scholarly journals Scaling production of GFP1-10 detector protein in E. coli for secretion screening by split GFP assay

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Carolin Müller ◽  
Chika L. Igwe ◽  
Wolfgang Wiechert ◽  
Marco Oldiges
Keyword(s):  
Inclusion Bodies ◽  
Shake Flask ◽  
Defined Medium ◽  
Laboratory Scale ◽  
Target Protein ◽  
Small Scale ◽  
Fluorescence Signal ◽  
Application Study ◽  
Shake Flask Cultivation ◽  
Independent Screening

Abstract Background The split GFP assay is a well-known technology for activity-independent screening of target proteins. A superfolder GFP is split into two non-fluorescent parts, GFP11 which is fused to the target protein and GFP1-10. In the presence of both, GFP1-10 and the GFP11-tag are self-assembled and a functional chromophore is formed. However, it relies on the availability and quality of GFP1-10 detector protein to develop fluorescence by assembly with the GFP11-tag connected to the target protein. GFP1-10 detector protein is often produced in small scale shake flask cultivation and purified from inclusion bodies. Results The production of GFP1-10 in inclusion bodies and purification was comprehensively studied based on Escherichia coli as host. Cultivation in complex and defined medium as well as different feed strategies were tested in laboratory-scale bioreactor cultivation and a standardized process was developed providing high quantity of GFP1-10 detector protein with suitable quality. Split GFP assay was standardized to obtain robust and reliable assay results from cutinase secretion strains of Corynebacterium glutamicum with Bacillus subtilis Sec signal peptides NprE and Pel. Influencing factors from environmental conditions, such as pH and temperature were thoroughly investigated. Conclusions GFP1-10 detector protein production could be successfully scaled from shake flask to laboratory scale bioreactor. A single run yielded sufficient material for up to 385 96-well plate screening runs. The application study with cutinase secretory strains showed very high correlation between measured cutinase activity to split GFP fluorescence signal proofing applicability for larger screening studies.

scholarly journals Influence of Light Intensity and Temperature on Cultivation of Microalgae Desmodesmus Communis in Flasks and Laboratory-Scale Stirred Tank Photobioreactor

2015 ◽  
Vol 52 (2) ◽  
pp. 59-70 ◽  
Author(s):  
J. Vanags ◽  
L. Kunga ◽  
K. Dubencovs ◽  
V. Galvanauskas ◽  
O. Grīgs
Keyword(s):  
Light Intensity ◽  
Shake Flask ◽  
Scale Up ◽  
Biomass Concentration ◽  
Biomass Productivity ◽  
Pilot Scale ◽  
Laboratory Scale ◽  
Stirred Tank ◽  
Light Limitation ◽  
Maximum Biomass

Abstract Optimization of the microalgae cultivation process and of the bioprocess in general traditionally starts with cultivation experiments in flasks. Then the scale-up follows, when the process from flasks is transferred into a laboratory-scale bioreactor, in which further experiments are performed before developing the process in a pilot-scale reactor. This research was done in order to scale-up the process from a 0.4 1 shake flask to a 4.0 1 laboratory-scale stirred-tank photobioreactor for the cultivation of Desmodesmus (D.) communis microalgae. First, the effect of variation in temperature (21-29 ºC) and in light intensity (200-600 μmol m-2s-1) was studied in the shake-flask experiments. It was shown that the best results (the maximum biomass concentration of 2.72 g 1-1 with a specific growth rate of 0.65 g g-1d-1) can be achieved at the cultivation temperature and light intensity being 25 °C and 300 μmol m2s-1, respectively. At the same time, D. communis cultivation under the same conditions in stirred-tank photobioreactor resulted in average volumetric productivities of biomass due to the light limitation even when the light intensity was increased during the experiment (the maximum biomass productivity 0.25 g 1-1d-1; the maximum biomass concentration 1.78 g 1-1).

scholarly journals First results of pipe pile static load test in small laboratory scale

2018 ◽  
Vol 251 ◽  
pp. 04038 ◽  
Author(s):  
Michal Baca ◽  
Jaroslaw Rybak
Keyword(s):  
Static Load ◽  
Axial Loading ◽  
Laboratory Scale ◽  
Load Test ◽  
Scale Model ◽  
Small Scale ◽  
Small Laboratory ◽  
Static Load Test ◽  
First Results ◽  
Load Tests

Presented laboratory testing program of tubular steel piles is a part of a bigger research program which contained static load tests in full scale and numerical simulations of conducted research. The main goal of the research is to compare static load tests with different working conditions of a shaft. The presented small scale model tests are the last part of the research. The paper contains the testing methodology description and first results of model pile axial loading. The static load tests in a small laboratory scale were conducted in a container filled with uniformly compacted medium sand (MSa). The first results of the investigation are presented in this paper, with the comparison of two pile capacities obtained for different roughness of the pile shaft (skin friction). The results are presented as load-displacement curves obtained by means of the Brinch-Hansen 80% method.

scholarly journals Versatile format of minichaperone-based protein fusion system

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Maria S. Yurkova ◽  
Olga A. Sharapova ◽  
Vladimir A. Zenin ◽  
Alexey N. Fedorov
Keyword(s):  
Recombinant Proteins ◽  
Inclusion Bodies ◽  
Target Protein ◽  
Fusion System ◽  
Protein Fusion ◽  
Gene Engineering ◽  
Apical Domain ◽  
Stable Forms ◽  
Protein Variants ◽  
Protein Moiety

Abstract Hydrophobic recombinant proteins often tend to aggregate upon expression into inclusion bodies and are difficult to refold. Producing them in soluble forms constitutes a common bottleneck problem. A fusion system for production of insoluble hydrophobic proteins in soluble stable forms with thermophilic minichaperone, GroEL apical domain (GrAD) as a carrier, has recently been developed. To provide the utmost flexibility of the system for interactions between the carrier and various target protein moieties a strategy of making permutated protein variants by gene engineering has been applied: the original N- and C-termini of the minichaperone were linked together by a polypeptide linker and new N- and C-termini were made at desired parts of the protein surface. Two permutated GrAD forms were created and analyzed. Constructs of GrAD and both of its permutated forms fused with the initially insoluble N-terminal fragment of hepatitis C virus’ E2 protein were tested. Expressed fusions formed inclusion bodies. After denaturation, all fusions were completely renatured in stable soluble forms. A variety of permutated GrAD variants can be created. The versatile format of the system provides opportunities for choosing an optimal pair between particular target protein moiety and the best-suited original or specific permutated carrier.

The Fracture and Aerosol Release of Impacted HLW Glasses and HLW Canisters

1988 ◽  
Vol 127 ◽  
Author(s):  
Hans G. Scheibel ◽  
V. Friehmelt ◽  
H. Froehlich
Keyword(s):  
Impact Velocity ◽  
Thermal Stresses ◽  
Full Scale ◽  
Laboratory Scale ◽  
Small Scale ◽  
Release Mechanism ◽  
Experimental Conditions ◽  
Drop Tests ◽  
Ground Plate ◽  
The Impact

ABSTRACTThe fracture and release mechanism of radioactive aerosols of HLW glass and HLW canisters are studied experimentally by laboratory scale and full scale drop tests. The experimental conditions model the conditions of accidental drops in a deep salt repository. The laboratory scale drop tests have a scaling factor of 1:10. Accelerated probes of simulated HLW glass impact on a ground plate and the size distributions of broken fines and released aerosols are measured by sieving and scanning electron microscopy (SEM) of aerosol samples.The impact velocity is determined as the dominating impact parameter. Further parameters tested, such as waste glass composition, cooling time (residual thermal stresses), probe temperature at impact, and ground characteristics, show no measurable influence. Source terms of released respirable aerosols are evaluated for two reference cases, borehole drop (impact velocity v = 80 m/s) and reloading hall drop (v = 14 m/s), the values being 0.1 % and to 2.10-4 % respectively of the glass probe mass. The full scale drop tests are performed with European Standard HLW canisters. The canisters keep their integrity in all tests up to drop heights of 14 m. On opening the canisters, the broken fines are analyzed by sieving. The results are in good agreement with the small scale tests and confirm their acceptability for use in a safety analysis.

scholarly journals Development of a semi-defined medium for high cell density cultivation of Escherichia coli in shake flask culture system: Part 2

2013 ◽  
Author(s):  
Wenfa Ng
Keyword(s):  
Cell Density ◽  
Shake Flask ◽  
High Cell Density ◽  
Defined Medium ◽  
Lag Phase ◽  
Overflow Metabolism ◽  
High Cell ◽  
High Cell Density Cultivation ◽  
Batch Mode ◽  
Diauxic Lag

The characteristics of the culture vessel determine, to a large extent, the type of growth medium suitable for use. For example, most growth media for high cell density cultivation are designed for expensive bioreactors operating either in continuous or fed-batch mode, where provision of additional nutrients and/or removal of metabolic waste products from the basal medium – comprising salts, buffer components and small amount of carbon and nitrogen sources - help increase biomass yield by maintaining culture conditions within the range conducive for growth. The inexpensive and ubiquitous shake flask, in contrast, is usually operated in batch mode and contains, at the outset, a comprehensive medium with all necessary nutrients for conversion into biomass and, also serves as a repository for secreted metabolites - some of which are detrimental for cell growth. Thus, designing medium for high cell density cultivation in shake-flask is an optimization process aiming to increase biomass formation while reducing toxic metabolite secretion. This preprint reports improvements to a previously reported semi-defined medium (Wenfa Ng, 2013, https://peerj.com/preprints/115v1) for high cell density aerobic cultivation of Escherichia coli DH5α (ATCC 53868) in shake flask. Specifically, by reducing the concentrations of glucose (from 6.0 to 4.0 g/L) and ammonium chloride (from 1.5 to 1.0 g/L), the following improvements were obtained: a shorter diauxic lag phase (3 versus 5 hours); a higher maximal optical density (12.0 versus 11.0) in a shorter total culture period (27 versus 48 hours), and smaller pH variation during cultivation (6.0 to 7.6 versus 5.5 to 7.8). Similar to the earlier study, glucose and yeast extract served as principal carbon sources in separate growth phases for E. coli in the improved formulated medium (FMimproved). Specifically, an OD600nm of 6.6 was attained after 9 hours of growth on glucose at 37 oC. Following a lag phase of 3 hours, growth resumed on yeast extract and the OD600nm reached 12.0 after 27 hours. The broth’s pH decreased from 7.1 to 6.0 during the first growth phase, whereupon it gradually rose to 7.6 at the end of culture. A smaller pH decrease along with higher biomass yield in the first growth phase suggested that the lower glucose concentration in FMimproved might have prevented overflow metabolism - and associated negative effects on growth - thus, resulting in a shorter diauxic lag phase and total culture period. Collectively, increase in cell yield, as well as decrease in total culture time and a shorter diauxic lag phase arise from a small reduction in glucose concentration - which suggested that an optimum exist, beyond which, occurrence of overflow metabolism would reduce cell yield and biomass formation.

Scale-up of agitated crystallizers; Cooling batch crystallization of potassium aluminium sulphate

1982 ◽  
Vol 47 (4) ◽  
pp. 1189-1194 ◽  
Author(s):  
Jaroslav Nývlt ◽  
Piotr Karpiński ◽  
Stanislav Žáček ◽  
Miloslav Karel ◽  
Jerzy Budz ◽  
...  
Keyword(s):  
Experimental Error ◽  
Scale Up ◽  
Laboratory Scale ◽  
Small Scale ◽  
Sieve Analysis ◽  
Aluminium Sulphate ◽  
Derived Relations ◽  
Batch Crystallization ◽  
Mean Size ◽  
The Mean

The crystallization of potassium aluminium sulphate was conducted by cooling a solution saturated at 70°C to a temperature of 25°C at three various cooling rates. The measurements were performed on a small scale (160 cm3) and a large laboratory scale (0.021 m3). The mean size of product crystals was determined by sieve analysis, and the system constant, BN, was calculated using previously derived relations. The BN value is the same, within experimental error, for all the experiments and scales, indicating that agitated-vessel cooling crystallizers can be modelled successfully even on a very small laboratory scale.

scholarly journals Indirect Dryers for Biomass Drying—Comparison of Experimental Characteristics for Drum and Rotary Configurations

2020 ◽  
Vol 4 (1) ◽  
pp. 18
Author(s):  
Jan Havlík ◽  
Tomáš Dlouhý
Keyword(s):  
Electric Heating ◽  
Pilot Scale ◽  
Laboratory Scale ◽  
Small Scale ◽  
Operating Characteristics ◽  
Drying Characteristics ◽  
Green Wood ◽  
Rotary Dryer ◽  
Specific Material ◽  
Drying Curves

This paper focuses on indirect biomass drying. It compares the operating characteristics of a laboratory-scale drum dryer and a pilot-scale rotary dryer. Before the design of an industrial dryer for a specific material, it is important to experimentally prove the process and to determine the drying characteristics of the material. To verify the portability of experimental results for indirect dryers, a drum dryer with indirect electric heating in a laboratory scale was designed and built to test and study the process of indirect drying. Based on the results obtained on a small-scale device, a prototype of a pilot steam-heated rotary dryer was designed and manufactured. A broad range of experiments with green wood chips and wet bark from open-air storage with moisture contents of 50 to 65 wt % were carried out on both dryers. The drying curves indicating the process, the square and volumetric evaporation capacities, and the drying energy consumption were obtained and compared, and the feasibility of indirect drying for these tested types of biomass was confirmed.

Spectral emission of flames from laboratory-scale vegetation fires

2009 ◽  
Vol 18 (7) ◽  
pp. 875 ◽  
Author(s):  
P. Boulet ◽  
G. Parent ◽  
A. Collin ◽  
Z. Acem ◽  
B. Porterie ◽  
...  
Keyword(s):  
Background Radiation ◽  
Reference Signal ◽  
Emission Spectra ◽  
Combustion Products ◽  
Infrared Emission ◽  
Laboratory Scale ◽  
Radiative Properties ◽  
Small Scale ◽  
Radiation Emission ◽  
Vegetation Fires

Outdoor experiments were conducted on a laboratory scale to study the infrared radiation emission of vegetation flames. Measurements were made in the spectral range 1000–4500 cm–1, using a compact and portable Fourier-transform infrared spectrometer including an HgCdTe/InSb dual detector. Flame emission was compared with the reference signal emitted by a blackbody surface at 1000 K. We carried out two different series of fire experiments: a series of fires in a 0.45 m-diameter steel tray and a series of wind-tunnel fires. Various types of wildland fuels were used: wood wool, vine branches, dry wood, and Kermes oak branches. From a qualitative observation of emission spectra, it appears that the main contribution comes from the hot gaseous combustion products, with a low-intensity background radiation from soot, as the small-scale flames in these experiments were optically thin. It was also found that, in the flaming combustion zone of the fuel bed, both phases contribute to infrared emission. Our results, in combination with existing data on the absorptivity of vegetation, give a better understanding of radiative transfer in vegetation fires and show how total radiative properties could be deduced from spectral measurements. We believe that this preliminary study provides pilot data for future studies in this area.

Evaluation of a Small-Scale Peanut Sheller

2016 ◽  
Vol 43 (1) ◽  
pp. 67-73 ◽  
Author(s):  
C.L. Butts ◽  
R.B. Sorensen ◽  
M.C. Lamb
Keyword(s):  
Developing Countries ◽  
Narrow Range ◽  
Laboratory Scale ◽  
Small Scale ◽  
Air Velocity ◽  
Small Seed

ABSTRACT Small-scale peanut shelling equipment has been designed and used to meet various needs and scales. A laboratory-scale sheller has been used by researchers to approximate the shelling outturns of a commercial shelling plant using 2 to 10 kg samples. A single commercial-sized sheller will have a shelling capacity up to 23 MT/hr. Commercial shelling operations utilize multiple shellers, each designed to shell a narrow range of peanut sizes. There are enterprises such as small seed processors or manufacturers in developing countries that need shelling equipment capable of processing 100 to 1000 kg of peanuts per hour with the capability of mechanically separating the hulls from the shelled material. A three-stage sheller was designed, fabricated, and tested to determine its throughput (kg/h), the efficiency of separating the hulls from the shelled peanut kernels, and sizing the shelled peanut kernels. The sheller had a maximum shelling rate in the first shelling stage of 1087 kg/h operating at 252 rpm. Approximately 93% of the peanuts were shelled in the first stage of shelling. An air velocity of 9.55 m/s was used to aspirate a mixed stream of peanuts and hulls and removed 97% of the hulls. The sheller was equipped with vibratory screens to separate the material into unshelled, edible sized peanut kernels, and oil stock.

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