Recent advances in developing and applying biosensors for synthetic biology

Zinkus-Boltz, Julia; Dickinson, Bryan C.

doi:10.1088/2399-1984/ab4b78

Cited by 12 publications

(10 citation statements)

References 270 publications

(323 reference statements)

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“…Characteristics such as high sensitivity and specificity to weak stimuli, robustness, orthogonality, continuous sensing, and scalability can be achieved. 39,40 In order to build stimuli-responsive ELMs, cells with such characteristics must modulate the physical or chemical properties of the material or modify the surrounding environment upon exposure to external stimuli.…”

Section: Introductionmentioning

confidence: 99%

Stimuli-responsive engineered living materials

2021

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Section: Introductionmentioning

confidence: 99%

Stimuli-responsive engineered living materials

2021

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“…39,40 Our new system relies on proximity-dependent split RNA polymerase (RNAP) biosensors, 41 greatly simplifying and streamlining the genetic sensing of PPIs in vivo compared with more traditional two-hybrid approaches. 42 Using these split RNAP biosensors, we designed a PACS system that links replicating bacteriophages to the binding of bacterial-expressed KRAS to phage-expressed RAF. After validating the system, we generated libraries of phage-encoded RAF variants and subjected them to PACS.…”

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confidence: 99%

A Phage-Assisted Continuous Selection Approach for Deep Mutational Scanning of Protein–Protein Interactions

2019

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Protein–protein interactions (PPIs) are critical for organizing molecules in a cell and mediating signaling pathways. Dysregulation of PPIs is often a key driver of disease. To better understand the biophysical basis of such disease processesand to potentially target themit is critical to understand the molecular determinants of PPIs. Deep mutational scanning (DMS) facilitates the acquisition of large amounts of biochemical data by coupling selection with high throughput sequencing (HTS). The challenging and labor-intensive design and optimization of a relevant selection platform for DMS, however, limits the use of powerful directed evolution and selection approaches. To address this limitation, we designed a versatile new phage-assisted continuous selection (PACS) system using our previously reported proximity-dependent split RNA polymerase (RNAP) biosensors, with the aim of greatly simplifying and streamlining the design of a new selection platform for PPIs. After characterization and validation using the model KRAS/RAF PPI, we generated a library of RAF variants and subjected them to PACS and DMS. Our HTS data revealed positions along the binding interface that are both tolerant and intolerant to mutations, as well as which substitutions are tolerated at each position. Critically, the “functional scores” obtained from enrichment data through continuous selection for individual variants correlated with K D values measured in vitro, indicating that biochemical data can be extrapolated from sequencing using our new system. Due to the plug and play nature of RNAP biosensors, this method can likely be extended to a variety of other PPIs. More broadly, this, and other methods under development support the continued development of evolutionary and high-throughput approaches to address biochemical problems, moving toward a more comprehensive understanding of sequence–function relationships in proteins.

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“…Future research on current Ca 2+ -binding proteins and dyes should focus on multicolor calcium sensors which achieve fast kinetic responses and Ca 2+ affinity optimized for a range that encompasses the various concentrations of Ca 2+ in cellular components and the cytosol [216]. Synthetic biology approaches towards biosensors necessarily focus on dynamic range, sensitivity, selectivity, and orthogonality [217], and numerous approaches including site-directed mutagenesis, directed evolution, rational design, and expression vector engineering have already guided us towards this path of optimization regarding calcium signaling research in recent decades.…”

Section: Discussionmentioning

confidence: 99%

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

Saha

2021

Biomolecules

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Since the 1970s, the emergence and expansion of novel methods for calcium ion (Ca2+) detection have found diverse applications in vitro and in vivo across a series of model animal systems. Matched with advances in fluorescence imaging techniques, the improvements in the functional range and stability of various calcium indicators have significantly enhanced more accurate study of intracellular Ca2+ dynamics and its effects on cell signaling, growth, differentiation, and regulation. Nonetheless, the current limitations broadly presented by organic calcium dyes, genetically encoded calcium indicators, and calcium-responsive nanoparticles suggest a potential path toward more rapid optimization by taking advantage of a synthetic biology approach. This engineering-oriented discipline applies principles of modularity and standardization to redesign and interrogate endogenous biological systems. This review will elucidate how novel synthetic biology technologies constructed for eukaryotic systems can offer a promising toolkit for interfacing with calcium signaling and overcoming barriers in order to accelerate the process of Ca2+ detection optimization.

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Recent advances in developing and applying biosensors for synthetic biology

Cited by 12 publications

References 270 publications

Stimuli-responsive engineered living materials

Stimuli-responsive engineered living materials

A Phage-Assisted Continuous Selection Approach for Deep Mutational Scanning of Protein–Protein Interactions

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

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