AutoBioTech-A Versatile Biofoundry for Automated Strain Engineering

Authors:
Tobias Michael Rosch, Julia Tenhaef, Tim Stoltmann, Till Redeker, Dominic Kösters, Niels Hollmann,Karin Krumbach, Wolfgang Wiechert, Michael Bott, Susana Matamouros, Jan Marienhagen,and Stephan Noack*
In:
Source: ACS Nano
Publication Date: (2024)
Issue: 13 (7): 2227-2237
Research Area:
Cancer Research/Cell Biology
Gene Expression
Basic Research
Molecular Biology
Drug Discovery
Platform:
4D-Nucleofector® 96-well Systems
Experiment

For electroporation, 20 µL of the previously generated competent C. glutamicum cells were transferred to each well of a 96-well Nucleocuvette Plate (Lonza Cologne GmbH, Cologne Germany). 3 µL of plasmid solution with a concentration of 20-50 ng µL-1 was added to each well. The electroporation plate was then placed in the 4DNucleofector 96-well Unit (Lonza Cologne GmbH, Cologne Germany) and the 4D-Nucleofector Core Unit (Lonza Cologne GmbH, Cologne Germany) was used to apply the bacterial pulse F to each well. Immediately after electroporation, 180 µL of BHISG medium prewarmed to 46 °C was added to each well. As much liquid as possible was transferred from the electroporation plate to a PCR plate and heat shocked at 46 °C for 6 min in a thermal cycler. 

Abstract

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.