Transcriptional analysis for off-target effects by sgRNA variants in E.coli BL21(DE3)
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE222051 | GEO | 2023/04/04
REPOSITORIES: GEO
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Project description:To identify potential single-guide RNA (sgRNAs) contaminants in the GMP HBG-sgRNA, we performed SMARTer smRNA-sequencing (Takara). 80.79% of the 5' spacer sequence perfectly matched the targeting sgRNA sequence and no evidence of contaminants sgRNAs that could target other genomic regions.
2024-08-21 | GSE275157 | GEO
Project description:Transcriptional analysis for off-target effects by sgRNA variants in E.coli BL21(DE3)
| PRJNA917517 | ENA
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(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:Transcriptional profiling of E. coli strain comparing control BL21 with recombineered HLT013. HLT013 strains was derived from BL21 and capable of producing thymidine.
2015-06-18 | GSE69963 | GEO
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:In many industrial sectors continuous processing is already the golden standard to maximize productivity. However, when working with living cells, subpopulation formation causes instabilities in long-term cultivations. In cascaded continuous cultivation, biomass formation and recombinant protein expression can be spatially separated. This cultivation mode was found to facilitate stable protein expression using microbial hosts, however mechanistic knowledge of this cultivation strategy is scarce. In this contribution we present a method workflow to reduce workload and accelerate the establishment of stable continuous processes with E. coli BL21(DE3) exclusively based on bioengineering methods.
| S-EPMC8169658 | biostudies-literature
Project description:tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the properties of tRNase ZL that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA) and that cytosolic tRNase ZL can modulate gene expression by cleaving mRNA under the direction of cellular 5M-bM-^@M-2-half-tRNA or microRNA as sgRNA. In order to estimate a number of potential therapeutic heptamer-type sgRNAs for hematological malignancies, we constructed an sgRNA library composed of 156 heptamer-type sgRNAs, and examined how the sgRNAs affect viability of leukemia and myeloma cells. And we found that 20 of the 156 sgRNAs can efficiently induce apoptosis in at least one of the cancer cell lines. Furthermore, we demonstrated that 4 of the 20 effective sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models. DNA microarray analysis for changes in an mRNA profile by these four heptamer-type sgRNAs suggested at least one candidate target mRNA that contains a potential tRNase ZL target site for each sgRNA. Changes in gene expression in HL60 cells were measured after 18-hour incubation in the absence or presence of one of five different heptamer-type sgRNAs. *Heptamer sequences requested but not provided by submitter
2014-06-30 | E-GEOD-49578 | biostudies-arrayexpress
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