PERSIA for Direct Fluorescence Measurements of Transcription, Translation, and Enzyme Activity in Cell-Free Systems

Wick, Scott; Walsh, David I.; Bobrow, Johanna; Hamad‐Schifferli, Kimberly; Kong, David S.; Thorsen, Todd; Mroszczyk, Keri; Carr, Peter A.

doi:10.1021/acssynbio.8b00450

Cited by 16 publications

(21 citation statements)

References 67 publications

(114 reference statements)

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“…To quantify rates of transcription and translation in real time, fluorescent reporters can be used at the RNA and/or protein level and measured directly. One example of such an assay, PERSIA, uses LETs in CFEs to rapidly characterize several biological phenomena during the reaction ( Wick et al, 2019 ). The activities of T7 promoter mutants have been efficiently characterized through the use of LETs by using the PCR products from an in vitro transcription screen directly in CFEs ( Komura et al, 2018 ).…”

Section: Applications Of Linear Expression Templatesmentioning

confidence: 99%

Effective Use of Linear DNA in Cell-Free Expression Systems

McSweeney

Styczynski

2021

Front. Bioeng. Biotechnol.

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Cell-free expression systems (CFEs) are cutting-edge research tools used in the investigation of biological phenomena and the engineering of novel biotechnologies. While CFEs have many benefits over in vivo protein synthesis, one particularly significant advantage is that CFEs allow for gene expression from both plasmid DNA and linear expression templates (LETs). This is an important and impactful advantage because functional LETs can be efficiently synthesized in vitro in a few hours without transformation and cloning, thus expediting genetic circuit prototyping and allowing expression of toxic genes that would be difficult to clone through standard approaches. However, native nucleases present in the crude bacterial lysate (the basis for the most affordable form of CFEs) quickly degrade LETs and limit expression yield. Motivated by the significant benefits of using LETs in lieu of plasmid templates, numerous methods to enhance their stability in lysate-based CFEs have been developed. This review describes approaches to LET stabilization used in CFEs, summarizes the advancements that have come from using LETs with these methods, and identifies future applications and development goals that are likely to be impactful to the field. Collectively, continued improvement of LET-based expression and other linear DNA tools in CFEs will help drive scientific discovery and enable a wide range of applications, from diagnostics to synthetic biology research tools.

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Section: Applications Of Linear Expression Templatesmentioning

confidence: 99%

Effective Use of Linear DNA in Cell-Free Expression Systems

McSweeney

Styczynski

2021

Front. Bioeng. Biotechnol.

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“…Fluorescent fusion proteins, such as GFP-fused proteins, are compatible with the high-throughput screen (Baumann et al, 2018). However, fluorescent fusion proteins sometimes inhibit the natural activity of target proteins (Wick et al, 2019). Since FLIM analysis of NAD(P)H does not require the fluorescent labelling of target proteins or their products, it might be applied to the screen of cells yielding the target protein in its natural form.…”

Section: Discussionmentioning

confidence: 99%

Fluorescence lifetime imaging of NAD(P)H upon oxidative stress in Kluyveromyces marxianus

Luo

Yang

et al. 2022

Front. Bioeng. Biotechnol.

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K. marxianus is a promising cell factory for producing heterologous proteins. Oxidative stresses were raised during overexpression of heterologous proteins, leading to the shift of the redox state. How to measure the redox state of live K. marxianus cells without perturbing their growth remains a big challenge. Here, a fluorescence lifetime imaging (FLIM)-based method was developed in live K. marxianus cells. During the early exponential growth, K. marxianus cells exhibited an increased mean fluorescence lifetime (τ-mean) of NAD(P)H compared with Saccharomyces cerevisiae cells, which was consistent with the preference for respiration in K. marxianus cells and that for fermentation in S. cerevisiae cells. Upon oxidative stresses induced by high temperature or H2O2, K. marxianus cells exhibited an increased τ-mean in company with decreased intracellular NAD(P)H/NAD(P)+, suggesting a correlation between an increased τ-mean and a more oxidized redox state. The relationship between τ-mean and the expression level of a heterologous protein was investigated. There was no difference between the τ-means of K. marxianus strains which were not producing a heterologous protein. The τ-mean of a strain yielding a high level of a heterologous protein was higher than that of a low-yielding strain. The results suggested the potential application of FLIM in the non-invasive screen of high-yielding cells.

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“…Communal synthetic biology resources and large collaboration, such as iGEM [40], demonstrate the power of interoperability and sharing of standard DNA parts. As we move towards more complex circuit characterisation technologies (i.e., for quantification of transcription and translation [9,41,42]), the use of standards for designing, executing, measuring, and logging experimental efforts will become yet more essential.…”

Section: Desirable Features Of Gene Expression Control Systemsmentioning

confidence: 99%

“…Prototyping using cell-free protein synthesis (CFPS) is rapidly becoming an important tool for biotechnology. Cell-free expression systems have been applied to quantify rates of transcription and translation [41] and to assess the burden associated with genetic networks [7,43]. CFPS allows prototyping and functional assessment of parts and circuits, measurement of their associated productivity and subsequent refactoring of the gene network to optimise performance.…”

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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PERSIA for Direct Fluorescence Measurements of Transcription, Translation, and Enzyme Activity in Cell-Free Systems

Cited by 16 publications

References 67 publications

Effective Use of Linear DNA in Cell-Free Expression Systems

Effective Use of Linear DNA in Cell-Free Expression Systems

Fluorescence lifetime imaging of NAD(P)H upon oxidative stress in Kluyveromyces marxianus

Contemporary Tools for Regulating Gene Expression in Bacteria

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