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The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations

Shuai Li, Zhixia Ye, Eirik A. Moreb, Romel Menacho-Melgar, View ORCID ProfileMichael D. Lynch
doi: https://doi.org/10.1101/2022.02.25.481916
Shuai Li
1Department of Chemistry, Duke University, Durham, NC
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Zhixia Ye
2Department of Biomedical Engineering, Duke University, Durham, NC
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Eirik A. Moreb
2Department of Biomedical Engineering, Duke University, Durham, NC
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Romel Menacho-Melgar
2Department of Biomedical Engineering, Duke University, Durham, NC
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Michael D. Lynch
2Department of Biomedical Engineering, Duke University, Durham, NC
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  • ORCID record for Michael D. Lynch
  • For correspondence: Michael.lynch@duke.edu
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Abstract

Cell based factories can be engineered to produce a wide variety of products. Advances in DNA synthesis and genome editing have greatly simplified the design and construction of these factories. It has never been easier to generate hundreds or even thousands of cell factory strain variants for evaluation. These advances have amplified the need for standardized, higher throughput means of evaluating these designs. Toward this goal, we have previously reported the development of engineered E. coli strains and associated 2-stage production processes to simplify and standardize strain engineering, evaluation and scale up. This approach relies on decoupling growth (stage 1), from production, which occurs in stationary phase (stage 2). Phosphate depletion is used as the trigger to stop growth as well as induce heterologous expression. Here, we describe in detail the development of optimal protocols used for the evaluation of engineered E. coli strains in 2-stage microfermentations. These protocols are readily adaptable to the evaluation of strains producing a wide variety of protein as well as small molecule products. Additionally, the development approach described is adaptable to additional cellular hosts, as well as other 2-stage processes with various additional triggers.

Competing Interest Statement

RMM and MDL have financial interests in Roke Biotechnologies, LLC. MDL and YE have a financial interest in DMC Biotechnologies, Inc.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted February 28, 2022.
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The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations
Shuai Li, Zhixia Ye, Eirik A. Moreb, Romel Menacho-Melgar, Michael D. Lynch
bioRxiv 2022.02.25.481916; doi: https://doi.org/10.1101/2022.02.25.481916
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The Development of 2-stage Microfermentation Protocols for High Throughput Cell Factory Evaluations
Shuai Li, Zhixia Ye, Eirik A. Moreb, Romel Menacho-Melgar, Michael D. Lynch
bioRxiv 2022.02.25.481916; doi: https://doi.org/10.1101/2022.02.25.481916

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