scholarly journals High-throughput microbioreactor provides a capable tool for early stage bioprocess development

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mathias Fink ◽  
Monika Cserjan-Puschmann ◽  
Daniela Reinisch ◽  
Gerald Striedner
Keyword(s):  
High Throughput ◽  
Process Development ◽  
Early Stage ◽  
Explanatory Power ◽  
Batch Mode ◽  
E Coli ◽  
Speed Up ◽  
Bioreactor System ◽  
Cell Densities ◽  
Microbial Systems

AbstractTremendous advancements in cell and protein engineering methodologies and bioinformatics have led to a vast increase in bacterial production clones and recombinant protein variants to be screened and evaluated. Consequently, an urgent need exists for efficient high-throughput (HTP) screening approaches to improve the efficiency in early process development as a basis to speed-up all subsequent steps in the course of process design and engineering. In this study, we selected the BioLector micro-bioreactor (µ-bioreactor) system as an HTP cultivation platform to screen E. coli expression clones producing representative protein candidates for biopharmaceutical applications. We evaluated the extent to which generated clones and condition screening results were transferable and comparable to results from fully controlled bioreactor systems operated in fed-batch mode at moderate or high cell densities. Direct comparison of 22 different production clones showed great transferability. We observed the same growth and expression characteristics, and identical clone rankings except one host-Fab-leader combination. This outcome demonstrates the explanatory power of HTP µ-bioreactor data and the suitability of this platform as a screening tool in upstream development of microbial systems. Fast, reliable, and transferable screening data significantly reduce experiments in fully controlled bioreactor systems and accelerate process development at lower cost.

scholarly journals High Dynamic Range Bacterial Cell Detection in a Novel “Lab-In-A-Comb” for High-Throughput Antibiotic Susceptibility Testing of Clinical Urine Samples

2017 ◽  
Author(s):  
Jeremy Pivetal ◽  
Martin J. Woodward ◽  
Nuno M. Reis ◽  
Alexander D. Edwards
Keyword(s):  
Cell Growth ◽  
High Throughput ◽  
Antibiotic Susceptibility ◽  
Dynamic Range ◽  
High Dynamic Range ◽  
Urine Samples ◽  
Cell Detection ◽  
E Coli ◽  
High Dynamic ◽  
Cell Densities

ABSTRACTAntibiotic resistance in urinary tract infection is a major global challenge, and improved cost-effective and rapid antibiotic susceptibility tests (AST) are urgently needed to inform correct antibiotic selection. Although microfluidic technology can miniaturise AST, the high dynamic range of pathogen density found in clinical urine samples makes direct testing of clinical samples – rather than testing colonies from overnight agar plates – extremely challenging. We evaluated for the first time how pathogen concentration in urine affects microfluidic AST using a novel microplate-compatible high-throughput microfluidic AST system termed “Lab-on-a-Comb”. When tested with clinical E. coli isolates at standardised density, these devices gave identical antibiotic susceptibility profiles to standard disc diffusion and microtitre plate tests. Bacterial detection directly in synthetic urine spiked with clinical E. coli UTI isolates was possible over a very large dynamic range of starting cell densities, from 103 – 108 CFU/mL which covers the range of pathogen cell densities found in patient urine. The lowest cell density where cell growth was reproducibly detected optically was 9.6x102 CFU/mL, corresponding to one single CFU detected in a 1 μL microcapillary-an unprecedented level of sensitivity. Cell growth kinetics followed a simple Monod model with fast growth limited by the substrate availability and an estimated doubling time of 24.5 min, indicating optimal E. coli growth conditions within these microfluidic devices. There was a trade-off between sensitivity and speed of detection, with 105 CFU/mL detection possible within 2h, but 6h incubation required at 103 CFU/mL.

Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents

2020 ◽  
Vol 17 (5) ◽  
pp. 716-724
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Antibacterial Agents ◽  
Sos Response ◽  
Luciferase Assay ◽  
Translational Machinery ◽  
E Coli ◽  
New Class

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

scholarly journals Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Laura Navone ◽  
Thomas Vogl ◽  
Pawarisa Luangthongkam ◽  
Jo-Anne Blinco ◽  
Carlos H. Luna-Flores ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Phytic Acid ◽  
Disulfide Bond ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
E Coli ◽  
Biochemical Characterisation ◽  
Disulfide Bond Isomerase ◽  
Microbial Systems ◽  
Protein Disulfide

Abstract Background Phytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market. Results In this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme. Conclusions Overall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

scholarly journals Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

scholarly journals A High-Throughput Approach To Identify Compounds That Impair Envelope Integrity in Escherichia coli

2016 ◽  
Vol 60 (10) ◽  
pp. 5995-6002 ◽  
Author(s):  
Kristin R. Baker ◽  
Bimal Jana ◽  
Henrik Franzyk ◽  
Luca Guardabassi
Keyword(s):  
Escherichia Coli ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gram Negative Bacteria ◽  
Chromogenic Substrate ◽  
Intrinsic Resistance ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Screening Platform

ABSTRACTThe envelope of Gram-negative bacteria constitutes an impenetrable barrier to numerous classes of antimicrobials. This intrinsic resistance, coupled with acquired multidrug resistance, has drastically limited the treatment options against Gram-negative pathogens. The aim of the present study was to develop and validate an assay for identifying compounds that increase envelope permeability, thereby conferring antimicrobial susceptibility by weakening of the cell envelope barrier in Gram-negative bacteria. A high-throughput whole-cell screening platform was developed to measureEscherichia colienvelope permeability to a β-galactosidase chromogenic substrate. The signal produced by cytoplasmic β-galactosidase-dependent cleavage of the chromogenic substrate was used to determine the degree of envelope permeabilization. The assay was optimized by using known envelope-permeabilizing compounds andE. coligene deletion mutants with impaired envelope integrity. As a proof of concept, a compound library comprising 36 peptides and 45 peptidomimetics was screened, leading to identification of two peptides that substantially increased envelope permeability. Compound 79 reduced significantly (from 8- to 125-fold) the MICs of erythromycin, fusidic acid, novobiocin and rifampin and displayed synergy (fractional inhibitory concentration index, <0.2) with these antibiotics by checkerboard assays in two genetically distinctE. colistrains, including the high-risk multidrug-resistant, CTX-M-15-producing sequence type 131 clone. Notably, in the presence of 0.25 μM of this peptide, both strains were susceptible to rifampin according to the resistance breakpoints (R> 0.5 μg/ml) for Gram-positive bacterial pathogens. The high-throughput screening platform developed in this study can be applied to accelerate the discovery of antimicrobial helper drug candidates and targets that enhance the delivery of existing antibiotics by impairing envelope integrity in Gram-negative bacteria.

scholarly journals Accelerating High-Throughput Screening for Structural Materials with Production Management Methods

Materials ◽  
2018 ◽  
Vol 11 (8) ◽  
pp. 1330 ◽  
Author(s):  
Alexander Bader ◽  
Finn Meiners ◽  
Kirsten Tracht
Keyword(s):  
High Throughput ◽  
Material Flow ◽  
High Throughput Screening ◽  
Production Management ◽  
Waiting Times ◽  
Complex Structure ◽  
New Drugs ◽  
Order Processing ◽  
Speed Up ◽  
Process Trial

High-throughput screenings are widely accepted for pharmaceutical developments for new substances and the development of new drugs with required characteristics by evolutionary studies. Current research projects transfer this principle of high-throughput testing to the development of metallic materials. In addition to new generating and testing methods, these types of high-throughput systems need a logistical control and handling method to reduce throughput time to get test results faster. Instead of the direct material flow found in classical high-throughput screenings, these systems have a very complex structure of material flow. The result is a highly dynamic system that includes short-term changes such as rerun stations, partial tests, and temporarily paced sequences between working systems. This paper presents a framework that divides the actions for system acceleration into three main sections. First, methods for special applications in high-throughput systems are designed or adapted to speed up the generation, treatment, and testing processes. Second, methods are needed to process trial plans and to control test orders, which can efficiently reduce waiting times. The third part of the framework describes procedures for handling samples. This reduces non-productive times and reduces order processing in individual lots.

scholarly journals CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics

The Analyst ◽  
2014 ◽  
Vol 139 (13) ◽  
pp. 3314-3323 ◽  
Author(s):  
Thomas Beneyton ◽  
Faith Coldren ◽  
Jean-Christophe Baret ◽  
Andrew D. Griffiths ◽  
Valérie Taly
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Recombinant Enzyme ◽  
Cell Analysis ◽  
E Coli ◽  
Cota Laccase

A high-throughput cell analysis and sorting platform using droplet-based microfluidics is introduced for directed evolution of recombinant CotA laccase expressed in E. coli.

scholarly journals Scale-down characterization of post-centrifuge flocculation processes for high-throughput process development

2014 ◽  
Vol 111 (12) ◽  
pp. 2486-2498 ◽  
Author(s):  
Georgina Espuny Garcia del Real ◽  
Jim Davies ◽  
Daniel G. Bracewell
Keyword(s):  
High Throughput ◽  
Process Development ◽  
Scale Down

Modular automated bottom-up proteomic sample preparation for high-throughput applications v1

2021 ◽  
Author(s):  
Yan Chen ◽  
Nurgul Kaplan Lease ◽  
Jennifer Gin ◽  
Tad Ogorzalek ◽  
Paul D. Adams ◽  
...  
Keyword(s):  
Sample Preparation ◽  
High Throughput ◽  
Tryptic Digestion ◽  
Gram Negative Bacteria ◽  
Bottom Up ◽  
Preparation Methods ◽  
Gram Negative ◽  
E Coli ◽  
Protocol Development ◽  
Set Up

Manual proteomic sample preparation methods limit sample throughput and often lead to poor data quality when thousands of samples must be analyzed. Automated workflows are increasingly used to overcome these issues for some (or even all) of the sample preparation steps. Here, we detail three optimised step-by-step protocols to: (A) lyse Gram-negative bacteria and fungal cells; (B) quantify the amount of protein extracted; and (C) normalize the amount of protein and set up tryptic digestion. These protocols have been developed to facilitate rapid, low variance sample preparation of hundreds of samples, be easily implemented on widely-available Beckman-Coulter Biomek automated liquid handlers, and allow flexibility for future protocol development. By using this workflow 50 micrograms of peptides for 96 samples can be prepared for tryptic digestion in under an hour. We validate these protocols by analyzing 47 E. coli and R. toruloides samples and show that this modular workflow provides robust, reproducible proteomic samples for high-throughput applications. The expected results from these protocols are 94 peptide samples from Gram-negative bacterial and fungal cells prepared for bottom-up quantitative proteomic analysis without the need for desalting column cleanup and with peptide variance (CVs) below 15%.

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