Please use this identifier to cite or link to this item: http://dx.doi.org/10.14279/depositonce-8744
Main Title: Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates
Author(s): Hans, Sebastian
Gimpel, Matthias
Glauche, Florian
Neubauer, Peter
Cruz-Bournazou, Mariano Nicolas
Type: Article
Language Code: en
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.
URI: https://depositonce.tu-berlin.de/handle/11303/9704
http://dx.doi.org/10.14279/depositonce-8744
Issue Date: 25-Jun-2018
Date Available: 2-Aug-2019
DDC Class: 570 Biowissenschaften; Biologie
660 Chemische Verfahrenstechnik
Subject(s): competent cells
Escherichia coli
turbidostat
automation
high throughput
chemostat
transformation
Sponsor/Funder: BMBF, 031L0018A, ERASysApp2 - Verbundprojekt: LEANPROT - Entwicklung einer Systembiologie-Plattform für die Entwicklung von lean-proteome-Escherichia coli-Stämmen - Deutsches Teilprojekt A
License: https://creativecommons.org/licenses/by/4.0/
Journal Title: Microorganisms
Publisher: MDPI
Publisher Place: Basel
Volume: 6
Issue: 3
Article Number: 60
Publisher DOI: 10.3390/microorganisms6030060
EISSN: 2076-2607
Appears in Collections:FG Bioverfahrenstechnik » Publications

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