Dataset Information

Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates.

ABSTRACT: Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in flask cultivations, parallelization becomes a challenging task when working with larger libraries and liquid handling stations as transformation efficiency depends on a distinct physiological state of the cells. We present a robust method for the preparation of competent cells and their transformation. The strength of the method is that all cells on the plate can be maintained at a high growth rate until all cultures have reached a defined cell density regardless of growth rate and lag phase variabilities. This allows sufficient transformation in automated high throughput facilities and solves important scheduling issues in wet-lab library screenings. We address the problem of different growth rates, lag phases, and initial cell densities inspired by the characteristics of continuous cultures. The method functions on a fully automated liquid handling platform including all steps from the inoculation of the liquid cultures to plating and incubation on agar plates. The key advantage of the developed method is that it enables cell harvest in 96 well plates at a predefined time by keeping fast growing cells in the exponential phase as in turbidostat cultivations. This is done by a periodic monitoring of cell growth and a controlled dilution specific for each well. With the described methodology, we were able to transform different strains in parallel. The transformants produced can be picked and used in further automated screening experiments. This method offers the possibility to transform any combination of strain- and plasmid library in an automated high-throughput system, overcoming an important bottleneck in the high-throughput screening and the overall chain of bioprocess development.

SUBMITTER: Hans S

PROVIDER: S-EPMC6163857 | biostudies-other | 2018 Jun

REPOSITORIES: biostudies-other

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Publications

Automated Cell Treatment for Competence and Transformation of <i>Escherichia coli</i> in a High-Throughput Quasi-Turbidostat Using Microtiter Plates.

Hans Sebastian S Gimpel Matthias M Glauche Florian F Neubauer Peter P Cruz-Bournazou Mariano Nicolas MN

Microorganisms 20180625 3

Metabolic engineering and genome editing strategies often lead to large strain libraries of a bacterial host. Nevertheless, the generation of competent cells is the basis for transformation and subsequent screening of these strains. While preparation of competent cells is a standard procedure in flask cultivations, parallelization becomes a challenging task when working with larger libraries and liquid handling stations as transformation efficiency depends on a distinct physiological state of th ...[more]

PMID: 29941834

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Project description:Background:Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35?mg/h per well (48-well-plate) at 37?°C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5?mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28?mmol/L/h should be achieved. Results:Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90?mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28?mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24-0.90?mg/h per well. Conclusions:The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.

Dataset Information

Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates.

Publications

Automated Cell Treatment for Competence and Transformation of <i>Escherichia coli</i> in a High-Throughput Quasi-Turbidostat Using Microtiter Plates.

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