Programmable cross-ribosome-binding sites to fine-tune the dynamic range of transcription factor-based biosensor

Ding, Nana; Yuan, Zhenqi; Zhang, Xiaojuan; Chen, Jing; Zhou, Shenghu; Deng, Yu

doi:10.1093/nar/gkaa786

Cited by 77 publications

(91 citation statements)

References 48 publications

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“…Both of these changes reduce the number of A nucleotides and increase the number of C nucleotides. We acknowledge, however, that the effect of spacer sequence on translation efficiency is idiosyncratic, as recent high-throughput studies identified efficient RBS sequences that deviate from this rule [ 19 , 20 ].…”

Section: Resultsmentioning

confidence: 99%

Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum

et al. 2021

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Background The bacteriophage T7 gene 10 ribosome binding site (g10RBS) has long been used for robust expression of recombinant proteins in Escherichia coli. This RBS consists of a Shine–Dalgarno (SD) sequence augmented by an upstream translational “enhancer” (Enh) element, supporting protein production at many times the level seen with simple synthetic SD-containing sequences. The objective of this study was to dissect the g10RBS to identify simpler derivatives that exhibit much of the original translation efficiency. Methods and results Twenty derivatives of g10RBS were tested using multiple promoter/reporter gene contexts. We have identified one derivative (which we call “CON_G”) that maintains 100% activity in E. coli and is 33% shorter. Further minimization of CON_G results in variants that lose only modest amounts of activity. Certain nucleotide substitutions in the spacer region between the SD sequence and initiation codon show strong decreases in translation. When testing these 20 derivatives in the alphaproteobacterium Agrobacterium fabrum, most supported strong reporter protein expression that was not dependent on the Enh. Conclusions The g10RBS derivatives tested in this study display a range of observed activity, including a minimized version (CON_G) that retains 100% activity in E. coli while being 33% shorter. This high activity is evident in two different promoter/reporter sequence contexts. The array of RBS sequences presented here may be useful to researchers in need of fine-tuned expression of recombinant proteins of interest.

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Section: Resultsmentioning

confidence: 99%

Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum

et al. 2021

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“…Such switches are now useful for paper-based systems and materials to sense specific input RNA stimuli and to drive gene expression [ 325 ]. Further examples of using AI include the prediction of ribosome-binding sites providing desired translation rates [ 326 ], the de novo design of promoter elements in E. coli [ 327 ], or the design of an optimized CRISPR guide RNA for high-fidelity Cas9 variants [ 328 ]. Such approaches to construct genetic tools with predictable functions are highly relevant for the design of more complex expression systems as required for synthetic gene networks [ 321 ] or for orchestrating complex metabolic fluxes [ 329 , 330 ].…”

Section: Discussionmentioning

confidence: 99%

Synthetic biology as driver for the biologization of materials sciences

Burgos-Morales

Gueye

Lacombe

et al. 2021

Materials Today Bio

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Materials in nature have fascinating properties that serve as a continuous source of inspiration for materials scientists. Accordingly, bio-mimetic and bio-inspired approaches have yielded remarkable structural and functional materials for a plethora of applications. Despite these advances, many properties of natural materials remain challenging or yet impossible to incorporate into synthetic materials. Natural materials are produced by living cells, which sense and process environmental cues and conditions by means of signaling and genetic programs, thereby controlling the biosynthesis, remodeling, functionalization, or degradation of the natural material. In this context, synthetic biology offers unique opportunities in materials sciences by providing direct access to the rational engineering of how a cell senses and processes environmental information and translates them into the properties and functions of materials. Here, we identify and review two main directions by which synthetic biology can be harnessed to provide new impulses for the biologization of the materials sciences: first, the engineering of cells to produce precursors for the subsequent synthesis of materials. This includes materials that are otherwise produced from petrochemical resources, but also materials where the bio-produced substances contribute unique properties and functions not existing in traditional materials. Second, engineered living materials that are formed or assembled by cells or in which cells contribute specific functions while remaining an integral part of the living composite material. We finally provide a perspective of future scientific directions of this promising area of research and discuss science policy that would be required to support research and development in this field.

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“…DcuS-DcuR is a two-component regulatory system that was identified in E. coli and functions as a classical two-component sensor-regulator in controlling gene expression. After optimizing the expression level of SK and RR and adjusting the kinase and phosphatase activity of SK by promoter engineering and DcuS engineering, respectively, the dynamic range (i.e., fold change in gene expression between the presence and absence of inducer [31]) was significantly improved. This work paves the way for high-throughput screening and dynamic regulation of fumaric acid cell factories.…”

Section: Introductionmentioning

confidence: 99%

Engineering a fumaric acid-responsive two-component biosensor for dynamic range improvement in Escherichia coli

Yang

Yang²,

Yan-Bo³

et al. 2022

Syst Microbiol and Biomanuf

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Due to the selective permeability of the cytomembrane, high-yield fumaric acid strains form a steep difference between intra-and extracellular concentrations. Intracellular biosensors cannot detect the real concentration change of extracellular fumaric acid. To overcome this limitation, a two-component biosensor (TCB) that could respond to extracellular fumaric acid was designed based on the DcuS-DcuR two-component system. The two-component system consists of a histidine kinase (SK) and response regulator. SK is a transmembrane histidine kinase sensor that can detect concentration changes in extracellular compounds. To improve the dynamic range of the constructed fumaric acid TCB, we optimized the expression ratio and expression intensity of dcuS and dcuR. We found that the optimum expression ratio of dcuS:dcuR was 46:54. Under this ratio, the higher was the expression level, the greater the dynamic range. In addition, we modified the ATP-binding site on the DcuS, and the final dynamic range of the TCB reached 6.6-fold. Overall, the obtained fumaric acid-responsive TCB with a high dynamic range is reported for the first time, providing a synthetic biology tool for high-throughput screening and dynamic metabolic regulation of fumaric acid cell factories.

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Programmable cross-ribosome-binding sites to fine-tune the dynamic range of transcription factor-based biosensor

Cited by 77 publications

References 48 publications

Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum

Translation initiation from sequence variants of the bacteriophage T7 g10RBS in Escherichia coli and Agrobacterium fabrum

Synthetic biology as driver for the biologization of materials sciences

Engineering a fumaric acid-responsive two-component biosensor for dynamic range improvement in Escherichia coli

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