Journal articles: 'Escherichia coli HT115 (DE3)'

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Ahn, Seung-Joon, Kelly Donahue, Youngho Koh, Robert R. Martin, and Man-Yeon Choi. "Microbial-Based Double-Stranded RNA Production to Develop Cost-Effective RNA Interference Application for Insect Pest Management." International Journal of Insect Science 11 (January 2019): 117954331984032. http://dx.doi.org/10.1177/1179543319840323.

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RNA interference (RNAi) is a convenient tool to identify and characterize biological functions in organisms. Recently, it has become an alternative to chemical insecticides as a biologically based control agent. This promising technology has the potential to avoid many problems associated with conventional chemical insecticides. In order for RNAi application to be practical for field use, a major hurdle is the development of a cost-effective system of double-stranded RNA (dsRNA) production for a large quantity of dsRNA. A handful of research reports has demonstrated microbial-based dsRNA production using L4440 vector and HT115 (DE3) Escherichia coli for application to vertebrate and invertebrate systems. However, the dsRNA yield, production efficiency, and biological purity from this in vitro system is still unclear. Thus, our study detailed biochemical and molecular tools for large-scale dsRNA production using the microbial system and investigated the production efficiency and yield of crude and purified dsRNAs. An unrelated insect gene, green fluorescent protein (GFP), and an insect neuropeptide gene, pyrokinin (PK) identified from Drosophila suzukii, were used to construct the recombinant L4440 to be expressed in the HT115 (DE3) cell. A considerable amount of dsRNA, 19.5 µg/mL of liquid culture, was isolated using ultrasonic disruption followed by phenol extraction. The sonication method was further evaluated to extract crude dsRNA without the additional phenol extraction and nuclease treatments and also to reduce potential bacterial viability. The results suggest that the ultrasonic method saved time and costs to isolate crude dsRNA directly from large volumes of cell culture without E coli contamination. We investigated whether the injection of PK dsRNA into flies resulted in increased adult mortality, but it was not statistically significant at 95% confidence level. In this study, the microbial-based dsRNA production has potential for applied RNAi technology to complement current insect pest management practices.

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Niño-Sánchez, Jonatan, Li-Hung Chen, Jorge Teodoro De Souza, Sandra Mosquera, and Ioannis Stergiopoulos. "Targeted Delivery of Gene Silencing in Fungi Using Genetically Engineered Bacteria." Journal of Fungi 7, no. 2 (February 9, 2021): 125. http://dx.doi.org/10.3390/jof7020125.

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Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens.

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Bernadus, Zefanya G., Fatimawali Fatimawali, and Beivy Kolondam. "TRANSFORMASI PLASMID YANG MENGANDUNG GEN merB PADA Escherichia coli BL21(DE3)." PHARMACON 8, no. 1 (February 28, 2019): 196. http://dx.doi.org/10.35799/pha.8.2019.29254.

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ABSTRACTDNA transformation is one of the methods for inserting DNA into bacterial cells. The current transformation method is widely used to transfer plasmids containing genetic material. This study aims to evaluate the results of plasmid transformation containing merB gene in Escherichia coli BL21(DE3) bacteria. The stages of the research carried out were preceded by the microbiological identification of the E. coli BL21(DE3) bacteria used as hosts. Then the plasmid transformation containing merB gene into the E. coli BL21(DE3) host cell using the heat shock method was carried out. The transformation results were evaluated by observing at the presence of E. coli BL21(DE3) colonies on agar Luria Bertani (LB) media containing ampicillin antibiotics. Plasmids in E. coli BL21(DE3) were isolated and analyzed by 1% agarose gel electrophoresis. The results showed the success of the transformation indicated by the growth of E. coli BL21(DE3) bacteria in agar LB media containing ampicillin and the visualization on agarose gel resulted that the plasmid which carried the merB gene could be transformed in to the E. coli BL21(DE3) bacteria.Keywords : Plasmids, merB genes, heat shock, Escherichia coli BL21(DE3)ABSTRAKTransformasi DNA merupakan salah satu metode untuk memasukkan DNA ke dalam sel bakteri. Metode transformasi saat ini dipakai secara luas untuk mentransfer plasmid yang mengandung bahan genetika. Penelitian ini bertujuan untuk mengevaluasi hasil transformasi plasmid yang mengandung gen merB pada bakteri Escherichia coli BL21(DE3). Tahapan penelitian didahului dengan identifikasi secara mikrobiologi bakteri E. coli BL21(DE3) yang digunakan sebagai inang. Selanjutnya dilakukan transformasi plasmid yang mengandung gen merB kedalam sel inang E. coli BL21(DE3) menggunakan metode heat shock. Hasil transformasi dievaluasi dengan melihat adanya koloni E. coli BL21(DE3) pada media agar Luria Bertani (LB) yang mengandung antibiotik ampisilin. Plasmid pada E. coli BL21(DE3) diisolasi dan dianalisis dengan elektroforesis gel agarose 1%. Hasil penelitian menunjukkan keberhasilan transformasi dengan adanya pertumbuhan bakteri E. coli BL21(DE3) pada media LB yang mengandung ampisillin dan hasil visualisasi pada agarose gel terlihat bahwa plasmid yang membawa gen merB dapat ditransformasikan ke dalam bakteri E. coli BL21(DE3).Kata Kunci : Plasmid, gen merB, heat shock, Escherichia coli BL21(DE3)

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Zhang, X., X. Liu, J. Ma, and J. Zhao. "Silencing of cytochrome P450 CYP6B6 gene of cotton bollworm (Helicoverpa armigera) by RNAi." Bulletin of Entomological Research 103, no. 5 (April 16, 2013): 584–91. http://dx.doi.org/10.1017/s0007485313000151.

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AbstractRNA interference (RNAi) induced through double-stranded RNA (dsRNA) has been used widely to study gene function in insects. In this paper we demonstrate the efficacy of RNAi in the cotton bollworm, Helicoverpa armigera. Using CYP6B6 as the target gene, which is expressed in the fat baby and midgut of the lepidopteran pest H. armigera, we constructed the vector which expressed dsRNA of CYP6B6. Northern blot analysis showed that dsRNA expressed in the Escherichia coli (HT115) was target gene. The results also showed that the gene expression level and protein expression level of H. armigera larvae fed with dsRNA expressed by E. coli were significantly lower than those of all controls, but the gene expression level was more obvious than that at the protein level; significant lethality differences were also found between HT115 bacteria containing L4440-dsC1 treatment and HT115 bacteria containing L4440 vector or CK (ddH2O) in instar larvae on 4 day when continuous feeding, 32.45% mortality was recorded in the group of feeding HT115 bacteria containing L4440-dsC1 on 10 day. Our results suggest that the RNAi pathway can be exploited to control insect pests.

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Noll, Stephan, Jan Reyelt, Thomas Rysiok, Roland Kellner, Detlef Güssow, Stefan Jäkel, Stefanie Hager, and Harald Kranz. "Gezielte Optimierung von Escherichia coli BL21(DE3)." BIOspektrum 19, no. 2 (March 2013): 211–13. http://dx.doi.org/10.1007/s12268-013-0292-2.

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Feng, Li Li, Jian Fei Zhang, Hui Luo, and Zheng Li. "Surface Modification of Acrylonitrile Fibers and Membrane by Nitrilase from Escherichia Coli BL21 (DE3)/pET-Nit." Advanced Materials Research 175-176 (January 2011): 651–55. http://dx.doi.org/10.4028/www.scientific.net/amr.175-176.651.

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The surface of polyacrylonitrile fibers and membrane were modified by nitrilase from Escherichia coli BL21 (DE3)/pET-Nit. Escherichia coli BL21(DE3)/pET-Nit was able to convert nitrile groups on PAN fibers and membrane to corresponding carboxylic acid as indicated by X-ray photoelectron spectroscopy (XPS). An increase of O/C atomic ratio on the fiber and membrane surface showed an increase in hydrophilicity and fabric-dyeing efficiency. Strength of treated fiber decreased by only 1.17%, because only surfacial nitrile groups of acrylic fibers were hydrolyzed by E.coli BL21(DE3)/pET-Nit.

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Liang, Rong, Chang Liu, Xiu Juan Meng, and Song Yi Lin. "Optimization of Proliferation Conditions of Recombinant Escherichia coli BL21(DE3)/pET-28b⁽⁺⁾-aroGM150." Advanced Materials Research 915-916 (April 2014): 887–90. http://dx.doi.org/10.4028/www.scientific.net/amr.915-916.887.

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Applying Escherichia coli (E. coli) for fermentation is a very common technology. However combined with genetic engineering techniques to construct the recombinant Escherichia coli and study their growth characteristics has become the hot spot now. The recombinant Escherichia coli BL21 (DE3) /pET-28baroGM150 had been constructed by our laboratory in the previous experiment. And the purpose of this study was to optimize the proliferation conditions of Escherichia coli BL21 (DE3) /pET-28baroGM150. In order to make the recombinant Escherichia coli grow stable under suitable conditions, using the density of bacteria and plasmid stability as indexes, three factors were tested including temperature, initial pH and loading volume. And the results indicated that the optimal proliferation temperature of the recombinant strain was 30°C, initial pH value was 6.5, loading volume was 150 mL medium of 1000 mL bottles.

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Kim, Sinyeon, Haeyoung Jeong, Eun-Youn Kim, Jihyun F. Kim, Sang Yup Lee, and Sung Ho Yoon. "Genomic and transcriptomic landscape of Escherichia coli BL21(DE3)." Nucleic Acids Research 45, no. 9 (March 31, 2017): 5285–93. http://dx.doi.org/10.1093/nar/gkx228.

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Pandey, Ramesh Prasad, Ha Young Jung, Prakash Parajuli, Thi Huyen Trang Nguyen, Puspalata Bashyal, and Jae Kyung Sohng. "A Synthetic Approach for Biosynthesis of Miquelianin and Scutellarin A in Escherichia coli." Applied Sciences 9, no. 2 (January 9, 2019): 215. http://dx.doi.org/10.3390/app9020215.

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Grapevine (Vitis vinifera) glycucuronosyltransferase (VvGT5) specifically catalyzes flavonol-3-O-glucuronosylation and the blue flowers of Veronica persica (Lamiales, Scrophulariaceae) uridine diphosphate (UDP)-dependent glycosyltransferase (UGT88D8) as flavonoid 7-O-specific glucuronosyltransferases, were chosen, codon optimized, and employed to synthesize the high valued flavonoids glucuronoids, miquelianin and scutellarin A in Escherichia coli. A single vector system was constructed to overexpress entire UDP-glucuronic acid biosynthesis pathway genes, along with a glucokinase gene in Escherichia coli BL21 (DE3). The newly generated E. coli BL21 (DE3) piBR181-glk.pgm2.galU.ugd.UGT88D8 strain produced 12 mg/L (28 µmol/L) of scutellarin A from apigenin, representing only 14% of maximum conversion percentage. Similarly, the strain E. coli BL21 (DE3) piBR181-glk.pgm2.galU.ugd.VvGT5 produced 30 mg/L (62 µmol/L) of miquelianin, representing a 31% conversion of quercetin. This production profile is a good starting point for further host engineering, and for production of respective compounds.

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Qin, Weitong, Xiaoqing Liu, Xiaoxia Yu, Xiaoyu Chu, Jian Tian, and Ningfeng Wu. "Identification of cadmium resistance and adsorption gene from Escherichia coli BL21 (DE3)." RSC Advances 7, no. 81 (2017): 51460–65. http://dx.doi.org/10.1039/c7ra10656d.

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Neve, Isaiah A. A., Jessica N. Sowa, Chih-Chun J. Lin, Priya Sivaramakrishnan, Christophe Herman, Youqiong Ye, Leng Han, and Meng C. Wang. "Escherichia coli Metabolite Profiling Leads to the Development of an RNA Interference Strain for Caenorhabditis elegans." G3: Genes|Genomes|Genetics 10, no. 1 (November 11, 2019): 189–98. http://dx.doi.org/10.1534/g3.119.400741.

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The relationship of genotypes to phenotypes can be modified by environmental inputs. Such crucial environmental inputs include metabolic cues derived from microbes living together with animals. Thus, the analysis of genetic effects on animals’ physiology can be confounded by variations in the metabolic profile of microbes. Caenorhabditis elegans exposed to distinct bacterial strains and species exhibit phenotypes different at cellular, developmental, and behavioral levels. Here we reported metabolomic profiles of three Escherichia coli strains, B strain OP50, K-12 strain MG1655, and B-K-12 hybrid strain HB101, as well as different mitochondrial and fat storage phenotypes of C. elegans exposed to MG1655 and HB101 vs. OP50. We found that these metabolic phenotypes of C. elegans are not correlated with overall metabolic patterning of bacterial strains, but their specific metabolites. In particular, the fat storage phenotype is traced to the betaine level in different bacterial strains. HT115 is another K-12 E. coli strain that is commonly utilized to elicit an RNA interference response, and we showed that C. elegans exposed to OP50 and HT115 exhibit differences in mitochondrial morphology and fat storage levels. We thus generated an RNA interference competent OP50 (iOP50) strain that can robustly and consistently knockdown endogenous C. elegans genes in different tissues. Together, these studies suggest the importance of specific bacterial metabolites in regulating the host’s physiology and provide a tool to prevent confounding effects when analyzing genotype-phenotype interactions under different bacterial backgrounds.

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Robichon, Carine, Jianying Luo, Thomas B. Causey, Jack S. Benner, and James C. Samuelson. "Engineering Escherichia coli BL21(DE3) Derivative Strains To Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography." Applied and Environmental Microbiology 77, no. 13 (May 20, 2011): 4634–46. http://dx.doi.org/10.1128/aem.00119-11.

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ABSTRACTRecombinant His-tagged proteins expressed inEscherichia coliand purified by immobilized metal affinity chromatography (IMAC) are commonly coeluted with nativeE. coliproteins, especially if the recombinant protein is expressed at a low level. TheE. colicontaminants display high affinity to divalent nickel or cobalt ions, mainly due to the presence of clustered histidine residues or biologically relevant metal binding sites. To improve the final purity of expressed His-tagged protein, we engineeredE. coliBL21(DE3) expression strains in which the most recurring contaminants are either expressed with an alternative tag or mutated to decrease their affinity to divalent cations. The current study presents the design, engineering, and characterization of twoE. coliBL21(DE3) derivatives, NiCo21(DE3) and NiCo22(DE3), which express the endogenous proteins SlyD, Can, ArnA, and (optionally) AceE fused at their C terminus to a chitin binding domain (CBD) and the protein GlmS, with six surface histidines replaced by alanines. We show that eachE. coliCBD-tagged protein remains active and can be efficiently eliminated from an IMAC elution fraction using a chitin column flowthrough step, while the modification of GlmS results in loss of affinity for nickel-containing resin. The “NiCo” strains uniquely complement existing methods for improving the purity of recombinant His-tagged protein.

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Isdiyono, Bima Wedana, Dudi Hardianto, and Fransiskus Xaverius Ivan. "PRODUKSI REKOMBINAN SEFALOSPORIN ASILASE SEBAGAI BIOKATALIS UNTUK PRODUKSI ASAM 7-AMINOSEFALOSPORANAT." Jurnal Bioteknologi & Biosains Indonesia (JBBI) 4, no. 1 (July 7, 2017): 28. http://dx.doi.org/10.29122/jbbi.v4i1.2059.

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Production of Cephalosporin Acylase Recombinant as Biocatalyst for 7-Aminocephalosporanic Acid Production7-aminocephalosporanic acid (7-ACA) is a precursor for the production of semisynthetic cephalosporin derivatives. The enzymatic 7-ACA production can use two-stage and one-step enzymatic methods. Two-stage enzymatic method uses D-amino acid oxidase (DAAO) enzyme to produce glutaryl-7-aminocephalosporanic acid (GL-7-ACA) in the first stage and glutaryl-7-aminocephalosporanic acid acylase to produce 7-ACA in the second stage. The one-stage enzymatic method using cephalosporin acylase (CPC acylase) converts the CPC to 7-ACA directly. The aim of this research was to produce recombinant CPC acylase in Escherichia coli BL21(DE3). Transformantion culture E. coli BL21(DE3) was induced with concentrations of IPTG 0; 0.25; 0.5; 0.75; 1; 2 mM for 5 hours. The induction time of IPTG was determined at 0, 1, 2, 3, 4, and 5 hours. The results showed that CPC acylase produced by E. coli BL21(DE3) with optimum condition of CPC acylase production was 0.5 mM IPTG and optimal induction time of IPTG was 5 hours.Keywords: Cephalosporin, cephalosporin acylase, 7-ACA, protein expression, Escherichia coli BL21(DE3) ABSTRAKAsam 7-aminosefalosporanat (7-ACA) merupakan prekursor untuk produksi turunan sefalosporin semisintetik. Produksi 7-ACA secara enzimatik dapat menggunakan metode dua tahap dan satu tahap enzimatik. Metode enzimatik secara dua tahap menggunakan enzim asam D-amino oksidase (DAAO) untuk menghasilkan asam glutaril-7-aminosefalosporinat (GL-7-ACA) pada tahap pertama dan menggunakan asam glutaril-7-aminosefalosporinat asilase untuk menghasilkan 7-ACA pada tahap kedua. Metode enzimatik satu tahap dengan sefalosporin asilase (CPC asilase) mengubah CPC menjadi 7-ACA secara langsung. Tujuan penelitian adalah memproduksi rekombinan CPC asilase di dalam sel Escherichia coli BL21(DE3). Kultur Transforman E. coli BL21(DE3) diinduksi dengan konsentrasi IPTG 0; 0,25; 0,5; 0,75; 1; 2 mM selama 5 jam. Waktu induksi IPTG ditentukan pada 0, 1, 2, 3, 4 dan 5 jam. Hasil penelitian menunjukan bahwa CPC asilase diproduksi oleh E. coli BL21(DE3) dengan kondisi optimal produksi CPC asilase adalah konsentrasi IPTG 0,5 mM dan waktu induksi IPTG optimal adalah 5 jam.

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Aon, Juan C., Richard J. Caimi, Alexander H. Taylor, Quinn Lu, Femi Oluboyede, Jennifer Dally, Michelle D. Kessler, et al. "Suppressing Posttranslational Gluconoylation of Heterologous Proteins by Metabolic Engineering of Escherichia coli." Applied and Environmental Microbiology 74, no. 4 (December 14, 2007): 950–58. http://dx.doi.org/10.1128/aem.01790-07.

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ABSTRACT Minimization of chemical modifications during the production of proteins for pharmaceutical and medical applications is of fundamental and practical importance. The gluconoylation of heterologously expressed protein which is observed in Escherichia coli BL21(DE3) constitutes one such undesired posttranslational modification. We postulated that formation of gluconoylated/phosphogluconoylated products of heterologous proteins is caused by the accumulation of 6-phosphogluconolactone due to the absence of phosphogluconolactonase (PGL) in the pentose phosphate pathway. The results obtained demonstrate that overexpression of a heterologous PGL in BL21(DE3) suppresses the formation of the gluconoylated adducts in the therapeutic proteins studied. When this E. coli strain was grown in high-cell-density fed-batch cultures with an extra copy of the pgl gene, we found that the biomass yield and specific productivity of a heterologous 18-kDa protein increased simultaneously by 50 and 60%, respectively. The higher level of PGL expression allowed E. coli strain BL21(DE3) to satisfy the extra demand for precursors, as well as the energy requirements, in order to replicate plasmid DNA and express heterologous genes, as metabolic flux analysis showed by the higher precursor and NADPH fluxes through the oxidative branch of the pentose phosphate shunt. This work shows that E. coli strain BL21(DE3) can be used as a host to produce three different proteins, a heterodimer of liver X receptors, elongin C, and an 18-kDa protein. This is the first report describing a novel and general strategy for suppressing this nonenzymatic modification by metabolic pathway engineering.

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Tseng, Hsien-Chung, Collin H. Martin, David R. Nielsen, and Kristala L. Jones Prather. "Metabolic Engineering of Escherichia coli for Enhanced Production of (R)- and (S)-3-Hydroxybutyrate." Applied and Environmental Microbiology 75, no. 10 (March 20, 2009): 3137–45. http://dx.doi.org/10.1128/aem.02667-08.

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ABSTRACT Synthetic metabolic pathways have been constructed for the production of enantiopure (R)- and (S)-3-hydroxybutyrate (3HB) from glucose in recombinant Escherichia coli strains. To promote maximal activity, we profiled three thiolase homologs (BktB, Thl, and PhaA) and two coenzyme A (CoA) removal mechanisms (Ptb-Buk and TesB). Two enantioselective 3HB-CoA dehydrogenases, PhaB, producing the (R)-enantiomer, and Hbd, producing the (S)-enantiomer, were utilized to control the 3HB chirality across two E. coli backgrounds, BL21Star(DE3) and MG1655(DE3), representing E. coli B- and K-12-derived strains, respectively. MG1655(DE3) was found to be superior for the production of each 3HB stereoisomer, although the recombinant enzymes exhibited lower in vitro specific activities than BL21Star(DE3). Hbd in vitro activity was significantly higher than PhaB activity in both strains. The engineered strains achieved titers of enantiopure (R)-3HB and (S)-3HB as high as 2.92 g liter−1 and 2.08 g liter−1, respectively, in shake flask cultures within 2 days. The NADPH/NADP+ ratio was found to be two- to three-fold higher than the NADH/NAD+ ratio under the culture conditions examined, presumably affecting in vivo activities of PhaB and Hbd and resulting in greater production of (R)-3HB than (S)-3HB. To the best of our knowledge, this study reports the highest (S)-3HB titer achieved in shake flask E. coli cultures to date.

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Serrano, Yunier, Susana Miraidys Brito, Elsa Pimienta, Alina Falero, and Karen Marrero. "Soluble production of a full-length human papillomavirus type 16 L1 protein by Escherichia coli." Bionatura 6, no. 2 (May 15, 2021): 1684–91. http://dx.doi.org/10.21931/rb/2021.06.02.4.

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Persistent infection with human papillomavirus type 16 (HPV16) causes the development of cervical cancer. Escherichia coli is a cost-effective host successfully used to develop a second-generation vaccine against HPV, based on the purification of soluble truncated L1 protein variants. Previous attempts to produce soluble full-length HPV16-L1 protein by E. coli have failed. This study was aimed at cloning a Cuban HPV16-L1 gene in E. coli and assessing its expression as a soluble full-length L1 protein by manipulating culture conditions. The L1 gene was amplified from a Cuban patient’s cervical sample and cloned into pET28a and pBAD/Myc-HisA vectors. Production and solubility of L1 protein were evaluated in E. coli TOP10 harboring pBADHPV16-L1 plasmid and E. coli BL21-(DE3), Rosetta-(DE3)/pLysS, and SHuffle® T7 Express lysY strains harboring pETHPV16-L1 plasmid, grown under arabinose (0.2%)- or isopropyl β-D-1-thiogalactopyranoside (IPTG, 100 µM)-induction or Super Broth-based auto-induction for 24 and 48 h. The recombinant plasmids pETHPV16-L1 and pBADHPV16-L1 were constructed. The HPV16-L1 protein was produced insoluble to high levels in conventionally IPTG-induced E. coli-pETHPV16-L1 cells. However, under auto-induction, soluble full-length HPV16-L1 protein was successfully produced at similar levels by E. coli BL21 (DE3), Rosetta (DE3) pLysS and SHuffle® T7 Express lysY cells, reaching up to 7.2 ± 0.5% and 14.3 ± 1.6% of the total proteins in the soluble fraction after growing for 24 and 48 h, respectively. It is concluded that the auto-induction procedure at 18 °C with 30 µM IPTG and 100 rev/min promotes soluble full-length HPV16-L1 protein production by E. coli.

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Dumon-Seignovert, Laurence, Guillaume Cariot, and Laurent Vuillard. "The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3)." Protein Expression and Purification 37, no. 1 (September 2004): 203–6. http://dx.doi.org/10.1016/j.pep.2004.04.025.

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Di, Zhi Xin, Jian Zhong Ma, and Yong Gang Wang. "Expression of a Fusion Protein of Human Proinsulin with Glutathione-S-Transferase in Escherichia coli." Advanced Materials Research 998-999 (July 2014): 248–51. http://dx.doi.org/10.4028/www.scientific.net/amr.998-999.248.

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A DNA sequence encoding for the human proinsulin was designed according to the codon bias of Escherichia coli and then chemically synthesized. The synthesized DNA fragment was subcloned into pGEX-3X for expression in E. coli BL21 (DE3) and E. coli BL21 Star (DE3), respectively. Conditions for the highest expression of the GST-proinsulin fusion proteins were optimized. These conditions are that cells of E. coli BL21 star (DE3) are incubated in 100mL of the LB medium with 2 mmol/L IPTG and 60μ?g/mL ampicillin at 26oCfor 4h. After disrupted E. coli cells with ultrasonication, inclusion bodies were precipitated from cell lysis and washed. Fusion proteins from the inclusion bodies were redissolved in 8mmol/L of urea. After dialysed in purified water, fusion proteins were analysed by SDS-PAGE. The purity of the fusion protein is about 80.5% in total. The fusion protein from SDS-PAGE was further identified by mass/mass spectrum. GST in the dyad protein is confirmed by the 9 matched sequences. However, the left part is proved a polypeptide of which is completely different from the human proinsulin.

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Iman P. Maksum, Astri Lestari, Retna P. Fauzia, Saadah D. Rachman, and Ukun M.S. Soedjanaatmadja. "Escherichia coli BL21(DE3) expression system using TorA signal peptide for Recombinant Human Albumin (rHA) secretion." International Journal of Research in Pharmaceutical Sciences 10, no. 4 (October 16, 2019): 3319–24. http://dx.doi.org/10.26452/ijrps.v10i4.1640.

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Human serum albumin (HSA) is the most abundant protein in blood plasm. This protein consisted of 585 amino acids with a molecular weight of 66 kDa and 17 disulfide bonds. HSA obtained from conventional technique allow viral or prion contamination. For that reason, recombinant DNA technology becomes a promising alternative. Because of its well-known genetic, simplicity, and capacity to accommodate many foreign protein, Escherichia coli remains the most widely used in the production of recombinant proteins. But, overproduction of protein may lead to the formation of inclusion bodies and proteolytic degradation. These problems can be overcome by using protease-deficient strain and protein secretion into periplasmic space. The objective of this research is to secrete recombinant HA on E. coli BL21(DE3) using TorA signal peptide and proved using SDS-PAGE. This research method begins with the preparation of competent cell and transformation of E. coli BL21(DE3), expression of recombinant HA in E. coli BL21(DE3), and characterization of expression result by using SDS-PAGE. The result of this study was rHSA can be secreted into extracellular medium using TorA signal peptide with a molecular weight of ± 66.5 kDa.

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Xia, Yanfei, Shen Li, Guohui Xu, Shanshan Xie, Xueting Liu, Xiaomin Lin, Huijun Wu, and Xuewen Gao. "The carB Gene of Escherichia coli BL21(DE3) is Associated with Nematicidal Activity against the Root-Knot Nematode Meloidogyne javanica." Pathogens 10, no. 2 (February 18, 2021): 222. http://dx.doi.org/10.3390/pathogens10020222.

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Biological nematicides have been widely used to lower the losses generated by phytoparasitic nematodes. The purpose of this study was to evaluate the nematicidal effects of Escherichia coli BL21(DE3) against Meloidogyne javanica and to identify nematicide-related genes. Culture filtrates of BL21(DE3) caused juvenile mortality and inhibited egg hatching in a dose-dependent manner. In the greenhouse, treatment of tomato seedlings with BL21(DE3) culture filtrates at 50 and 100% concentrations not only reduced the amount of M. javanica egg masses and galls, but improved plant root and shoot fresh weight. Culture filtrate analysis indicated that the nematicidal active ingredients of strain BL21(DE3) were non-proteinaceous, heat and cold resistant, sensitive to pH and volatile. To identify the genes associated with nematicidal activity, a BL21(DE3) library of 5000 mutants was produced using Tn5 transposase insertion. The culture filtrate of the MB12 mutant showed no nematicidal activity after 72 h of treatment and thermal asymmetrical interlaced PCR demonstrated that the carB gene was disrupted. Nematicidal activity was restored when the pH of the MB12 culture filtrate was adjusted to the original pH value (4.15) or following MB12 complementation with the carB gene, confirming a role for carB in mediating pH value and nematicidal activity. The outcomes of this pilot study indicate that BL21(DE3) is a potential microorganism for the continuable biological control of root-knot nematode in tomato and that carB affects the nematicidal activity of BL21(DE3) by modulating the pH environment.

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Ratelade, Julien, Marie-Caroline Miot, Emmett Johnson, Jean-Michel Betton, Philippe Mazodier, and Nadia Benaroudj. "Production of Recombinant Proteins in the lon-Deficient BL21(DE3) Strain of Escherichia coli in the Absence of the DnaK Chaperone." Applied and Environmental Microbiology 75, no. 11 (April 3, 2009): 3803–7. http://dx.doi.org/10.1128/aem.00255-09.

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ABSTRACT To eliminate unavoidable contamination of purified recombinant proteins by DnaK, we present a unique approach employing a BL21(DE3) ΔdnaK strain of Escherichia coli. Selected representative purified proteins remained soluble, correctly assembled, and active. This finding establishes DnaK dispensability for protein production in BL21(DE3), which is void of Lon protease, key to eliminating unfolded proteins.

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Cheng, Jun, Guochao Xu, Ruizhi Han, Jinjun Dong, and Ye Ni. "Efficient access to l-phenylglycine using a newly identified amino acid dehydrogenase from Bacillus clausii." RSC Advances 6, no. 84 (2016): 80557–63. http://dx.doi.org/10.1039/c6ra17683f.

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Razali, Rafida, Vijay Kumar Subbiah, and Cahyo Budiman. "Technical Data of Heterologous Expression and Purification of SARS-CoV-2 Proteases Using Escherichia coli System." Data 6, no. 9 (September 16, 2021): 99. http://dx.doi.org/10.3390/data6090099.

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The SARS-CoV-2 coronavirus expresses two essential proteases: firstly, the 3Chymotrypsin-like protease (3CLpro) or main protease (Mpro), and secondly, the papain-like protease (PLpro), both of which are considered as viable drug targets for the inhibition of viral replication. In order to perform drug discovery assays for SARS-CoV-2, it is imperative that efficient methods are established for the production and purification of 3CLpro and PLpro of SARS-CoV-2, designated as 3CLpro-CoV2 and PLpro-CoV2, respectively. This article expands the data collected in the attempts to express SARS-CoV-2 proteases under different conditions and purify them under single-step chromatography. Data showed that the use of E. coli BL21(DE3) strain was sufficient to express 3CLpro-CoV2 in a fully soluble form. Nevertheless, the single affinity chromatography step was only applicable for 3CLpro-CoV2 expressed at 18 °C, with a yield and purification fold of 92% and 49, respectively. Meanwhile, PLpro-CoV2 was successfully expressed in a fully soluble form in either BL21(DE3) or BL21-CodonPlus(DE3) strains. In contrast, the single affinity chromatography step was only applicable for PLpro-CoV2 expressed using E. coli BL21-CodonPlus(DE3) at 18 or 37 °C, with a yield and purification fold of 86% (18 °C) or 83.36% (37 °C) and 112 (18 °C) or 71 (37 °C), respectively. The findings provide a guide for optimizing the production of SARS-CoV-2 proteases of E. coli host cells.

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Di Lorenzo, Mirella, Aurelio Hidalgo, Michael Haas, and Uwe T. Bornscheuer. "Heterologous Production of Functional Forms of Rhizopus oryzae Lipase in Escherichia coli." Applied and Environmental Microbiology 71, no. 12 (December 2005): 8974–77. http://dx.doi.org/10.1128/aem.71.12.8974-8977.2005.

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ABSTRACT To date, expression of the lipase from Rhizopus oryzae (ROL) in Escherichia coli always led to the formation of inclusion bodies and inactive protein. However, the production of active ROL and its precursor ProROL in soluble form was achieved when E. coli Origami(DE3) and pET-11d were used as expression systems.

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Vu, Thi Bich Ngoc, Thi Thao Nguyen, Thi Hoa Chu, and Thi Tuyen Do. "Cloning and expression of recombinant thrombin in Escherichia coli JM109 (DE3)." Journal of Vietnamese Environment 8, no. 1 (January 15, 2017): 21–25. http://dx.doi.org/10.13141/jve.vol8.no1.pp21-25.

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Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Prothrombin is a thrombin precursor playing the important role in the coagulation physiological as well as pathological condition. Thrombin is the key to convert the fibrinogen into fibrin by switching activation of XIII factor, pushed plasminogen into plasmin, the develope of the fibroblast and helps the stabilization of thrombolysis. In this study, the prothrombin gene was 936 bp in lengths and encoded 312 amino acids from bovine lung was optimized codon, was cloned in pET21a+ vector and expression in E. coli, in order to replace traditional bandages having slow affect, reduce the cost of products, cater the community health. The results showed that initially the successful cloning and expression of recombinant prothrombin in E. coli JM109 (DE3). Prothrombin, 1 glycoprotein huyết tương liên quan tới quá trình đông máu gồm 2 vùng Gla, 2 vùng Kringle và 1 vùng serine protease. Prothrombin là tiền chất của thrombin có vai trò quan trọng trong sinh lý đông máu cũng như tình trạng bệnh lý. Thrombin được xem như chìa khóa để chuyển hóa fibrinogen thành fibrin bằng cách hoạt hóa các yếu tố đông máu như XIII, thúc đẩy chuyển plasminogen thành plasmin và kích thích tăng sinh các tế bào tơ (fibroblast), giúp ổn định quá trình làm tan huyết khối. Trong nghiên cứu, các gen prothrombin được tách dòng từ phổi bỏ có kích thước 936 bp, mã hóa cho 312 axit amin được tối ưu hóa codon, nhân dòng vào vector pET21a+ và biểu hiện trong E. coli. Mục đích của nghiên cứu nhằm tạo ra băng gạc cầm máu nhanh, giá thành rẻ, phục vụ sức khỏe cộng đồng và thay thể băng gạc truyền thống. Kết quả nghiên cứu bước đầu cho thấy đã nhân dòng và biểu hiện thành công prothrombin tái tổ hợp ở chủng E. coli JM109 (DE3).

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Tian, Zi-gang, Tian-tang Dong, Ya-lin Yang, Da Teng, and Jian-hua Wang. "Expression of antimicrobial peptide LH multimers in Escherichia coli C43(DE3)." Applied Microbiology and Biotechnology 83, no. 1 (May 2009): 143–49. http://dx.doi.org/10.1007/s00253-009-1893-z.

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Jeong, Haeyoung, Valérie Barbe, Choong Hoon Lee, David Vallenet, Dong Su Yu, Sang-Haeng Choi, Arnaud Couloux, et al. "Genome Sequences of Escherichia coli B strains REL606 and BL21(DE3)." Journal of Molecular Biology 394, no. 4 (December 2009): 644–52. http://dx.doi.org/10.1016/j.jmb.2009.09.052.

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Schlegel, Susan, John Löfblom, Chiara Lee, Anna Hjelm, Mirjam Klepsch, Marc Strous, David Drew, Dirk Jan Slotboom, and Jan-Willem de Gier. "Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)." Journal of Molecular Biology 423, no. 4 (November 2012): 648–59. http://dx.doi.org/10.1016/j.jmb.2012.07.019.

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Zhao, Mei Na, Zongbao Zheng, and Tao Chen. "Expressing Xylanases in Escherichia Coli by Cell Surface Display." Advanced Materials Research 634-638 (January 2013): 965–69. http://dx.doi.org/10.4028/www.scientific.net/amr.634-638.965.

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In this research, xylan was utilized by a recombinant whole cell biocatalyst, which was developed by expressing three xylanases — β-xylosidase, endoxylanase, and α-arabinofuranosidase — on the surface of the E. coli BL21 (DE3). The xylanases were displayed on the surface of the cells by fusing with anchor proteins, Blc. The assimilation of xylan by cell surface display was the first step in the consolidated bioprocessing (CBP). This result shows that the engineering strains could be endowed with the ability to assimilate xylan. The co-display engineering strains utilized xylan and expressed less metabolic burden than the engineering strains secreting extracellular xylanases.

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Li, Da, Fangling Ji, Chundong Huang, and Lingyun Jia. "High Expression Achievement of Active and Robust Anti-β2 microglobulin Nanobodies via E.coli Hosts Selection." Molecules 24, no. 16 (August 7, 2019): 2860. http://dx.doi.org/10.3390/molecules24162860.

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Nanobodies (VHHs) overcome many of the drawbacks of conventional antibodies, and the related technologies represent state-of-the-art and advanced applications in scientific research, pharmaceuticals, and therapies. In terms of productivity and economic cost, the cytoplasmic expression of VHHs in Escherichia coli (E. coli) is a good process for their recombinant production. The cytoplasmic environment of the host is critical to the affinity and stability of the recombinant VHHs in soluble form, yet the effects have not been studied. For this purpose, recombinant anti-β2 microglobulin VHHs were constructed and expressed in four commercialized E. coli hosts, including BL21 (DE3), Rosetta-gami B (DE3) pLysS, Origami 2 (DE3) and SHuffle T7 Express. The results showed that anti-β2 microglobulin (β2MG) VHHs expressed in different hosts exhibited distinctive differences in the affinity and structural characteristics. The VHHs expressed in Rosetta-gami B (DE3) pLysS possessed not only the greatest affinity of (equilibrium dissociation constant) KD = 4.68 × 10−8 M but also the highest yields compared with the VHHs expressed in BL21 (DE3), Origami 2 (DE3) and SHuffle T7 Express. In addition, the VHHs expressed in Rosetta-gami B (DE3) pLysS were more stable than the VHHs expressed in the rest three hosts. Thus far, we have successfully realized the high expression of the active and robust anti-β2MG VHHs in Rosetta-gami B (DE3) pLysS. The underlying principle of our study is able to guide the expression strategies of nanobodies on the context of industrial large-scale production.

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Papić, Ljubomir, José Rivas, Soledad Toledo, and Jaime Romero. "Double-stranded RNA production and the kinetics of recombinant Escherichia coli HT115 in fed-batch culture." Biotechnology Reports 20 (December 2018): e00292. http://dx.doi.org/10.1016/j.btre.2018.e00292.

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Trung, Do Minh, Do Hai Quynh, Tran Viet Tien, Nguyen Duy Bac, Do Thi Tuyen, and Nguyen Thuy Duong. "Cloning and expression of pigC gene in Escherichia coli." Vietnam Journal of Biotechnology 16, no. 4 (August 8, 2020): 757–65. http://dx.doi.org/10.15625/1811-4989/16/4/13488.

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Prodigiosin (Pg), which is particularly of interest because of anticancer and antimicrobial activities, can be produced through the PigC-catalyzed condensation reaction of 4-methoxy-2, 2’-bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-amylpyrrole (MAP). Therefore, the PigC protein plays an important role in prodigiosin biosynthetic pathway. However, studies related to PigC protein have not been carried out in Vietnam yet. In this work, the pigC gene was cloned and expressed in Escherichia coli DH10B and BL21 (DE3), respectively. Using PCR and universal primers, we amplified a fragment of 3 kb covering entire coding region of the pigC gene from Serratia sp. strain M5. The pigC gene was inserted into pJET1.2 vector, and then transformed into E. coli DH10B. The sequence of a recombinant vector pJET1.2/pigC was evaluated by using whole colony PCR amplification. Sequence alignment results revealed that the obtained pigC gene possesses 71.5% and 75.4% of nucleotide identity in comparison with two strains, Serratia 39006 and Serratia sp. AS9 published in GenBank with their respective accession numbers of AJ833001 and CP002773. The recombinant vector pJET1.2/pigC was used to reamplify pigC, and the acquired amplicon was inserted into pET22b vector at the site of HindIII and XhoI. The clone E. coli BL21 (DE3) containing recombinant vector pET22b/pigC was expressed in the auto-induced medium. The presence of PigC protein in the lysate was identified as a 100 kDa band through Western Blot analysis using anti his-tag antibody. Afterward, the PigC protein was purified by Ni-NTA column, and its expression level was quantified through SDS-PAGE analysis. The results of our study provide a potential material for producing prodigiosin from recombinant protein in Vietnam.

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Villarreal, D. M., C. L. Phillips, A. M. Kelley, S. Villarreal, A. Villaloboz, P. Hernandez, J. S. Olson, and D. P. Henderson. "Enhancement of Recombinant Hemoglobin Production in Escherichia coli BL21(DE3) Containing the Plesiomonas shigelloides Heme Transport System." Applied and Environmental Microbiology 74, no. 18 (August 1, 2008): 5854–56. http://dx.doi.org/10.1128/aem.01291-08.

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ABSTRACT To produce recombinant hemoglobin in Escherichia coli, sufficient intracellular heme must be present, or the protein folds improperly and is degraded. In this study, coexpression of human hemoglobin genes and Plesiomonas shigelloides heme transport genes enhanced recombinant hemoglobin production in E. coli BL21(DE3) grown in medium containing heme.

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Le, Xuan Mai Huong, To Dinh Le, Thao Thi Phuong Dang, and Thuoc Linh Tran. "Cloning and expression of recombinant human leptin in Escherichia coli." Science and Technology Development Journal 16, no. 1 (March 31, 2013): 5–12. http://dx.doi.org/10.32508/stdj.v16i1.1391.

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Leptin, a peptide hormone, is produced by mature adipocytes and functions primarily in the hypothalamus to reduce food intake and body weight. Recombinant h-leptin has been shown to be effective in obesity treatment. To overexpression of recombinant human leptin in Escherichia coli, the human leptin gene (hob gene) was cloned into the vector pET-28a. When analysis expression of human leptin in E. coli BL21(DE3) strain, it was found that recombinant vector pET-hob expressed h-leptinproteins in cytoplasm, and mainly as insoluble inclusion bodies. This result will be the premise for researching to produce recombinant human leptin protein.

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Yadava, Ramesh Singh, Ravindra Kumar, and Pramod Kumar Yadava. "Expression of lexA targeted ribozyme in Escherichia coli BL-21 (DE3) cells." Molecular and Cellular Biochemistry 271, no. 1-2 (March 2005): 197–203. http://dx.doi.org/10.1007/s11010-005-6340-6.

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Lee, Jae Hyeon, Suman Lama, Jung Rae Kim, and Sung Hoon Park. "Production of 1,3-Propanediol from Glucose by Recombinant Escherichia coli BL21(DE3)." Biotechnology and Bioprocess Engineering 23, no. 2 (March 2018): 250–58. http://dx.doi.org/10.1007/s12257-018-0017-y.

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Andreishcheva, Ekaterina N., and Willie F. Vann. "Escherichia coli BL21(DE3) chromosome contains a group II capsular gene cluster." Gene 384 (December 2006): 113–19. http://dx.doi.org/10.1016/j.gene.2006.07.020.

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Wang, Mengmeng, Qinghua Liu, Fen Li, Jiawei Tang, Xuesong Xiong, Yingying Yang, Pei Ju, Ziyi Wang, Robert G. Gilbert, and Liang Wang. "The dynamic changes of glycogen molecular structure in Escherichia coli BL21(DE3)." Carbohydrate Polymers 259 (May 2021): 117773. http://dx.doi.org/10.1016/j.carbpol.2021.117773.

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Hien Trang, Nguyen Thi, Le Thanh Hoang, and Do Thi Tuyen. "Optimization of L-asparaginase production from Escherichia coli using response surface methodology." Vietnam Journal of Biotechnology 16, no. 4 (August 8, 2020): 767–75. http://dx.doi.org/10.15625/1811-4989/16/4/10861.

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Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. In previous study, the L-asparaginase from Erwinia chrysanthermy was expressed in Escherichia coli BL21(DE3). The recombinant L-asparaginase was produced from recombinant E.coli BL21(DE3) under different cultivation conditions (inducer concentration, inoculum concentration and KH2PO4 concentration). The optimized conditions by response surface methodology using face centered central composite design. The analysis of variance coupled with larger value of R2 (0.9) showed that the quadratic model used for the prediction was highly significant (p < 0.05). Under the optimized conditions, the model produced L-asparaginase activity of 123.74 U/ml at 1.03 mM IPTG, 3% (v/v) inoculum and 0.5% (w/v) KH2PO4. Recombinant protein was purified by two step using gel filtration and DEAE chromatography. The purified L-asparaginase had a molecular mass of 37 kDa with specific activity of 462 U/mg and identified by MALDI-TOF mass spectrometry. Results of MALDI-TOF analysis confirmed that recombinant protein was L-asparaginase II. Recombinant L-asparaginase has antiproliferative activity with K562 cell line. In conclusion, this study has innovatively developed cultivation conditions for better production of recombinant L-asparaginase in shake flask culture.

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Hanh, Vu Thi. "EXPRESSION OF GLUTARYL7-AMINOCEPHALOSPORANIC ACID ACYLASE IN ESCHERICHIA COLI BL21(DE3) AND IMMOBILIZATION OF RECOMBINANT ENZYME ON NANOPOROUS MATERIALS." Vietnam Journal of Science and Technology 54, no. 4A (March 21, 2018): 123. http://dx.doi.org/10.15625/2525-2518/54/4a/12012.

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The synthesis of 7-ACA from cephalosporin C (CPC) by a two-step bioconversion using D-amino acid oxidase (DAAO) and glutaryl 7-ACA acylase (GLA) has been effectively and largely applied in pharmaceutical industry. In this study, the gene gla coding for 720-amino acid GLA from plasmid pUC57::gla was analyzed and successfully inserted into vector pET22b(+) to form expression vector pET22b(+)::gla. The newly constructed expression vector pET22b(+)::gla was cloned and then transformed into Escherichia coli BL21(DE3) to generate recombinant strain E. coli BL21(DE3)[pET22b(+)::gla]. The suitable conditions for expression of gla gene were in LB medium at 30 oC and induced by 0.4 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 hours. Under the chosen culturing parameters, expression of gla gene by E. coli BL21(DE3)/[pET22b(+)::gla] resulted in a recombinant GLA (rGLA) with molecular weight of 83 kDa and catalytic activity of 2.7 U/mg of total protein. Experimental research on immobilization of rGLA onto ten nanoporous materials were showed that, SBA-15 was the best one for immobilization of rGLA, reaching activity of immobilized enzyme of 22.2 U/g matrix. Furthermore, optimal conditions of procedure for immobilizing rGLA on nanomaterials (SBA-15) were determined as follows: temperature is 25 °C, pH7.0 and immobilization time –60 minutes. Therefore the results reported in this study revealed the successfully heterologous expression of GLA in recombinant E. coli and potential immobilization of enzyme on inorganic nano-materials.

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Li, Qi, Bingbing Sun, Jun Chen, Yiwen Zhang, Yu Jiang, and Sheng Yang. "A modified pCas/pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli." Acta Biochimica et Biophysica Sinica 53, no. 5 (March 25, 2021): 620–27. http://dx.doi.org/10.1093/abbs/gmab036.

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Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (Cas9)-based genome editing tool pCas/pTargetF system that we established previously has been widely used in Escherichia coli MG1655. However, this system failed to manipulate the genome of E. coli BL21(DE3), owing to the potential higher leaky transcription of the gRNA-pMB1 specific to pTargetF in this strain. In this study, we modified the pCas/pTargetF system by replacing the promoter of gRNA-pMB1 with a tightly regulated promoter PrhaB, changing the replicon of pCas to a nontemperature-sensitive replicon, adding the sacB gene into pCas, and replacing the original N20-specific sequence of pTargetF with ccdB gene. We call this updated system as pEcCas/pEcgRNA. We found that gRNA-pMB1 indeed showed a slightly higher leaky expression in the pCas/pTargetF system compared with pEcCas/pEcgRNA. We also confirmed that genome editing can successfully be performed in BL21(DE3) by pEcCas/pEcgRNA with high efficiency. The application of pEcCas/pEcgRNA was then expanded to the E. coli B strain BL21 StarTM (DE3), K-12 strains MG1655, DH5α, CGMCC3705, Nissle1917, W strain ATCC9637, and also another species of Enterobacteriaceae, Tatumella citrea DSM13699, without any specific modifications. Finally, the plasmid curing process was optimized to shorten the time from $\sim$60 h to $\sim$32 h. The entire protocol (including plasmid construction, editing, electroporation and mutant verification, and plasmid elimination) took only $\sim$5.5 days per round in the pEcCas/pEcgRNA system, whereas it took $\sim$7.5 days in the pCas/pTargetF system. This study established a faster-acting genome editing tool that can be used in a wider range of E. coli strains and will also be useful for other Enterobacteriaceae species.

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Thu, Nguyễn Minh, Nguyễn Minh Giang, Phạm Hải Như, Đinh Nho Thái, Đỗ Thị Huyền, and Trương Nam Hải. "BIỂU HIỆN GEN XBXS14 MÃ HÓA XYLAN 1,4-Β-XYLOSIDASE CÓ NGUỒN GỐC TỪ VI KHUẨN RUỘT MỐI COPTOTERMES GESTROI TRONG TẾ BÀO ESCHERICHIA COLI ROSETTA (DE3)." Vietnam Journal of Biotechnology 15, no. 3 (December 14, 2018): 555–61. http://dx.doi.org/10.15625/1811-4989/15/3/13392.

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Khung đọc mở GL0112518 gồm 1077 nucleotide (còn gọi là Xbxs14) mã hóa xylan 1,4-β-xylosidase kiềm đã được khai thác từ dữ liệu giải trình tự DNA metagenome của vi sinh trong ruột mối. Gen được tổng hợp nhân tạo và gắn vào vector pET22b(+) để tiến hành biểu hiện enzyme dạng tái tổ hợp cho đánh giá tính chất nzyme. Kết quả cho thấy gen được biểu hiện tốt trong ba chủng E. coli BL21, Rosetta (DE3) và JM109 (DE3), trong đó hai chủng E. coli BL21, Rosetta (DE3) thu được sinh khối lớn nhưng hầu hết enzyme nằm ở pha không tan. Bằng cách thay đổi điều kiện biểu hiện bao gồm nồng độ chất cảm ứng IPTG, nhiệt độ nuôi cấy biểu hiện gen và thời gian thu mẫu, một phần nhỏ enzyme đã được biểu hiện ở dạng tan. Điều kiện thích hợp cho việc biểu hiện Xbxs14 trong chủng E. coli Rosetta (DE3) là nuôi cấy lắc trong môi trường LB, cảm ứng ở IPTG 0,05 mM, nhiệt độ 20oC trong 2 ngày. Enzyme tái tổ hợp được biểu hiện ở pha tan (soluble fraction) có hoạt tính của xylosidase, phân cắt liên kết β(1→4) glycoside ở cơ chất pNPX tạo màu vàng đặc trưng của nitrophenyl. Trong 1 lít môi trường nuôi cấy thu được 3,15 U enzyme.

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Grishin, D. V., Ju A. Gladilina, D. D. Zhdanov, M. V. Pokrovskaya, I. Yu Toropygin, S. S. Aleksandrova, V. S. Pokrovskiy, and N. N. Sokolov. "Preparation and characterization of a new mutant homolog of chemotaxis protein CheY from anaerobic hyperthermophilic microorganism Thermotoga naphthophila." Biomeditsinskaya Khimiya 65, no. 1 (January 2019): 41–50. http://dx.doi.org/10.18097/pbmc20196501041.

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Using genetic engineering methods the expression vectors structures have been designed to produce recombinant proteins TnaCheY and Tna CheY-mut, the homologues of the chemotaxis protein CheY from the hyperthermophilic organism Thermotoga naphthophila in Escherichia coli BL21(DE3) cells. The cultivation conditions of transformed strains were optimized. The influence of episomal expression of the heterologous chemotaxis protein CheY on growth kinetics parameters of the culture of mesophilic bacteria E. coli was studied. The optimal purification flowchart of the obtained proteins using thermolysis is proposed. Using the E. coli BL21(DE3) laboratory strain as an example, the possibility of employment the episomal expression of such proteins to control the cultivation and production time of pharmaceutically and industrially valuable metabolites due to the impact on some stages of the bacterial chemotaxis is experimentally proved.

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Giap, Ho Ta, Phan Ngoc Han, Tran Le Duy Phuong, Phung Thi Thu Phung, and Vu Van Van. "Cloning, expression and purification of fructosyl amino acid oxidase (FAOX) in Escherichia Coli." ENGINEERING AND TECHNOLOGY 11, no. 1 (March 24, 2021): 21–29. http://dx.doi.org/10.46223/hcmcoujs.tech.en.11.1.1238.2021.

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Introduction: The level of serum HbA1c is an indicator of the average blood sugar level in the last three months. HbA1c can be quantified using assays involving the enzyme fructosyl amino acid oxidase (FAOX). This study aims to produce GST-tagged FAOX-TE (GST/FAOX-TE), a thermal stable and specific variant of FAOX, for future application studies. Materials and methods: The E. coli strains DH5α and BL21 (DE3) were used as cloning and expression hosts, respectively. The FAOX-TE sequence was synthesized at IDT (US) and clonned into pGEX-4T3 vector, which was confirmed by Colony PCR. The expression was induced at 16°C, 0.5 mM IPTG in LB media containing 50 µg/ml ampicilin. The protein expression profile was analyzed by SDS-PAGE. The cell pellet was sonicated and purified by Glutathione Sepharose 4 Fast Flow (Cytiva, US). The catalytic activity of GST/FAOX-TE with fructosyl valine was determined using high performance anion exchange chromatography with pulsed amperometry detection (HPAEC-PAD). Results: The fusion protein was successfully expressed in Escherichia coli using the plasmid pGEX-4T3 and purified to high purity 93%. Recombinant GST/FAOX-TE was shown to be active on fructosyl valine. Conclusions: Active GST/FAOX-TE was successfully expressed in E. coli BL21 (DE3) and purified, which will be used for future development of biosensors for fructosyl valine quantification.

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Chai, Pengdi, Xiuying Pu, Jianqiang Li, Xiaoyu Xia, Jun Ge, Amiao Luo, Hui Su, Weijie Zhang, and Jianzhong Ma. "Expression and Purification of Tetanus Toxin Fragment C in Escherichia coli BL21(DE3)." Protein & Peptide Letters 27, no. 11 (November 16, 2020): 1132–40. http://dx.doi.org/10.2174/0929866527666200528113327.

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Background: Tetanus is an infectious disease caused by Clostridium secreting tetanus toxin in anaerobic environment. The fragment C of Tetanus toxin (TTc) has been widely studied as a candidate vaccine to replace the existing tetanus toxoid vaccine. Objective: In this study, we established a simple method to purify recombinant protein TTc with ion-exchange chromatography from Escherichia coli expression systems. Methods: The TTc gene sequence was cloned into pET26b (+) vector and transferred to E. coli BL21 (DE3) for expression. The fermentation conditions (IPTG concentration, Induction temperature, Induction time) were optimized to obtain more soluble proteins. The soluble proteins were purified by Anion exchange chromatography and Cation exchange chromatography. The sequence of columns in the purification process was discussed. Finally, the stability of purified TTc protein were determined, the secondary structure of the purified TTc protein was determined by circular dichroism. The molecular weight of the purified TTc protein was determined by liquid chromatograph- mass spectrometer. Furthermore, we verified the immunogenicity of the purified protein in mice. Results: The purity of TTc improved from 34% to 88% after the first anion exchange column, and the final yield of recombinant TTc (purity > 95%) can reach 84.79% after the following cation exchange chromatography. The recombinant TTc had a molecular weight of 51.737 KDa, was stable at 4 °C and weak alkaline environment, was a β-sheet secondary structure, and had strong immunogenicity. Conclusion: The purification method we developed might be an efficient method for the industrial production of tetanus recombinant TTc vaccine.

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Akhtar, M. Kalim, and Patrik R. Jones. "Construction of a synthetic YdbK-dependent pyruvate:H2 pathway in Escherichia coli BL21(DE3)." Metabolic Engineering 11, no. 3 (May 2009): 139–47. http://dx.doi.org/10.1016/j.ymben.2009.01.002.

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Tegel, Hanna, Samuel Tourle, Jenny Ottosson, and Anja Persson. "Increased levels of recombinant human proteins with the Escherichia coli strain Rosetta(DE3)." Protein Expression and Purification 69, no. 2 (February 2010): 159–67. http://dx.doi.org/10.1016/j.pep.2009.08.017.

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Liu, Changqing, Kai Zheng, Ying Xu, Lacmata Tamekou Stephen, Jiming Wang, Hongwei Zhao, Tongqing Yue, et al. "Expression and characterization of soybean seed coat peroxidase in Escherichia coli BL21(DE3)." Preparative Biochemistry & Biotechnology 47, no. 8 (July 18, 2017): 768–75. http://dx.doi.org/10.1080/10826068.2017.1342258.

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Pinske, Constanze, Markus Bönn, Sara Krüger, Ute Lindenstrauß, and R. Gary Sawers. "Metabolic Deficiences Revealed in the Biotechnologically Important Model Bacterium Escherichia coli BL21(DE3)." PLoS ONE 6, no. 8 (August 3, 2011): e22830. http://dx.doi.org/10.1371/journal.pone.0022830.

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Chaudhary, Amit Kumar, and Eun Yeol Lee. "Tightly regulated and high level expression vector construction for Escherichia coli BL21 (DE3)." Journal of Industrial and Engineering Chemistry 31 (November 2015): 367–73. http://dx.doi.org/10.1016/j.jiec.2015.07.011.

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Journal articles on the topic 'Escherichia coli HT115 (DE3)'

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