Rapid Characterization of Genetic Parts with Cell-free Systems.

John B McManus, Casey B Bernhards, Caitlin E Sharpes, David C Garcia, Stephanie D Cole, Richard M Murray, Peter A Emanuel, Matthew W Lux

Journal of visualized experiments : JoVE 2021 Aug 30

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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 rapid and cost-effective 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 and DNA at precise concentrations. Results obtained from cell-free screening can be used directly in applications of cell-free systems or, in some cases, as a way to predict function in whole cells.

Citation

John B McManus, Casey B Bernhards, Caitlin E Sharpes, David C Garcia, Stephanie D Cole, Richard M Murray, Peter A Emanuel, Matthew W Lux. Rapid Characterization of Genetic Parts with Cell-free Systems. Journal of visualized experiments : JoVE. 2021 Aug 30(174)