Dual transcriptional-translational cascade permits cellular level tuneable expression control

Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa M.; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

doi:10.1093/nar/gkv912

Cited by 38 publications

(57 citation statements)

References 46 publications

(72 reference statements)

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“…While there has been great progress in improving the dynamic range of RNA regulators by engineering mechanisms that control a single gene expression process (3,4,13), only several studies have explored the idea of engineering multiple genetic control processes for tighter regulation (14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

“…Specifically, Morra et al recently combined transcriptional and translational control with two distinct mechanisms -inducible promoters and orthogonal translational riboswitches -to achieve tight control of fluorescent proteins (14). Horbal and Luzhetskyy also recently used a similar approach to control pamamycin production in Streptomyces albus (15).…”

Section: Introductionmentioning

confidence: 99%

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Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator

Westbrook

Lucks

2016

Preprint

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RNA transcriptional regulators are emerging as versatile components for genetic circuit construction.However, RNA transcriptional regulators suffer from incomplete repression, making their dynamic range less than that of their protein counterparts. This incomplete repression can cause expression leak, which impedes the construction of larger RNA synthetic regulatory networks. Here we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and RBS sequestration increases repression from 85% to 98% and activation from 10 fold to over 900 fold in response to cognate antisense RNAs. We also show that orthogonal versions of this mechanism can be created through engineering minimal antisense RNAs.Finally, to demonstrate the utility of this dual control mechanism, we use it to reduce circuit leak in an RNA-only transcriptional cascade that activates gene expression as a function of a small molecule input. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of larger and more sophisticated circuits.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator

Westbrook

Lucks

2016

Preprint

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“…Five E. coli strains were used in this study, coded as wild, PET, EGFP, iL3PET, and iL3EGFP, a detailed description of these strains can be found in [18]. All strains were streak plated three times on LB agar prior to every experiment to ensure the purity of the stocks.…”

Section: Methodsmentioning

confidence: 99%

“…One dataset (denoted as riboswitch in this paper), was obtained from an experiment to investigate the metabolic effects of producing enhanced green fluorescent protein (eGFP) as a recombinant protein in Escherichia coli ( E. coli ) cells [18]. A two-factor, full-factorial experimental design was employed and the metabolic profiles of five different E. coli strains, namely BL21(DE3) (wild-type), BL21(DE3) pET-empty (PET), BL21(DE3) pET-eGFP (EGFP), BL21(IL3) pET-empty (iL3PET), BL21(IL3) pETeGFP (iL3EGFP), under four different inducer conditions (control, lac inducer Isopropyl β- d -1-thiogalactopyranoside (IPTG), pyimido[4,5- d ] pyrimidine-2,4-diamine (PPDA), and IPTG + PPDA) were measured by Fourier transform infrared (FT-IR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) on cell extracts (the details of this experiment and a more detailed description of the strains can be found in [19]).…”

Section: Introductionmentioning

confidence: 99%

Partial Least Squares with Structured Output for Modelling the Metabolomics Data Obtained from Complex Experimental Designs: A Study into the Y-Block Coding

Muhamadali

Sayqal

et al. 2016

Metabolites

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Partial least squares (PLS) is one of the most commonly used supervised modelling approaches for analysing multivariate metabolomics data. PLS is typically employed as either a regression model (PLS-R) or a classification model (PLS-DA). However, in metabolomics studies it is common to investigate multiple, potentially interacting, factors simultaneously following a specific experimental design. Such data often cannot be considered as a "pure" regression or a classification problem. Nevertheless, these data have often still been treated as a regression or classification problem and this could lead to ambiguous results. In this study, we investigated the feasibility of designing a hybrid target matrix Y that better reflects the experimental design than simple regression or binary class membership coding commonly used in PLS modelling. The new design of Y coding was based on the same principle used by structural modelling in machine learning techniques. Two real metabolomics datasets were used as examples to illustrate how the new Y coding can improve the interpretability of the PLS model compared to classic regression/classification coding.

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“…(B) In vitro cell-free protein expression can be applied for prototyping and optimisation of genetic regulatory networks [46,48,138], enzymes [49], and biosensors [50,51]. (C) By engineering and constructing novel genetic architectures, native sensory pathways and regulatory networks can be used for an array of dynamic sensing, computation, and regulatory applications [20,23,89].…”

Section: Regulation Of Transcription Initiationmentioning

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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Dual transcriptional-translational cascade permits cellular level tuneable expression control

Cited by 38 publications

References 46 publications

Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator

Achieving large dynamic range control of gene expression with a compact RNA transcription-translation regulator

Partial Least Squares with Structured Output for Modelling the Metabolomics Data Obtained from Complex Experimental Designs: A Study into the Y-Block Coding

Contemporary Tools for Regulating Gene Expression in Bacteria

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