scholarly journals The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

2020 ◽  
Vol 7 (1) ◽  
pp. 8 ◽  
Author(s):  
Johanna Hausjell ◽  
Regina Kutscha ◽  
Jeannine D. Gesson ◽  
Daniela Reinisch ◽  
Oliver Spadiut
Keyword(s):  
Expression System ◽  
Stable Process ◽  
Product Formation ◽  
Lac Repressor ◽  
Process Conditions ◽  
Expression Systems ◽  
Iptg Induction ◽  
Lactose Induction ◽  
A Genome ◽  
Uptake Rates

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

scholarly journals Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

2020 ◽  
Author(s):  
Artur Schuller ◽  
Monika Cserjan-Puschmann ◽  
Christopher Tauer ◽  
Johanna Jarmer ◽  
Martin Wagenknecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Cellular Level ◽  
Lac Repressor ◽  
Expression Systems ◽  
E Coli ◽  
Basal Expression

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences. Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level. Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

scholarly journals Escherichia coli σ70 promoters allow expression rate control at the cellular level in genome-integrated expression systems

2019 ◽  
Author(s):  
Artur Schuller ◽  
Monika Cserjan-Puschmann ◽  
Christopher Tauer ◽  
Johanna Jarmer ◽  
Martin Wagenknecht ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Protein Production ◽  
Protein Production ◽  
Expression System ◽  
Lac Repressor ◽  
Expression Systems ◽  
E Coli ◽  
Basal Expression ◽  
T7 Expression System

Abstract Background The genome-integrated T7 expression system offers significant advantages, in terms of productivity and product quality, even when expressing the gene of interest (GOI) from a single copy of. Compared to plasmid-based expression systems, this system does not incur a plasmid-mediated metabolic load, and it does not vary the dosage of the GOI during the production process. However, long-term production with T7 expression system leads to a rapidly growing non-producing population, because the T7 RNA polymerase (RNAP) is prone to mutations. The present study aimed to investigate whether two σ 70 promoters, which were recognized by the Escherichia coli host RNAP, might be suitable in genome-integrated expression systems. We applied a promoter engineering strategy that allowed control of expressing the model protein, GFP, by introducing lac operators ( lacO ) into the constitutive T5 and A1 promoter sequences.Results We showed that, in genome-integrated E. coli expression systems that used σ 70 promoters, the number of lacO sites must be well balanced. Promoters containing three and two lacO sites exhibited low basal expression, but resulted in a complete stop in recombinant protein production in partially induced cultures. In contrast, expression systems regulated by a single lacO site and the lac repressor element, lacI Q , on the same chromosome caused very low basal expression, were highly efficient in recombinant protein production, and enables fine-tuning of gene expression levels on a cellular level.Conclusions Based on our results, we hypothesized that this phenomenon was associated with the autoregulation of the lac repressor protein, LacI. We reasoned that the affinity of LacI for the lacO sites of the GOI must be lower than the affinity of LacI to the lacO sites of the endogenous lac operon; otherwise, LacI autoregulation could not take place, and the lack of LacI autoregulation would lead to a disturbance in lac repressor-mediated regulation of transcription. By exploiting the mechanism of LacI autoregulation, we created a novel E. coli expression system for use in recombinant protein production, synthetic biology, and metabolic engineering applications.

scholarly journals Indicative Marker Microbiome Structures Deduced from the Taxonomic Inventory of 67 Full-Scale Anaerobic Digesters of 49 Agricultural Biogas Plants

2021 ◽  
Vol 9 (7) ◽  
pp. 1457
Author(s):  
Julia Hassa ◽  
Johanna Klang ◽  
Dirk Benndorf ◽  
Marcel Pohl ◽  
Benedikt Hülsemann ◽  
...  
Keyword(s):  
Stable Process ◽  
Process Condition ◽  
Full Scale ◽  
Process Models ◽  
Process Efficiency ◽  
Process Conditions ◽  
Rrna Gene ◽  
General Process ◽  
Specific Distribution ◽  
Biogas Plants

There are almost 9500 biogas plants in Germany, which are predominantly operated with energy crops and residues from livestock husbandry over the last two decades. In the future, biogas plants must be enabled to use a much broader range of input materials in a flexible and demand-oriented manner. Hence, the microbial communities will be exposed to frequently varying process conditions, while an overall stable process must be ensured. To accompany this transition, there is the need to better understand how biogas microbiomes respond to management measures and how these responses affect the process efficiency. Therefore, 67 microbiomes originating from 49 agricultural, full-scale biogas plants were taxonomically investigated by 16S rRNA gene amplicon sequencing. These microbiomes were separated into three distinct clusters and one group of outliers, which are characterized by a specific distribution of 253 indicative taxa and their relative abundances. These indicative taxa seem to be adapted to specific process conditions which result from a different biogas plant operation. Based on these results, it seems to be possible to deduce/assess the general process condition of a biogas digester based solely on the microbiome structure, in particular on the distribution of specific indicative taxa, and without knowing the corresponding operational and chemical process parameters. Perspectively, this could allow the development of detection systems and advanced process models considering the microbial diversity.

scholarly journals A genome-scale metabolic model of Saccharomyces cerevisiae that integrates expression constraints and reaction thermodynamics

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Omid Oftadeh ◽  
Pierre Salvy ◽  
Maria Masid ◽  
Maxime Curvat ◽  
Ljubisa Miskovic ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Expression System ◽  
Biomass Composition ◽  
Industrial Biotechnology ◽  
Overflow Metabolism ◽  
Cellular Expression ◽  
A Genome ◽  
Wide Range ◽  
Eukaryotic Organisms ◽  
Genome Scale

AbstractEukaryotic organisms play an important role in industrial biotechnology, from the production of fuels and commodity chemicals to therapeutic proteins. To optimize these industrial systems, a mathematical approach can be used to integrate the description of multiple biological networks into a single model for cell analysis and engineering. One of the most accurate models of biological systems include Expression and Thermodynamics FLux (ETFL), which efficiently integrates RNA and protein synthesis with traditional genome-scale metabolic models. However, ETFL is so far only applicable for E. coli. To adapt this model for Saccharomyces cerevisiae, we developed yETFL, in which we augmented the original formulation with additional considerations for biomass composition, the compartmentalized cellular expression system, and the energetic costs of biological processes. We demonstrated the ability of yETFL to predict maximum growth rate, essential genes, and the phenotype of overflow metabolism. We envision that the presented formulation can be extended to a wide range of eukaryotic organisms to the benefit of academic and industrial research.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

2020 ◽  
Author(s):  
Youngbin Oh ◽  
Hyeonjin Kim ◽  
Bora Lee ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Binary Vector ◽  
Expression System ◽  
Plant Genome ◽  
Nicotiana Attenuata ◽  
Specific Sequence ◽  
Editing Efficiency ◽  
Guide Rna ◽  
Assembly Method ◽  
A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

scholarly journals Analysis of Melt Pool Characteristics and Process Parameters Using a Coaxial Monitoring System during Directed Energy Deposition in Additive Manufacturing

Materials ◽  
2019 ◽  
Vol 12 (2) ◽  
pp. 308 ◽  
Author(s):  
Christian Kledwig ◽  
Holger Perfahl ◽  
Martin Reisacher ◽  
Frank Brückner ◽  
Jens Bliedtner ◽  
...  
Keyword(s):  
Stable Process ◽  
Base Material ◽  
Industrial Applications ◽  
Process Conditions ◽  
Melt Pool ◽  
Temporal Variance ◽  
Laser Deposition Welding ◽  
Working Distance ◽  
Melt Pool Characteristics ◽  
Pool Area

The growing number of commercially available machines for laser deposition welding show the growing acceptance and importance of this technology for industrial applications. Their increasing usage in research and production requires process stability and user-friendly handling. A commercially available DMG MORI LT 65 3D hybrid machine used in combination with a CCD-based coaxial temperature measurement system was utilized in this work to investigate what information relating to the intensity distribution of melt pool surfaces could be appropriate to draw conclusions about process conditions. In this study it is shown how the minimal required specific energy for a stable process can be determined, and it is indicated that the evolution of a plasma plume depends on thermal energy within the base material. An estimated melt pool area—calculated by the number of pixels (NOP) with intensities larger than a fixed, predefined threshold—builds the main measure in analysing images from the process camera. The melt pool area and its temporal variance can also serve as an indicator for an increased working distance.

scholarly journals Effect of His-Tag on Expression, Purification, and Structure of Zinc Finger Protein, ZNF191(243-368)

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Dongxin Zhao ◽  
Zhongxian Huang
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression System ◽  
Zinc Finger Proteins ◽  
Hereditary Diseases ◽  
Expression Systems ◽  
His Tag ◽  
Finger Protein ◽  
Inclusion Body Formation ◽  
Structure And Functions

Zinc finger proteins are associated with hereditary diseases and cancers. To obtain an adequate amount of zinc finger proteins for studying their properties, structure, and functions, many protein expression systems are used. ZNF191(243-368) is a zinc finger protein and can be fused with His-tag to generate fusion proteins such as His6-ZNF191(243-368) and ZNF191(243-368)-His8. The purification of His-tag protein using Ni-NTA resin can overcome the difficulty of ZNF191(243-368) separation caused by inclusion body formation. The influences of His-tag on ZNF191(243-368) properties and structure were investigated using spectrographic techniques and hydrolase experiment. Our findings suggest that insertion of a His-tag at the N-terminal or C-terminal end of ZNF191(243-368) has different effects on the protein. Therefore, an expression system should be considered based on the properties and structure of the protein. Furthermore, the hydrolase activity of ZNF191(243-368)-His8has provided new insights into the design of biological functional molecules.

Oxygen Transfer Efficiency Measurements Using Off-Gas Techniques

1989 ◽  
Vol 21 (10-11) ◽  
pp. 1295-1300 ◽  
Author(s):  
W. C. Boyle ◽  
B. G. Hellstrom ◽  
L. Ewing
Keyword(s):  
Oxygen Uptake ◽  
Uptake Rate ◽  
Oxygen Transfer ◽  
Oxygen Uptake Rate ◽  
Transfer Efficiency ◽  
Theoretical Development ◽  
Process Conditions ◽  
Uptake Rates ◽  
Oxygen Transfer Efficiency ◽  
On Line

The off-gas technique for measuring oxygen transfer efficiency in aeration tanks under process conditions was proposed as an accurate technique for calibrating or verifying on-line methods used to estimate oxygen uptake rates. The theoretical development of the off-gas technique was presented. Application of this technique in verifying or calibrating existing on-line methods for estimating oxygen uptake rate was presented. Direct application as an on-line method for estimating oxygen uptake rate was also discussed.

scholarly journals Production of Human IFNγ Protein in Nicotiana benthamiana Plant through an Enhanced Expression System Based on Bamboo mosaic Virus

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 509 ◽  
Author(s):  
Min-Chao Jiang ◽  
Chung-Chi Hu ◽  
Na-Sheng Lin ◽  
Yau-Heiu Hsu
Keyword(s):  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Expression System ◽  
Therapeutic Protein ◽  
Biologically Active ◽  
Total Soluble Protein ◽  
Expression Vectors ◽  
Human Interferon ◽  
Protein Accumulation ◽  
Expression Systems

Plant-based systems are safe alternatives to the current platforms for the production of biologically active therapeutic proteins. However, plant-based expression systems face certain major challenges, including the relatively low productivity and the generation of target proteins in biologically active forms. The use of plant virus-based expression systems has been shown to enhance yields, but further improvement is still required to lower the production cost. In this study, various strategies were employed to increase the yields of an important therapeutic protein, human interferon gamma (IFNγ), in Nicotiana benthamiana through modifications of expression vectors based on potexviruses. Among these, the vector based on a coat protein (CP)-deficient Bamboo mosaic virus (BaMV), pKB△CHis, was shown to exhibit the highest expression level for the unmodified IFNγ. Truncation of the N-terminal signal peptide of IFN (designated mIFNγ) resulted in a nearly seven-fold increase in yield. Co-expression of a silencing suppressor protein by replacing the coding sequence of BaMV movement protein with that of P19 led to a 40% increase in mIFNγ accumulation. The fusion of endoplasmic reticulum (ER) retention signal with mIFNγ significantly enhanced the accumulation ratio of biologically active dimeric mIFNγ to 87% relative to the non-active monomeric form. The construct pKB19mIFNγER, employing the combination of all the above enhancement strategies, gave the highest level of protein accumulation, up to 119 ± 0.8 μg/g fresh weight, accounting for 2.5% of total soluble protein (TSP) content. These findings advocate the application of the modified BaMV-based vector as a platform for high-level expression of therapeutic protein in N. benthamiana.

scholarly journals Monitoring N : P Ratio and Cd, Cu, Pb, and Zn Contents in Different Types of Anaerobic Digestates: A Six-Year Study Case

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Christina-Luise Roß ◽  
Kerstin Nielsen ◽  
Jorita Krieger ◽  
Marieke Hoffmann ◽  
Karen Sensel-Gunke ◽  
...  
Keyword(s):  
Stable Process ◽  
Zinc Content ◽  
Pig Manure ◽  
Process Conditions ◽  
Pig Slurry ◽  
Large Variability ◽  
Lead And Zinc ◽  
German Legislation ◽  
Biogas Plants

Depending on the quality of the input substrates, process parameters, and postfermentation treatments, digestates may contain a broad spectrum of potentially toxic elements. We suspected that these contents may vary on a broad scale even under seemingly stable process conditions at the biogas plant. Digestates from four biogas plants were therefore continuously analyzed for their contents of phosphorus, nitrogen, cadmium, copper, lead, and zinc over a period of six years. The input substrates varied between the plants (e.g., cattle and pig slurry and rye and maize silage), but were the same for each plant over the whole period. The N : P ratio of the digestates ranged from 2 to 24, with the digestate coming from cofermentation of pig slurry and energy crops (“DG Pig”) having the widest range of N : P ratio over the years. Heavy metal loads of all digestates and during all evaluations did not exceed the limits set by European or German legislation, but as previously expected, showed a large variability especially if cattle or pig manure were used as substrates. Copper content of Cattle slurry before digestion was 897.7 mg kg−1 DM in one case, and zinc content of DG Pig reached 590.2 mg kg−1 DM also once during the investigation. As a result, we strongly recommend to monitor especially phosphorus, copper, and zinc contents in digestates very closely and in short intervals.

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