Escherichia coli BL21(DE3)
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ABSTRACT:
PROVIDER: PRJNA510019 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8316940.fastq.gz | Fastqsanger.gz | |||
| SRR8316950.fastq.gz | Fastqsanger.gz | |||
| SRR8316941.fastq.gz | Fastqsanger.gz | |||
| SRR8316951.fastq.gz | Fastqsanger.gz | |||
| SRR8316942.fastq.gz | Fastqsanger.gz |
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Project description:Escherichia coli BL21(DE3) has long served as a model organism for scientific research, as well as a workhorse for biotechnology. Here we present the most current genome annotation of E. coli BL21(DE3) based on the transcriptome structure of the strain that was determined for the first time. The genome was annotated using multiple automated pipelines and compared to the current genome annotation of the closely related strain, E. coli K-12. High-resolution tiling array data of E. coli BL21(DE3) from several different stages of cell growth in rich and minimal media were analyzed to characterize the transcriptome structure and to provide supporting evidence for open reading frames. This new integrated analysis of the genomic and transcriptomic structure of E. coli BL21(DE3) has led to the correction of translation initiation sites for 88 coding DNA sequences and provided updated information for most genes. Additionally, 37 putative genes and 66 putative non-coding RNAs were also identified. The panoramic landscape of the genome and transcriptome of E. coli BL21(DE3) revealed here will allow us to better understand the fundamental biology of the strain and also advance biotechnological applications in industry.
| S-EPMC5435950 | biostudies-literature
Project description:Escherichia coli BLR(DE3) is a commercially available recA-deficient derivative of BL21(DE3), one of the most widely used strains for recombinant protein expression. Here, we present the full-genome sequence of BLR(DE3) and highlight additional differences with its parent strain BL21(DE3) which were previously unreported but may affect its physiology.
| S-EPMC5454202 | biostudies-literature
Project description:Experimentally mapped transcriptome structure of Escherichia coli BL21(DE3) by hybridizing total RNA (including RNA species <200 nt) to genome-wide high-density tiling arrays (60 mer probes tiled every 10 nt).
2017-01-13 | GSE93565 | GEO
Project description:BACKGROUND:Escherichia coli is one of the most widely used hosts for recombinant protein production in academia and industry. Strain BL21(DE3) is frequently employed due to its advantageous feature of lacking proteases which avoids degradation of target protein. Usually it is used in combination with the T7-pET system where induction is performed by one point addition of IPTG. We recently published a few studies regarding lactose induction in BL21(DE3) strains. BL21(DE3) can only take up the glucose-part of the disaccharide when fed with lactose. However, initially additional glucose has to be supplied as otherwise the ATP-related lactose uptake barely happens. Yet, as lactose is an inexpensive compound compared to glucose and IPTG, a new induction strategy by a lactose-only feed during induction seems attractive. Thus, we investigated this idea in the galactose metabolizing strain HMS174(DE3). RESULTS:We show that strain HMS174(DE3) can be cultivated on lactose as sole carbon source during induction. We demonstrate that strain HMS174(DE3) exhibits higher product and biomass yields compared to BL21(DE3) when cultivated in a lactose fed-batch. More importantly, HMS174(DE3) cultivated on lactose even expresses more product than BL21(DE3) in a standard IPTG induced glucose fed-batch at the same growth rate. Finally, we demonstrate that productivity in HMS174(DE3) lactose-fed batch cultivations can easily be influenced by the specific lactose uptake rate (qs,lac). This is shown for two model proteins, one expressed in soluble form and one as inclusion body. CONCLUSIONS:As strain HMS174(DE3) expresses even slightly higher amounts of target protein in a lactose fed-batch than BL21(DE3) in a standard cultivation, it seems a striking alternative for recombinant protein production. Especially for large scale production of industrial enzymes cheap substrates are essential. Besides cost factors, the strategy allows straight forward adjustment of specific product titers by variation of the lactose feed rate.
| S-EPMC6206895 | biostudies-literature
Project description:Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that the system increased the BL21's defense ability against phage infection. Furthermore, the targeted loci on phage genome played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 h of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% of the un-infected control, which was significantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21's to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection.
| S-EPMC7332536 | biostudies-literature
Project description:When producing recombinant proteins, the use of Escherichia coli strain BL21(DE3) in combination with the T7-based pET-expression system is often the method of choice. In a recent study we introduced a mechanistic model describing the correlation of the specific glucose uptake rate (qs,glu) and the corresponding maximum specific lactose uptake rate (qs,lac,max) for a pET-based E. coli BL21(DE3) strain producing a single chain variable fragment (scFv). We showed the effect of qs,lac,max on productivity and product location underlining its importance for recombinant protein production. In the present study we investigated the mechanistic qs,glu/qs,lac,max correlation for four pET-based E. coli BL21(DE3) strains producing different recombinant products and thereby proved the mechanistic model to be platform knowledge for E. coli BL21(DE3). However, we found that the model parameters strongly depended on the recombinant product. Driven by this observation we tested different dynamic bioprocess strategies to allow a faster investigation of this mechanistic correlation. In fact, we succeeded and propose an experimental strategy comprising only one batch cultivation, one fed-batch cultivation as well as one dynamic experiment, to reliably determine the mechanistic model for qs,glu/qs,lac,max and get trustworthy model parameters for pET-based E. coli BL21(DE3) strains which are the basis for bioprocess development.
| S-EPMC5362885 | biostudies-literature
Project description:BackgroundStyrene is a versatile commodity petrochemical used as a monomer building-block for the synthesis of many useful polymers. Although achievements have been made on styrene biosynthesis in microorganisms, several bottleneck problems limit factors for further improvement in styrene production.ResultsA two-step styrene biosynthesis pathway was developed and introduced into Escherichia coli BL21(DE3). Systematic optimization of styrene biosynthesis, such as enzyme screening, codon and plasmid optimization, metabolic flow balance, and in situ fermentation was performed. Candidate isoenzymes of the rate-limiting enzyme phenylalanine ammonia lyase (PAL) were screened from Arabidopsis thaliana (AtPAL2), Fagopyrum tataricum (FtPAL), Petroselinum crispum (PcPAL), and Artemisia annua (AaPAL). After codon optimization, AtPAL2 was found to be the most effective one, and the engineered strain was able to produce 55 mg/L styrene. Subsequently, plasmid optimization was performed, which improved styrene production to 103 mg/L. In addition, two upstream shikimate pathway genes, aroF and pheA, were overexpressed in the engineered strain, which resulted in styrene production of 210 mg/L. Subsequently, combined overexpression of tktA and ppsA increased styrene production to 275 mg/L. Finally, in situ product removal was used to ease the burden of end-product toxicity. By using isopropyl myristate as a solvent, styrene production reached a final titer of 350 mg/L after 48 h of shake-flask fermentation, representing a 636% improvement, which compared with that achieved in the original strain.ConclusionsThis present study achieved the highest titer of de novo production of styrene in E. coli at shake-flask fermentation level. These results obtained provided new insights for the development of microbial production of styrene in a sustainable and environment friendly manner.
| S-EPMC5784704 | biostudies-literature
Project description:The Escherichia coli B strain BL21(DE3) has had a profound impact on biotechnology through its use in the production of recombinant proteins. Little is understood, however, regarding the physiology of this important E. coli strain. We show here that BL21(DE3) totally lacks activity of the four [NiFe]-hydrogenases, the three molybdenum- and selenium-containing formate dehydrogenases and molybdenum-dependent nitrate reductase. Nevertheless, all of the structural genes necessary for the synthesis of the respective anaerobic metalloenzymes are present in the genome. However, the genes encoding the high-affinity molybdate transport system and the molybdenum-responsive transcriptional regulator ModE are absent from the genome. Moreover, BL21(DE3) has a nonsense mutation in the gene encoding the global oxygen-responsive transcriptional regulator FNR. The activities of the two hydrogen-oxidizing hydrogenases, therefore, could be restored to BL21(DE3) by supplementing the growth medium with high concentrations of Ni²⁺ (Ni²⁺-transport is FNR-dependent) or by introducing a wild-type copy of the fnr gene. Only combined addition of plasmid-encoded fnr and high concentrations of MoO₄²⁻ ions could restore hydrogen production to BL21(DE3); however, to only 25-30% of a K-12 wildtype. We could show that limited hydrogen production from the enzyme complex responsible for formate-dependent hydrogen evolution was due solely to reduced activity of the formate dehydrogenase (FDH-H), not the hydrogenase component. The activity of the FNR-dependent formate dehydrogenase, FDH-N, could not be restored, even when the fnr gene and MoO₄²⁻ were supplied; however, nitrate reductase activity could be recovered by combined addition of MoO₄²⁻ and the fnr gene. This suggested that a further component specific for biosynthesis or activity of formate dehydrogenases H and N was missing. Re-introduction of the gene encoding ModE could only partially restore the activities of both enzymes. Taken together these results demonstrate that BL21(DE3) has major defects in anaerobic metabolism, metal ion transport and metalloprotein biosynthesis.
| S-EPMC3149613 | biostudies-literature
Project description:Escherichia coli BL21(DE3) is an industrial model microbe for the mass-production of bioproducts such as biofuels, biorefineries, and recombinant proteins. However, despite its important role in scientific research and biotechnological applications, a high-quality metabolic network model for metabolic engineering is yet to be developed. Here, we present the comprehensive metabolic network model of E. coli BL21(DE3), named iHK1487, based on the latest genome reannotation and phenome analysis. The metabolic model consists of 1,164 unique metabolites, 2,701 metabolic reactions, and 1,487 genes. The model was validated and improved by comparing the simulation results with phenome data from phenotype microarray tests. Previous transcriptome profile data was incorporated during model reconstruction, and flux prediction was simulated using the model. iHK1487 was simulated to explore the metabolic features of BL21(DE3) such as broad spectrum amino acid utilization and enhanced flux through the upper glycolytic pathway and TCA cycle. iHK1487 will contribute to systematic understanding of cellular physiology and metabolism of E. coli BL21(DE3) and highlight its biotechnological applications.
| S-EPMC6150544 | biostudies-literature
Project description:Heterologous expression systems based on promoters inducible with isopropyl-?-D-1-thiogalactopyranoside (IPTG), e.g., Escherichia coli BL21(DE3) and cognate LacI(Q)/P(lacUV5)-T7 vectors, are commonly used for production of recombinant proteins and metabolic pathways. The applicability of such cell factories is limited by the complex physiological burden imposed by overexpression of the exogenous genes during a bioprocess. This burden originates from a combination of stresses that may include competition for the expression machinery, side-reactions due to the activity of the recombinant proteins, or the toxicity of their substrates, products and intermediates. However, the physiological impact of IPTG-induced conditional expression on the recombinant host under such harsh conditions is often overlooked.The physiological responses to IPTG of the E. coli BL21(DE3) strain and three different recombinants carrying a synthetic metabolic pathway for biodegradation of the toxic anthropogenic pollutant 1,2,3-trichloropropane (TCP) were investigated using plating, flow cytometry, and electron microscopy. Collected data revealed unexpected negative synergistic effect of inducer of the expression system and toxic substrate resulting in pronounced physiological stress. Replacing IPTG with the natural sugar effector lactose greatly reduced such stress, demonstrating that the effect was due to the original inducer's chemical properties.IPTG is not an innocuous inducer; instead, it exacerbates the toxicity of haloalkane substrate and causes appreciable damage to the E. coli BL21(DE3) host, which is already bearing a metabolic burden due to its content of plasmids carrying the genes of the synthetic metabolic pathway. The concentration of IPTG can be effectively tuned to mitigate this negative effect. Importantly, we show that induction with lactose, the natural inducer of P lac , dramatically lightens the burden without reducing the efficiency of the synthetic TCP degradation pathway. This suggests that lactose may be a better inducer than IPTG for the expression of heterologous pathways in E. coli BL21(DE3).
| S-EPMC4687329 | biostudies-literature