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A Method for Cost-Effective and Rapid Characterization of Genetic Parts

McManus, John B. and Bernhards, Casey B. and Sharpes, Caitlin E. and Garcia, David C. and Cole, Stephanie D. and Murray, Richard M. and Emanuel, Peter A. and Lux, Matthew W. (2021) A Method for Cost-Effective and Rapid Characterization of Genetic Parts. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20210506-075939102

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Abstract

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing. Here, we show a cost-effective and rapid method for characterizing genetic parts. Our method utilizes cell-free lysate, prepared in-house, as a medium to evaluate parts via the expression of a reporter protein. Template DNA is prepared by PCR-amplification using inexpensive primers to add variant parts to the reporter gene, and the template is added to the reaction as linear DNA without cloning. Parts that can be added in this way include promoters, operators, ribosome binding sites, insulators, and terminators. This approach, combined with the incorporation of an acoustic liquid handler and 384-well plates, allows the user to carry out high-throughput evaluations of genetic parts in a single day. By comparison, cell-based screening approaches require time-consuming cloning and have longer testing times due to overnight culture and culture density normalization steps. Further, working in cell-free lysate allows the user to exact tighter control over the expression conditions through the addition of exogenous components, or by titrating DNA concentrations rather than relying on limited plasmid copy numbers. Because this method retains a cell-like environment, the function of the genetic part will typically mimic its function in whole cells.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/2021.04.30.440836DOIDiscussion Paper
ORCID:
AuthorORCID
Murray, Richard M.0000-0002-5785-7481
Emanuel, Peter A.0000-0001-5475-7349
Lux, Matthew W.0000-0002-2773-742X
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. This version posted May 1, 2021. This work was made possible by the Office of the Secretary of Defense’s Applied Research for the Advancement of Science and Technology Priorities program. We thank Scott Walper (Naval Research Laboratory) for providing the stock of sfGFP used, and Zachary Sun and Abel Chiao (Tierra Biosciences) for fruitful discussions related to prototyping with cell-free systems and related troubleshooting of acoustic liquid handling. Competing Interest Statement: RMM has a financial stake in Tierra Biosciences, a private company that makes use of cell-free technologies such as those described in this article for protein expression and screening. The other authors have nothing to disclose.
Funders:
Funding AgencyGrant Number
Department of DefenseUNSPECIFIED
Subject Keywords:Synthetic Biology, TXTL, CFPS, Cell-free protein synthesis, T7 RNA Polymerase, genetic parts, genetic circuits
Record Number:CaltechAUTHORS:20210506-075939102
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20210506-075939102
Official Citation:A Method for Cost-Effective and Rapid Characterization of Genetic Parts. John B. McManus, Casey B. Bernhards, Caitlin E. Sharpes, David C. Garcia, Stephanie D. Cole, Richard M. Murray, Peter A. Emanuel, Matthew W. Lux. bioRxiv 2021.04.30.440836; doi: https://doi.org/10.1101/2021.04.30.440836
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:108981
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:06 May 2021 18:55
Last Modified:06 May 2021 18:55

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