Vector redesign and in‐droplet cell‐growth improves enrichment and recovery in live Escherichia coli

Eenink, Bernard D. G., Kaminski, Tomasz S., Bornberg‐Bauer, Erich, Jose, Joachim, Hollfelder, Florian and Van Loo, Bert (2022) Vector redesign and in‐droplet cell‐growth improves enrichment and recovery in live Escherichia coli. Microbial Biotechnology, 15 (11). pp. 2845-2853. ISSN 1751-7915

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Official URL: https://doi.org/10.1111/1751-7915.14144

Abstract

Directed evolution (DE) is a widely used method for improving the function of biomolecules via multiple rounds of mutation and selection. Microfluidic droplets have emerged as an important means to screen the large libraries needed for DE, but this approach was so far partially limited by the need to lyse cells, recover DNA, and retransform into cells for the next round, necessitating the use of a high-copy number plasmid or oversampling. The recently developed live cell recovery avoids some of these limitations by directly regrowing selected cells after sorting. However, repeated sorting cycles used to further enrich the most active variants ultimately resulted in unfavourable recovery of empty plasmid vector-containing cells over those expressing the protein of interest. In this study, we found that engineering of the original expression vector solved the problem of false positives (i.e. plasmids lacking an insert) cells containing empty vectors. Five approaches to measure activity of cell-displayed enzymes in microdroplets were compared. By comparing various cell treatment methods prior to droplet sorting two things were found. Substrate encapsulation from the start, that is prior to expression of enzyme, showed no disadvantage to post-induction substrate addition by pico-injection with respect to recovery of true positive variants. Furthermore in-droplet cell growth prior to induction of enzyme production improves the total amount of cells retrieved (recovery) and proportion of true positive variants (enrichment) after droplet sorting.

Item Type: Article
Additional Information: Funding information: This project has received funding from the EU under the Horizon 2020 Research and Innovation Framework Programme No 722610 to FH and EBB through which BDGE was supported. FH and EBB acknowledge support from HFSP grant RGP006/2013. TSK was supported as an EU H2020 Marie Skłodowska-Curie Fellow No 750772. FH is an ERC Advanced Investigator (695669).
Subjects: C500 Microbiology
Department: Faculties > Health and Life Sciences > Applied Sciences
Depositing User: John Coen
Date Deposited: 14 Sep 2022 11:44
Last Modified: 09 Dec 2022 10:15
URI: https://nrl.northumbria.ac.uk/id/eprint/50123

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