Next Article in Journal
The Microbial Community of a Terrestrial Anoxic Inter-Tidal Zone: A Model for Laboratory-Based Studies of Potentially Habitable Ancient Lacustrine Systems on Mars
Next Article in Special Issue
Inclusion Body Bead Size in E. coli Controlled by Physiological Feeding
Previous Article in Journal
Sulfate-Reducing Naphthalene Degraders Are Picky Eaters
Previous Article in Special Issue
Application of Continuous Culture Methods to Recombinant Protein Production in Microorganisms
Article

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

Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Ackerstraße 76, D-13357 Berlin, Germany
*
Author to whom correspondence should be addressed.
Microorganisms 2018, 6(3), 60; https://doi.org/10.3390/microorganisms6030060
Received: 5 May 2018 / Revised: 10 June 2018 / Accepted: 22 June 2018 / Published: 25 June 2018
(This article belongs to the Special Issue Recombinant Protein Expression in Microorganisms)
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. View Full-Text
Keywords: competent cells; Escherichia coli; turbidostat; automation; high throughput; chemostat; transformation competent cells; Escherichia coli; turbidostat; automation; high throughput; chemostat; transformation
Show Figures

Graphical abstract

MDPI and ACS Style

Hans, S.; Gimpel, M.; Glauche, F.; Neubauer, P.; Cruz-Bournazou, M.N. Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates. Microorganisms 2018, 6, 60. https://doi.org/10.3390/microorganisms6030060

AMA Style

Hans S, Gimpel M, Glauche F, Neubauer P, Cruz-Bournazou MN. Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates. Microorganisms. 2018; 6(3):60. https://doi.org/10.3390/microorganisms6030060

Chicago/Turabian Style

Hans, Sebastian, Matthias Gimpel, Florian Glauche, Peter Neubauer, and Mariano N. Cruz-Bournazou 2018. "Automated Cell Treatment for Competence and Transformation of Escherichia coli in a High-Throughput Quasi-Turbidostat Using Microtiter Plates" Microorganisms 6, no. 3: 60. https://doi.org/10.3390/microorganisms6030060

Find Other Styles
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Back to TopTop