Synthetic Biology Applications Research Articles

Translational activation by a synthetic PPR protein elucidates control of psbA translation in Arabidopsis chloroplasts.

Translation initiation on chloroplast psbA mRNA in plants scales with light intensity, providing its gene product, D1, as needed to replace photodamaged D1 in Photosystem II. The psbA translational activator HIGH CHLOROPHYLL FLUORESCENCE 173 (HCF173) has been hypothesized to mediate this regulation. HCF173 belongs to the short-chain dehydrogenase/reductase superfamily, associates with the psbA 5'-untranslated region (5'-UTR), and has been hypothesized to enhance translation by binding an RNA segment that would otherwise pair with and mask the ribosome binding region. To test these hypotheses, we examined whether a synthetic pentatricopeptide repeat (sPPR) protein can substitute for HCF173 when bound to the HCF173 binding site. We show that an sPPR designed to bind HCF173's footprint in the psbA 5'-UTR bound the intended site in vivo and partially substituted for HCF173 to activate psbA translation. However, sPPR-activated translation did not respond to light. These results imply that HCF173 activates translation, at least in part, by sequestering the RNA it binds to maintain an accessible ribosome binding region, and that HCF173 is also required to regulate psbA translation in response to light. Translational activation can be added to the functions that can be programmed with sPPR proteins for synthetic biology applications in chloroplasts.

Enhancing Escherichia coli abiotic stress resistance through ornithine lipid formation

Escherichia coli is a common host for biotechnology and synthetic biology applications. During growth and fermentation, the microbes are often exposed to stress conditions, such as variations in pH or solvent concentrations. Bacterial membranes play a key role in response to abiotic stresses. Ornithine lipids (OLs) are a group of membrane lipids whose presence and synthesis have been related to stress resistance in bacteria. We wondered if this stress resistance could be transferred to bacteria not encoding the capacity to form OLs in their genome, such as E. coli. In this study, we engineered different E. coli strains to produce unmodified OLs and hydroxylated OLs by expressing the synthetic operon olsFC. Our results showed that OL formation improved pH resistance and increased biomass under phosphate limitation. Transcriptome analysis revealed that OL-forming strains differentially expressed stress- and membrane-related genes. OL-producing strains also showed better growth in the presence of the ionophore carbonyl cyanide 3-chlorophenylhydrazone (CCCP), suggesting reduced proton leakiness in OL-producing strains. Furthermore, our engineered strains showed improved heterologous violacein production at phosphate limitation and also at low pH. Overall, this study demonstrates the potential of engineering the E. coli membrane composition for constructing robust hosts with an increased abiotic stress resistance for biotechnology and synthetic biology applications.Key points• Ornithine lipid production in E. coli increases biomass yield under phosphate limitation.• Engineered strains show an enhanced production phenotype under low pH stress.• Transcriptome analysis and CCCP experiments revealed reduced proton leakage.

The effect of pseudoknot base pairing on cotranscriptional structural switching of the fluoride riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium.

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonia, they have a great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have improved genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants or symbiotic association with non-legumes.

High-throughput visualization mutation screening technology to enhance the specificity of CadR based whole-cell cadmium biosensor

As a heavy metal pollutant, Cd2+ often enters the human body through the food chain causing great harm to human health. Whole-cell biosensor is an emerging technology for rapid on-site detection of heavy metals with the advantages of inexpensive, fast to mass-produce, and strong in anti-interference resistance, but suffering from insatisfactory specificity. In this study, a strategy of Adjacent Site Saturation Mutation (ASSM) was designed to improve the specificity of transcription factors CadR, which acted as the recognition element and determined the specificity of whole cell Cd2+ biosensors. A specific saturated library was constructed using the strategy of adjacent mutation. After two rounds of high-throughput visual screening, a whole-cell biosensor with good response to Cd2+, and with significant weakened Hg2+ interference was obtained. The optimized whole-cell biosensor showed a linear dynamic concentration range from 500 nM to 100 μM, a detection limit of 0.079 μM, and has satisfactory specificity and anti-interference. The ASSM strategy proposed in this study can provide a new method for the application of synthetic biology in food safety detection, indicating the importance of whole-cell biosensors for the detection of heavy metals.

Development of SynBio Tools for Pseudomonas chlororaphis: A Versatile Non-Pathogenic Bacterium Host

Pseudomonas chlororaphis ATCC 9446 is a non-pathogenic bacterium associated with the rhizosphere. It is commonly used as a biocontrol agent against agricultural pests. This organism can grow on a variety of carbon sources, has a robust secondary metabolism, and produces secondary metabolites with antimicrobial properties. This makes it an alternative host organism for synthetic biology applications. However, as a novel host there is a need for well-characterized molecular tools that allow fine control of gene expression and exploration of its metabolic potential. In this work we developed and characterized expression vectors for P. chlororaphis. We used two different promoters: the exogenously induced lac-IPTG promoter, and LuxR-C6-AHL, which we evaluated for its auto-inducible capacities, as well as using an external addition of C6-AHL. The expression response of these vectors to the inducer concentration was characterized by detecting a reporter fluorescent protein (YFP: yellow fluorescent protein). Furthermore, the violacein production operon was evaluated as a model heterologous pathway. We tested violacein production in shake flasks and a 3 L fermenter, showing that P. chlororaphis possesses a vigorous aromatic amino acid metabolism and was able to produce 1 g/L of violacein in a simple batch reactor experiment with minimal medium using only glucose as the carbon source. We compared the experimental results with the predictions of a modified genome scale model. The presented results show the potential of P. chlororaphis as a novel host organism for synthetic biology applications.

An RNA origami robot that traps and releases a fluorescent aptamer.

RNA nanotechnology aims to use RNA as a programmable material to create self-assembling nanodevices for application in medicine and synthetic biology. The main challenge is to develop advanced RNA robotic devices that both sense, compute, and actuate to obtain enhanced control over molecular processes. Here, we use the RNA origami method to prototype an RNA robotic device, named the "Traptamer," that mechanically traps the fluorescent aptamer, iSpinach. The Traptamer is shown to sense two RNA key strands, acts as a Boolean AND gate, and reversibly controls the fluorescence of the iSpinach aptamer. Cryo-electron microscopy of the closed Traptamer structure at 5.45-angstrom resolution reveals the mechanical mode of distortion of the iSpinach motif. Our study suggests a general approach to distorting RNA motifs and a path forward to build sophisticated RNA machines that through sensing, computing, and actuation modules can be used to precisely control RNA functionalities in cellular systems.

The roles of SARP family regulators involved in secondary metabolism in Streptomyces.

Streptomyces species are best known for their ability to produce abundant secondary metabolites with versatile bioactivities and industrial importance. These metabolites are usually biosynthesized through metabolic pathways encoded by cluster-situated genes. These genes are also known as biosynthetic gene clusters (BGCs) of secondary metabolites. The expression of BGCs is intricately controlled by pyramidal transcriptional regulatory cascades, which include various regulators. Streptomyces antibiotic regulatory proteins (SARPs), a genus-specific family of regulators, are widely distributed and play important roles in regulating the biosynthesis of secondary metabolites in Streptomyces. Over the past decade, the biological functions of SARPs have been extensively investigated. Here, we summarized the recent advances in characterizing the roles of SARPs involved in Streptomyces secondary metabolism from the following three aspects. First, the classification and domain organization of SARPs were summarized according to their size variation. Second, we presented a detailed description of the regulatory mechanisms and modes of action of SARPs involved in secondary metabolism. Finally, the biotechnological application of SARPs was illustrated by improving the production of target secondary metabolites and discovering novel bioactive natural products. This review will help researchers to comprehensively understand the roles of SARPs in secondary metabolite biosynthesis in Streptomyces, which will contribute to building a solid foundation for their future application in synthetic biology.

Between the lab and the Wild: Establishing the Potential of Gene Drive Mosquitoes for Malaria Control

ABSTRACT Malaria control has been one of the defining goals in global health. Recently, strategies that aim to control the insect-borne disease by altering mosquito biology have gained interest. One such strategy currently in development is to engineer mosquitoes equipped with so-called gene drives: elements that increase the likelihood of a specific set of genes to be passed on to the next generation. The strategy encapsulates hopes of technological solutions in global health, as well as expectations for the applications of synthetic biology. Hence, gene drive research operates with the potential of genetically modified mosquitoes: to survive in the wild, to reproduce, and to perform the work expected of them in some future timeline. I examine gene drive research with the concept of potentiality and how establishing it has guided gene drive research. Knowledge and uncertainties about mosquitoes and mosquito ecologies emerged through working with the mosquito as a research animal in the laboratory, as population data, and as a regulatable biotechnology. Mathematical modelling made it possible to establish the potential to move from the lab to the wild. In modelling, the mosquito as a research object could be made to work as part of future projections that assembled different kinds of data. As gene drive research progresses, I suggest that the researchers are also modelling how biotechnology handles uncertainty that is related to the ways producing genetic modification in the lab and producing knowledge about their possible environmental effects and efficacy translates into landscapes and mosquito ecologies in the wild.

Expanding the Biosynthetic Toolbox: The Potential and Challenges of In Vitro Type II Polyketide Synthase Research

Type II polyketide synthase (PKS) systems are a rich source of structurally diverse polycyclic aromatic compounds with clinically relevant antibiotic and chemotherapeutic properties. The enzymes responsible for synthesizing the polyketide core, known collectively as the minimal cassette, hold potential for applications in synthetic biology. The minimal cassette provides polyketides of different chain lengths, which interact with other enzymes that are responsible for the varied cyclization patterns. Additionally, the type II PKS enzyme clusters offer a wide repertoire of tailoring enzymes for oxidations, glycosylations, cyclizations, and rearrangements. This review begins with the variety of chemical space accessible with type II PKS systems including the recently discovered highly reducing variants that produce polyalkenes instead of the archetypical polyketide motif. The main discussion analyzes the previous approaches with an emphasis on further research that is needed to characterize the minimal cassette enzymes in vitro. Finally, the potential type II PKS systems hold the potential to offer new tools in biocatalysis and synthetic biology, particularly in the production of novel antibiotics and biofuels.

'Seeing' the electromagnetic spectrum: spotlight on the cryptochrome photocycle.

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

Revealing the grammar of small RNA secretion using interpretable machine learning

Small non-coding RNAs can be secreted through a variety of mechanisms, including exosomal sorting, in small extracellular vesicles, and within lipoprotein complexes. However, the mechanisms that govern their sorting and secretion are not well understood. Here, we present ExoGRU, a machine learning model that predicts small RNA secretion probabilities from primary RNA sequences. We experimentally validated the performance of this model through ExoGRU-guided mutagenesis and synthetic RNA sequence analysis. Additionally, we used ExoGRU to reveal cis and trans factors that underlie small RNA secretion, including known and novel RNA-binding proteins (RBPs), e.g., YBX1, HNRNPA2B1, and RBM24. We also developed a novel technique called exoCLIP, which reveals the RNA interactome of RBPs within the cell-free space. Together, our results demonstrate the power of machine learning in revealing novel biological mechanisms. In addition to providing deeper insight into small RNA secretion, this knowledge can be leveraged in therapeutic and synthetic biology applications.

Structure-Based Engineering of a Sesquiterpene Cyclase to Generate an Alcohol Product: Conversion of epi-Isozizaene Synthase into α-Bisabolol Synthase.

The sesquiterpene cyclase epi-isozizaene synthase (EIZS) from Streptomyces coelicolor catalyzes the metal-dependent conversion of farnesyl diphosphate (FPP) into the complex tricyclic product epi-isozizaene. This remarkable transformation is governed by an active site contour that serves as a template for catalysis, directing the conformations of multiple carbocation intermediates leading to the final product. Mutagenesis of residues defining the active site contour remolds its three-dimensional shape and reprograms the cyclization cascade to generate alternative cyclization products. In some cases, mutagenesis enables alternative chemistry to quench carbocation intermediates, e.g., through hydroxylation. Here, we combine structural and biochemical data from previously characterized EIZS mutants to design and prepare F95S-F198S EIZS, which converts EIZS into an α-bisabolol synthase with moderate fidelity (65% at 18 °C, 74% at 4 °C). We report the complete biochemical characterization of this double mutant as well as the 1.47 Å resolution X-ray crystal structure of its complex with three Mg2+ ions, inorganic pyrophosphate, and the benzyltriethylammonium cation, which partially mimics a carbocation intermediate. Most notably, the two mutations together create an active site contour that stabilizes the bisabolyl carbocation intermediate and positions a water molecule for the hydroxylation reaction. Structural comparison with a naturally occurring α-bisabolol synthase reveals common active site features that direct α-bisabolol generation. In showing that EIZS can be redesigned to generate a sesquiterpene alcohol product instead of a sesquiterpene hydrocarbon product, we have expanded the potential of EIZS as a platform for the development of designer cyclases that could be utilized in synthetic biology applications.

Peptide nucleic acid-assisted generation of targeted double-stranded DNA breaks with T7 endonuclease I.

Gene-editing technologies have revolutionized biotechnology, but current gene editors suffer from several limitations. Here, we harnessed the power of gamma-modified peptide nucleic acids (γPNAs) to facilitate targeted, specific DNA invasion and used T7 endonuclease I (T7EI) to recognize and cleave the γPNA-invaded DNA. Our data show that T7EI can specifically target PNA-invaded linear and circular DNA to introduce double-strand breaks (DSBs). Our PNA-Guided T7EI (PG-T7EI) technology demonstrates that T7EI can be used as a programmable nuclease capable of generating single or multiple specific DSBs in vitro under a broad range of conditions and could be potentially applied for large-scale genomic manipulation. With no protospacer adjacent motif (PAM) constraints and featuring a compact protein size, our PG-T7EI system will facilitate and expand DNA manipulations both in vitro and in vivo, including cloning, large-fragment DNA assembly, and gene editing, with exciting applications in biotechnology, medicine, agriculture, and synthetic biology.

The landscape of transcription factor promoter activity during vegetative development in Marchantia.

Transcription factors (TFs) are essential for the regulation of gene expression and cell fate determination. Characterising the transcriptional activity of TF genes in space and time is a critical step towards understanding complex biological systems. The vegetative gametophyte meristems of bryophytes share some characteristics with the shoot apical meristems of flowering plants. However, the identity and expression profiles of TFs associated with gametophyte organization are largely unknown. With only ∼450 putative TF genes, Marchantia (Marchantia polymorpha) is an outstanding model system for plant systems biology. We have generated a near-complete collection of promoter elements derived from Marchantia TF genes. We experimentally tested reporter fusions for all the TF promoters in the collection and systematically analysed expression patterns in Marchantia gemmae. This allowed us to build a map of expression domains in early vegetative development and identify a set of TF-derived promoters that are active in the stem cell zone. The cell markers provide additional tools and insight into the dynamic regulation of the gametophytic meristem and its evolution. In addition, we provide an online database of expression patterns for all promoters in the collection. We expect that these promoter elements will be useful for cell-type-specific expression, synthetic biology applications, and functional genomics.

Machine Learning and Deep Learning in Synthetic Biology: Key Architectures, Applications, and Challenges.

Machine learning (ML), particularly deep learning (DL), has made rapid and substantial progress in synthetic biology in recent years. Biotechnological applications of biosystems, including pathways, enzymes, and whole cells, are being probed frequently with time. The intricacy and interconnectedness of biosystems make it challenging to design them with the desired properties. ML and DL have a synergy with synthetic biology. Synthetic biology can be employed to produce large data sets for training models (for instance, by utilizing DNA synthesis), and ML/DL models can be employed to inform design (for example, by generating new parts or advising unrivaled experiments to perform). This potential has recently been brought to light by research at the intersection of engineering biology and ML/DL through achievements like the design of novel biological components, best experimental design, automated analysis of microscopy data, protein structure prediction, and biomolecular implementations of ANNs (Artificial Neural Networks). I have divided this review into three sections. In the first section, I describe predictive potential and basics of ML along with myriad applications in synthetic biology, especially in engineering cells, activity of proteins, and metabolic pathways. In the second section, I describe fundamental DL architectures and their applications in synthetic biology. Finally, I describe different challenges causing hurdles in the progress of ML/DL and synthetic biology along with their solutions.

CRISPR/Cas Technology: The Unique Synthetic Biology Genome-Editing Tool Shifting the Paradigm in Viral Diagnostics, Defense, and Therapeutics.

The emergence of the COVID-19 pandemic has starkly exposed our significantly limited ability to promptly identify and respond to emergent biological threats. Consequently, there is an urgent need to advance biotechnological methods for addressing both known and unforeseen biological hazards. Recently, the CRISPR/Cas system has revolutionized genetic engineering, enabling precise and efficient synthetic biology applications. Therefore, this review aims to provide a comprehensive introduction to the fundamental principles underlying the CRISPR/Cas system and assess the advantages and limitations of various CRISPR/Cas-based techniques applicable to the detection of, defense against, and treatment of viral infections. These techniques include viral diagnostics, the development of antiviral vaccines, B cell engineering for antibody production, viral activation/interference, and epigenetic modifications. Furthermore, this review delves into the challenges and bioethical considerations associated with use of the CRISPR/Cas system. With the continuous evolution of technology, the CRISPR/Cas system holds considerable promise for addressing both existing and unforeseen biological threats. Expected final online publication date for the Annual Review of Biomedical Engineering, Volume 26 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Light Color‐Controlled pH‐Adjustment of Aqueous Solutions Using Engineered Proteoliposomes

AbstractControlling the pH at the microliter scale can be useful for applications in research, medicine, and industry, and therefore represents a valuable application for synthetic biology and microfluidics. The presented vesicular system translates light of different colors into specific pH changes in the surrounding solution. It works with the two light‐driven proton pumps bacteriorhodopsin and blue light‐absorbing proteorhodopsin Med12, that are oriented in opposite directions in the lipid membrane. A computer‐controlled measuring device implements a feedback loop for automatic adjustment and maintenance of a selected pH value. A pH range spanning more than two units can be established, providing fine temporal and pH resolution. As an application example, a pH‐sensitive enzyme reaction is presented where the light color controls the reaction progress. In summary, light color‐controlled pH‐adjustment using engineered proteoliposomes opens new possibilities to control processes at the microliter scale in different contexts, such as in synthetic biology applications.

Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve.

Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.

Whispering-Gallery Mode Optoplasmonic Microcavities: From Advanced Single-Molecule Sensors and Microlasers to Applications in Synthetic Biology.

Optical microcavities, specifically, whispering-gallery mode (WGM) microcavities, with their remarkable sensitivity to environmental changes, have been extensively employed as biosensors, enabling the detection of a wide range of biomolecules and nanoparticles. To push the limits of detection down to the most sensitive single-molecule level, plasmonic nanorods are strategically introduced to enhance the evanescent fields of WGM microcavities. This advancement of optoplasmonic WGM sensors allows for the detection of single molecules of a protein, conformational changes, and even atomic ions, marking significant contributions in single-molecule sensing. This Perspective discusses the exciting research prospects in optoplasmonic WGM sensing of single molecules, including the study of enzyme thermodynamics and kinetics, the emergence of thermo-optoplasmonic sensing, the ultrasensitive single-molecule sensing on WGM microlasers, and applications in synthetic biology.