A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

Lavickova, Barbora; Maerkl, Sebastian J.

doi:10.1021/acssynbio.8b00427

Cited by 111 publications

(115 citation statements)

References 31 publications

(67 reference statements)

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“…Although the third approach reached protein synthesis levels comparable to PUREfrex, in all three of these approaches it is not possible to rapidly modify protein levels or omit proteins. We recently demonstrated that all proteins, except ribosomes, can be prepared from individual strains in a single co-culture and purification step called the OnePot PURE system, which achieves a similar protein synthesis yield as commercial PURExpress (Lavickova and Maerkl, 2019) (Figure 2A).…”

Section: Recombinant Systemsmentioning

confidence: 99%

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

Laohakunakorn

Grasemann

Lavickova

et al. 2020

Front. Bioeng. Biotechnol.

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100

101

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Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner.

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Section: Recombinant Systemsmentioning

confidence: 99%

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

Laohakunakorn

Grasemann

Lavickova

et al. 2020

Front. Bioeng. Biotechnol.

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100

101

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“…This in vitro prototyping provides a useful method for the rapid exploration and optimisation of the large experimental spaces associated with complex genetic systems ( Figure 2B). Given recent improvement in CFPS lysate production, this tool is likely to see gains in popularity in the future [52][53][54][55]. (A) Introduction of heterologous genes into a production strain leads to competition for resources.…”

Section: Cell-free Technology For Prototyping Gene Regulatory Networkmentioning

confidence: 99%

Contemporary Tools for Regulating Gene Expression in Bacteria

Kent

Dixon

2020

Trends in Biotechnology

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Insights from novel mechanistic paradigms in gene expression control have led to the development of new gene expression systems for bioproduction, control, and sensing applications. Coupled with a greater understanding of synthetic burden and modern creative biodesign approaches, contemporary bacterial gene expression tools and systems are emerging that permit fine-tuning of expression, enabling greater predictability and maximisation of specific productivity, while minimising deleterious effects upon cell viability. These advances have been achieved by using a plethora of regulatory tools, operating at all levels of the so-called 'central dogma' of molecular biology. In this review, we discuss these gene regulation tools in the context of their design, prototyping, integration into expression systems, and biotechnological application. From Overexpression to Precise Regulation: Engineering Gene Expression Control Historically, researchers have relied on a relatively small number of mechanisms to regulate heterologous gene expression [1]. These traditional systems have focussed on massive overexpression of genes in an effort to maximise product yield (see Glossary). Often systems developed for overexpression are adapted ad hoc for integration into genetic circuits. While suited for the rapid accumulation of high levels of protein, these tools are often insufficient for developing the more precise systems required for many modern applications in sensing, computation and production. Recent years have seen improvements in the functionality of gene regulation tools, enhancing utility for dynamic, tuneable regulation. With any bioproduction effort, there is often an important tradeoff between yield, biomass accumulation, and titre that must be considered (Figure 1A). This balance is important for an array of biotechnological applications to ensure process viability and stability. As our ability to engineer more complex systems increases, the importance of harmonising gene expression with the metabolic capacity of the host organism, or chassis, by reducing the burden associated with the engineered function only increases. In the case of recombinant protein production, aberrant protein synthesis can lead to an overload in cellular capacity, such as synthesis or secretion. Both resource limitations, such as limited amino acid and ribosome availability [2], and symptoms of cellular capacity overload, such as impaired protein folding and secretion capacity [3-5], can impact cellular viability. Resource demand and overload also present a challenge to metabolic engineering efforts where substrate, intermediate, and product concentration can all have deleterious effects on cell viability [6]. This issue is exacerbated when central metabolites and/or cofactors are consumed, creating further competition for cellular resources.

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“…Lastly, as the redox reagent used in the PURE system is known to degrade rapidly, we eliminated 1,4-dithiothreitol (DTT) in the energy solution and instead added tris(2 carboxyethyl)phosphine (TCEP) to the energy and protein solutions. To allow PURE system modification and omission of protein components we produced our own PURE system based on the original formulation [36,23]. For each protein regenerated, we produced a ∆PURE system lacking that particular protein or proteins.…”

Section: Experimental Designmentioning

confidence: 99%

“…First, synthesis capacity of the system in terms of its protein synthesis rate must be sufficient to regenerate the necessary components. This problem is exacerbated by the fact that protein synthesis capacity drastically decreases in a non-optimal system [23,24,25]. Second, the components being regenerated must be functionally synthesized which may require chaperones, and modifying enzymes.…”

Section: Introductionmentioning

confidence: 99%

A self-regenerating synthetic cell model

Lavickova

Laohakunakorn

Maerkl

2020

Preprint

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AbstractSelf-regeneration is a fundamental function of all living systems. Here we demonstrate molecular self-regeneration in a synthetic cell model. By implementing a minimal transcription-translation system within microfluidic reactors, the system was able to regenerate essential protein components from DNA templates and sustained synthesis activity for over a day. By mapping genotype-phenotype landscapes combined with computational modeling we found that minimizing resource competition and optimizing resource allocation are both critically important for achieving robust system function. With this understanding, we achieved simultaneous regeneration of multiple proteins by determining the required DNA ratios necessary for sustained self-regeneration. This work introduces a conceptual and experimental framework for the development of a self-replicating synthetic cell.

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A Simple, Robust, and Low-Cost Method To Produce the PURE Cell-Free System

Cited by 111 publications

References 31 publications

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

Contemporary Tools for Regulating Gene Expression in Bacteria

A self-regenerating synthetic cell model

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