cloned into pTOLT plasmid and expressed in E. coli BL21 (DE3) pLysE cells. Since the high-level expression of the protein caused the protein molecules to aggregate in the inclusion bodies, co-expression of MMLV RT and chaperone plasmids (pG-KJE8, pGro7, pKJE7, pGTf2, pTf16) was performed to obtain the MMLV RT protein in soluble form. Contrary to our expectations, because it could not be obtained in soluble form, the protein was recovered from the inclusion bodies using refolding process. Finally, the protein was purified by affinity chromatography and the activity of the protein was checked using RT-PCR technique.

Sweet basil (Ocimum basilicum L.), a well-known medicinal and aromatic herb, rich in essential oils and antioxidants (contributed by phenolics), is widely used in traditional medicine. The biosynthesis of phytochemicals occurs via different biochemical pathways, and the expression of selected genes encoding enzymes involved in the formation of phenolic compounds is regulated in response to environmental factors. The synthesis of the compounds is closely interrelated: usually, the products formed in the first reaction steps are used as substrates for the next reactions. The current study attempted a comprehensive overview of the effect of aromatic amino acid composition (AAAs) in Ocimum basilicum in respect to the expression of genes related to the biosynthesis of phenolic compound and their content. The transcript expression levels of EOMT, PAL, CVOMT, HPPR, C4L, EGS, and FLS increased depending on the AAAs concentration compared to the control plants. The highest mRNA accumulation was obtained in EOMT, FLS, and HPPR in the leaves of sweet basil. The expression of the TAT gene in the leaves significantly reduced in response to all AAAs applications compared to untreated groups and it had the lowest transcript accumulation. Eleven individual phenolic compounds were determined in the basil leaves, and the contents of chicoric acid, methyl chavicol, caffeic acid, and vanillic acid increased depending on administered concentration to control (p

Gluten is a protein group found in wheat, barley, rye, and oats, known as cereals. When this vegetable protein is introduced into the body, celiac disease can occurs. The use of bacterial and fungal oligopeptidase to ensure the cleavage of gluten into non-toxic fragments are considered a promising alternative for celiac disease. In this study, the Aspergillus niger Prolyl EndoPeptidase (AN-PEP) enzyme was cloned into pET22b vector and recombinantly produced in BL21 (DE3) pLysE cells. PEP enzyme expressed as inclusion body and was recovered by refolding. And N-terminal His-tagged recombinant protein was purified by nickel affinity chromatography. 280 mg AN-PEP enzyme from 1L bacterial culture was purified at very high yield, and this protein was 90% purity. As a result; It has been determined that the recombinantly produced PEP enzyme can digest gluten. This study shows that recombinantly produced AN-PEP (rAN-PEP) has great potential to use in the production processes of gluten-free foods.

Hematite (alpha-Fe2O3) nanoparticles have received significant attention from the researchers due to their favorable and desirable properties in diverse applications. In the present study, single-crystalline hematite pseudo-nanocubes and porous nanorods were synthesized by the hydrothermal route without the usage of any surfactant agents, which were then decorated with gold nanoparticles hidrothermally in an aqueous solution in order to increase the solar energy conversion efficiency. The photocatalytic activities of synthesized nanoparticles were studied against Rhodamine B (RhB) under the illumination of AM 1.5 solar simulator. The excellent photocatalytic efficiency was obtained by changing morphological features of hematite nanostructures, along with the photocatalytic performance enhancement up to 25% thanks to the surface plasmon resonances for the gold nanoparticles-decorated hematite nanostructures. For instance, gold-nanoparticles decorated hematite pseudo-nanocubes almost completely degraded all the RhB after 30 min of illumination. It was also observed that all hematite products, with and without gold decoration, exhibited an impressive antibacterial effect and showed the lethal effect in E.coli. The same nanoparticles being photocatalytically active for organic pollutant degradation and having antibacterial effect may have a good potential for waste water remediation applications. (C) 2020 Elsevier B.V. All rights reserved.

Phenolic compounds mainly benefit human health and have many biological activities. Their activities are related to their structure, which allows them to interact with enzymes. The inhibition potencies of synthesized polyphenolic compounds (3a and 3b) were investigated on cholinesterases, α‑Gly, and tyrosinase activities. The structures of 3a and 3b were determined based on spectral data (NMR, UV-vis, XRD pattern, SEM, and EDX). The compounds have effective inhibitory potential with IC

Genom düzenleme, hedef gende istenilen değişiklikleri yapmak için kullanılan bir yöntemdir. Günümüzde genom düzenleme çalışmaları için çeşitli yöntemler kullanılmaktadır; bunlar arasında en yaygın kullanılan yöntemlerden biri kümelenmiş, düzenli aralıklarla yerleştirilmiş kısa palindromik tekrarlardır (CRISPR). CRISPR ile ilişkili (Cas) genleri ve bunlara karşılık gelen CRISPR dizileri, CRISPR-Cas sistemlerini oluşturur. Basitliği nedeniyle, CRISPR-Cas sisteminin insanlarda ex vivogen terapisi çalışmalarında etkili bir şekilde kullanılmaya başlanmıştır veCRISPR taşıyıcı sistemlerin önemi giderek artmaktadır.Genom düzenleme çalışmalarında dağıtım yöntemleri olarak viral ve viral olmayan sistemler kullanılmaktadır. Nanopartiküllerin ayarlanabilir boyutları, yüzeyleri, şekilleri ve biyolojik davranışları nedeniyle gen terapisi için en umut verici araçlar olduğukanıtlanmıştır. Polimerik taşıyıcı sistem, azaltılmış immünojenisitesi ve patojenitesi nedeniyle gen aktarımı için viral olmayan ana ikame haline gelmiştir. Bu derlemede, viral olmayan CRISPR dağıtım sistemlerinde kullanılan polimerik taşıyıcılarla ilgiligüncel çalışmalar hakkında bilgiler sunulmaktadır., Genome editing is a method used to make desired changes in the target gene. Today, various methods are used for genome-editing studies; among them, one of the most widely used methods is the clustered, regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated (Cas) genes and their corresponding CRISPR sequences constitute CRISPR-Cas systems. Due to its simplicity, it is likely that the CRISPR–Cas system could be used effectively in ex vivo gene therapy studies in humans. If this happens, the importance of CRISPR carrier systems will gradually increase. Viral and non-viral systems are used as delivery modalities in genome-editing studies. It has been proven that nanoparticles are the most promising tools for gene therapy due to their adjustable size, surface, shape, and biological behaviours. The polymeric carrier system has become the main non-viral substitute for gene delivery due to its reduced immunogenicity and pathogenicity. In this review, information about current studies related to polymeric carriers used in non-viral CRISPR delivery systems is presented.

Single-stranded DNA-binding (SSB) proteins play an important role in DNA metabolism involving DNA replication, recombination, and repair in all living beings. In molecular biology, SSB proteins are used as enhancers to increase the efficiency and specificity of PCR. Thermostable SSB protein eliminates secondary structure or dimer formation and significantly increase the effectiveness of amplification of DNA fragments. In this study, it was ensured that the SSB gene of thermophilic bacteria Thermus aquaticus (T. aquaticus) was cloned into the pET28b vector and expressed in E. coli BL21 (DE3) PLysE cells. Then, the purification of the SSB protein produced in E. coli BL21 (DE3) PLysE cells was performed. 20 mg SSB protein was obtained from 1L bacterial culture, and its purity was more than 90%. It was shown by the PCR experiment that the SSB protein produced in this study could increase the amplification efficiency.

Aquamarine proteini siyan (mavi) floresan protein ailesinin bir üyesi olup ve Aequorea victoria'dan elde edilen GFP (yeşil floresan protein) türevi floresan bir proteindir. Aquamarine benzeri floresan proteinler genetik mühendisliği teknikleri ile geliştirilmiş özellikleri sayesinde canlı hücrelerin biyolojik olarak görüntülenmesi ve hücre içerisinde lokalizasyon çalışmaları için sıklıkla kullanılmaktadır. Bu çalışmada Escherichia coli BL21(AI) hücreleri pROEX Aqua plasmid DNA’sı ile transforme edilmiş ve %0.04 konsantrasyonda arabinoz ilavesi ile protein ekspresyonu indüklenmiştir. 3L kültür hacimli biyoreaktörde yüksek ekspresyon seviyesinde üretilen Histidin etiketli hedef protein Ni+2 afinite kromotografisi ile saflaştırılmıştır. Saflaştırılan protein konsantrasyonu bir litre bakteri kültürü için 80 mg olarak belirlenmiştir. Rekombinant Aquamarine’ne ait fotolüminesans özellikler florometre ile analiz edilmiştir. SDS-PAGE analizi rekombinant Aquamarine proteine ait tek bir bandın olduğunu ve saflaştırılan proteinin ileri çalışmalarda kullanılmak üzere yüksek verimde ve saflıkta (>%95) elde edildiğini göstermektedir. Aquamarine is a member of the cyan fluorescent protein (CFP) protein family and is a GFP (green fluorescent protein) derived fluorescent protein from Aequorea victoria. Aquamarine-like fluorescence proteins are often used for biological imaging and localization studies of living cells due to their properties developed by genetic engineering techniques. In this study, Escherichia coli BL21 (AI) cells were transformed with pROEX Aqua plasmid DNA and protein expression was induced by the addition of arabinose at a concentration of 0.04%. Histidine-labeled target protein produced at high expression levels in the bioreactor that has 3L culture volume was purified by Ni+2 affinity chromatography. About 80 mg of the purified protein was yielded per liter of bacterial culture. The photoluminescence properties of the Aquamarine were analyzed by fluorimeter. SDS-PAGE analysis shows a single band corresponding to the recombinant Aquamarine that was obtained high yield and purity (>95%) for further studies.