Protocols for Implementing an &lt;em&gt;Escherichia coli&lt;/em&gt; Based TX-TL Cell-Free Expression System for Synthetic Biology

Sun, Zachary Z.; Hayes, Clarmyra A.; Shin, Jinwoo; Caschera, Filippo; Murray, Richard M.; Noireaux, Vincent

doi:10.3791/50762

Cited by 336 publications

(638 citation statements)

References 34 publications

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“…Cells grown in this media were collected at both early, (OD 600 2.8) and mid-log phase (OD 600 4.0), and are referred to as YT-E and YT-M, respectively. The 2xYPTG condition, collected in early-log phase growth, is commonly used for CFPS (22). Cells collected early in log phase growth have the greatest specific growth rate, a parameter that is suggested to influence CFPS capabilities and may affect the abundance of glycolytic enzymes (23,24).…”

Section: Resultsmentioning

confidence: 99%

Elucidating the potential of crude cell extracts for producing pyruvate from glucose

et al. 2018

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Living systems possess a rich biochemistry that can be harnessed through metabolic engineering to produce valuable therapeutics, fuels and fine chemicals. In spite of the tools created for this purpose, many organisms tend to be recalcitrant to modification or difficult to optimize. Crude cellular extracts, made by lysis of cells, possess much of the same biochemical capability, but in an easier to manipulate context. Metabolic engineering in crude extracts, or cell-free metabolic engineering, can harness these capabilities to feed heterologous pathways for metabolite production and serve as a platform for pathway optimization. However, the inherent biochemical potential of a crude extract remains ill-defined, and consequently, the use of such extracts can result in inefficient processes and unintended side products. Herein, we show that changes in cell growth conditions lead to changes in the enzymatic activity of crude cell extracts and result in different abilities to produce the central biochemical precursor pyruvate when fed glucose. Proteomic analyses coupled with metabolite measurements uncover the diverse biochemical capabilities of these different crude extract preparations and provide a framework for how analytical measurements can be used to inform and improve crude extract performance. Such informed developments can allow enrichment of crude extracts with pathways that promote or deplete particular metabolic processes and aid in the metabolic engineering of defined products.

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Section: Resultsmentioning

confidence: 99%

Elucidating the potential of crude cell extracts for producing pyruvate from glucose

et al. 2018

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“…Significantly, a number of costly and therefore undesirable components are present in this original protocol, such as Staphylococcus nuclease and pyruvate kinase. To try a different low-cost strategy, we prepared a S. venezuelae cell-extract using the original Streptomyces method and tested its activity with a 3-phosphoglyceric acid (3-PGA) energy regeneration buffer derived from E. coli TX-TL [22]. Cell-extracts were tested for activity using sfGFP reporter coupled to a high-activity kasOp* promoter.…”

Section: Optimizing a High-activity S Venezuelae Cell-extractmentioning

confidence: 99%

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

Moore

Lai

Needham

et al. 2017

Biotechnology Journal

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Streptomyces venezuelae is a promising chassis in synthetic biology for fine chemical and secondary metabolite pathway engineering. The potential of S. venezuelae could be further realized by expanding its capability with the introduction of its own in vitro transcription-translation (TX-TL) system. TX-TL is a fast and expanding technology for bottom-up design of complex gene expression tools, biosensors and protein manufacturing. Herein, we introduce a S. venezuelae TX-TL platform by reporting a streamlined protocol for cell-extract preparation, demonstrating high-yield synthesis of a codon-optimized sfGFP reporter and the prototyping of a synthetic tetracycline-inducible promoter in S. venezuelae TX-TL based on the tetO-TetR repressor system. The aim of this system is to provide a host for the homologous production of exotic enzymes from Actinobacteria secondary metabolism in vitro. As an example, the authors demonstrate the soluble synthesis of a selection of enzymes (12-70 kDa) from the Streptomyces rimosus oxytetracycline pathway.

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“…Such approaches have been employed to study cellular systems including the characterization of membrane proteins 1 , the probing of protein interactions 2 , and the exploration of fundamental aspects of translation [3][4][5][6][7] .…”

Section: Introductionmentioning

confidence: 99%

Sealable Femtoliter Chamber Arrays for Cell-free Biology

Norred

Caveney

Retterer

et al. 2015

JoVE

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Cell-free systems provide a flexible platform for probing specific networks of biological reactions isolated from the complex resource sharing (e.g., global gene expression, cell division) encountered within living cells. However, such systems, used in conventional macro-scale bulk reactors, often fail to exhibit the dynamic behaviors and efficiencies characteristic of their living micro-scale counterparts. Understanding the impact of internal cell structure and scale on reaction dynamics is crucial to understanding complex gene networks. Here we report a microfabricated device that confines cell-free reactions in cellular scale volumes while allowing flexible characterization of the enclosed molecular system. This multilayered poly(dimethylsiloxane) (PDMS) device contains femtoliter-scale reaction chambers on an elastomeric membrane which can be actuated (open and closed). When actuated, the chambers confine Cell-Free Protein Synthesis (CFPS) reactions expressing a fluorescent protein, allowing for the visualization of the reaction kinetics over time using time-lapse fluorescent microscopy. Here we demonstrate how this device may be used to measure the noise structure of CFPS reactions in a manner that is directly analogous to those used to characterize cellular systems, thereby enabling the use of noise biology techniques used in cellular systems to characterize CFPS gene circuits and their interactions with the cell-free environment.

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Protocols for Implementing an <em>Escherichia coli</em> Based TX-TL Cell-Free Expression System for Synthetic Biology

Cited by 336 publications

References 34 publications

Elucidating the potential of crude cell extracts for producing pyruvate from glucose

Elucidating the potential of crude cell extracts for producing pyruvate from glucose

Streptomyces venezuelae TX‐TL – a next generation cell‐free synthetic biology tool

Sealable Femtoliter Chamber Arrays for Cell-free Biology

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