Validation of a high-throughput fermentation system based on online monitoring of biomass and fluorescence in continuously shaken microtiter plates

Abstract

Background: An advanced version of a recently reported high-throughput fermentation system with online measurement, called BioLector, and its validation is presented. The technology combines high-throughput screening and high-information content by applying online monitoring of scattered light and fluorescence intensities in continuously shaken microtiter plates. Various examples in calibration of the optical measurements, clone and media screening and promoter characterization are given.

Combinatorial Strategies for Improving Multiple-Stress Resistance in Industrially Relevant Escherichia coli Strains

“…These combinations of conditions were chosen because E. coli produces acidic by-products (primarily acetate) during aerobic fermentation which need to be neutralized by a base such as NaOH, which can result in simultaneous acid and osmotic stress under nonideal mixing conditions (1,2,42). The BioLector minibioreactor system, which allows for sufficient oxygen transfer rates to support fully aerobic growth of E. coli (43), was employed for all growth measurements. This system also enables relative quantification of biomass at optical densities above the linear range achievable by most absorbance measurements, due to its use of light backscatter as a measure of cell density (43).…”

“…The BioLector minibioreactor system, which allows for sufficient oxygen transfer rates to support fully aerobic growth of E. coli (43), was employed for all growth measurements. This system also enables relative quantification of biomass at optical densities above the linear range achievable by most absorbance measurements, due to its use of light backscatter as a measure of cell density (43). Averaged background-subtracted growth curves between three replicate cultures for each strain and condition are provided in the supplemental material (see Fig.…”

The two-component system ChrSA is crucial for haem tolerance and interferes with HrrSA in haem-dependent gene regulation in Corynebacterium glutamicum

“…For promoter fusion studies, the eYFP chromophore was excited with 510 nm and emission was measured at 532 nm (signal gain factor of 50). The specific fluorescence (au) was calculated by the eYFP fluorescence signal per backscatter signal (Kensy et al, 2009).…”

Flavin Mononucleotide-Based Fluorescent Reporter Proteins Outperform Green Fluorescent Protein-Like Proteins as Quantitative In Vivo Real-Time Reporters

“…After a decrease of more than 6-fold during the first 8 h of cell growth, YFP fluorescence subsequently increased 2.5-fold during the late stationary growth phase. Thus, YFP accumulated at constant levels in all phases of bacterial cell growth ( We further analyzed YFP and FbFP fluorescence in the respective E. coli DH5␣ expression strains batch cultured in Luria-Bertani (LB) medium (13) at 37°C in a 48-well MTP by using the BioLector to determine potential changes in YFP fluorescence during cell growth caused by oxygen limitation. As described previously, the cultivation of E. coli in unbuffered complex LB medium leads to a fast exhaustion of carbon sources and a rapid basification of the culture medium, resulting in an early inhibition of cell growth (14).…”

“…Subsequently, the recombinant E. coli strains were cultivated in TB medium (13) under defined conditions (37°C, 700 rpm shaking frequency, 3 mm shaking diameter) in a BioLector microbioreactor system (m2p-labs GmbH, Aachen, Germany) over 16 h. To allow aeration of the cultures, a 48-well microtiter plate (MTP) (Greiner bio-one GmbH, Frickenhausen, Germany) was sealed with air-permeable sealing tape (AB-0718; ABgene, Epsom, United Kingdom). During cultivation, the BioLector provides quantitative online data of (i) biomass via measuring of scattered light at an excitation wavelength of 620 nm, (ii) dissolved oxygen tension (DOT) using an immobilized oxygen-sensitive fluorescent dye (m2p-labs GmbH, Aachen, Germany), and (iii) FP-mediated fluorescence in a continuously shaken MTP, as described previously (11,13,17). YFP fluorescence was measured at an excitation wavelength of 510 nm and an emission wavelength of 540 nm, whereas FbFP fluorescence was monitored at 492 nm upon excitation at 460 nm.…”