Whole-cell biosensors have several advantages for the detection of biological substances and have proven to be useful analytical tools. However, several hurdles have limited whole-cell biosensor ...application in the clinic, primarily their unreliable operation in complex media and low signal-to-noise ratio. We report that bacterial biosensors with genetically encoded digital amplifying genetic switches can detect clinically relevant biomarkers in human urine and serum. These bactosensors perform signal digitization and amplification, multiplexed signal processing with the use of Boolean logic gates, and data storage. In addition, we provide a framework with which to quantify whole-cell biosensor robustness in clinical samples together with a method for easily reprogramming the sensor module for distinct medical detection agendas. Last, we demonstrate that bactosensors can be used to detect pathological glycosuria in urine from diabetic patients. These next-generation whole-cell biosensors with improved computing and amplification capacity could meet clinical requirements and should enable new approaches for medical diagnosis.
Libraries of well-characterised components regulating gene expression levels are essential to many synthetic biology applications. While widely available for the Gram-negative model bacterium ...Escherichia coli, such libraries are lacking for the Gram-positive model Bacillus subtilis, a key organism for basic research and biotechnological applications. Here, we engineered a genetic toolbox comprising libraries of promoters, Ribosome Binding Sites (RBS), and protein degradation tags to precisely tune gene expression in B. subtilis We first designed a modular Expression Operating Unit (EOU) facilitating parts assembly and modifications and providing a standard genetic context for gene circuits implementation. We then selected native, constitutive promoters of B. subtilis and efficient RBS sequences from which we engineered three promoters and three RBS sequence libraries exhibiting ∼14 000-fold dynamic range in gene expression levels. We also designed a collection of SsrA proteolysis tags of variable strength. Finally, by using fluorescence fluctuation methods coupled with two-photon microscopy, we quantified the absolute concentration of GFP in a subset of strains from the library. Our complete promoters and RBS sequences library comprising over 135 constructs enables tuning of GFP concentration over five orders of magnitude, from 0.05 to 700 μM. This toolbox of regulatory components will support many research and engineering applications in B. subtilis.
Synthetic biological circuits are promising tools for developing sophisticated systems for medical, industrial, and environmental applications. So far, circuit implementations commonly rely on gene ...expression regulation for information processing using digital logic. Here, we present a different approach for biological computation through metabolic circuits designed by computer-aided tools, implemented in both whole-cell and cell-free systems. We first combine metabolic transducers to build an analog adder, a device that sums up the concentrations of multiple input metabolites. Next, we build a weighted adder where the contributions of the different metabolites to the sum can be adjusted. Using a computational model fitted on experimental data, we finally implement two four-input perceptrons for desired binary classification of metabolite combinations by applying model-predicted weights to the metabolic perceptron. The perceptron-mediated neural computing introduced here lays the groundwork for more advanced metabolic circuits for rapid and scalable multiplex sensing.
In
Drosophila melanogaster, Hox genes are organized in an anterior and a posterior cluster, called Antennapedia complex and bithorax complex, located on the same chromosome arm and separated by 10 Mb ...of DNA. Both clusters are repressed by Polycomb group (PcG) proteins. Here, we show that genes of the two Hox complexes can interact within nuclear PcG bodies in tissues where they are corepressed. This colocalization increases during development and depends on PcG proteins. Hox gene contacts are conserved in the distantly related
Drosophila virilis species and they are part of a large gene interaction network that includes other PcG target genes. Importantly, mutations on one of the loci weaken silencing of genes in the other locus, resulting in the exacerbation of homeotic phenotypes in sensitized genetic backgrounds. Thus, the three-dimensional organization of Polycomb target genes in the cell nucleus stabilizes the maintenance of epigenetic gene silencing.
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► A subset of Polycomb target genes frequently colocalize within
Drosophila nuclei ► Hox gene contacts depend on Polycomb proteins and are evolutionarily conserved ► Specific regulatory DNA elements affect Hox gene contacts ► Decrease of Hox gene contacts reduces silencing
Engineered bacteria promise to revolutionize diagnostics and therapeutics, yet many applications are precluded by the limited number of detectable signals. Here we present a general framework to ...engineer synthetic receptors enabling bacterial cells to respond to novel ligands. These receptors are activated via ligand-induced dimerization of a single-domain antibody fused to monomeric DNA-binding domains (split-DBDs). Using E. coli as a model system, we engineer both transmembrane and cytosolic receptors using a VHH for ligand detection and demonstrate the scalability of our platform by using the DBDs of two different transcriptional regulators. We provide a method to optimize receptor behavior by finely tuning protein expression levels and optimizing interdomain linker regions. Finally, we show that these receptors can be connected to downstream synthetic gene circuits for further signal processing. The general nature of the split-DBD principle and the versatility of antibody-based detection should support the deployment of these receptors into various hosts to detect ligands for which no receptor is found in nature.
Summary
Living cells have evolved to detect and process various signals and can self‐replicate, presenting an attractive platform for engineering scalable and affordable biosensing devices. Microbes ...are perfect candidates: they are inexpensive and easy to manipulate and store. Recent advances in synthetic biology promise to streamline the engineering of microbial biosensors with unprecedented capabilities. Here we review the applications of microbially‐derived biosensors with a focus on environmental monitoring and healthcare applications. We also identify critical challenges that need to be addressed in order to translate the potential of synthetic microbial biosensors into large‐scale, real‐world applications.
Living cells have evolved to detect and process various signals and can self‐replicate, presenting an attractive platform for engineering scalable and affordable biosensing devices. Here we review the applications of microbial‐derived biosensors to environmental monitoring and healthcare applications. We also discuss the challenges to address in order to accelerate the transition of these technologies into real‐world applications.
Tools to systematically reprogram cellular behavior are crucial to address pressing challenges in manufacturing, environment, or healthcare. Recombinases can very efficiently encode Boolean and ...history-dependent logic in many species, yet current designs are performed on a case-by-case basis, limiting their scalability and requiring time-consuming optimization. Here we present an automated workflow for designing recombinase logic devices executing Boolean functions. Our theoretical framework uses a reduced library of computational devices distributed into different cellular subpopulations, which are then composed in various manners to implement all desired logic functions at the multicellular level. Our design platform called CALIN (Composable Asynchronous Logic using Integrase Networks) is broadly accessible via a web server, taking truth tables as inputs and providing corresponding DNA designs and sequences as outputs (available at http://synbio.cbs.cnrs.fr/calin). We anticipate that this automated design workflow will streamline the implementation of Boolean functions in many organisms and for various applications.
In this issue, Broussard et al. (2013) report genetic switches that regulate cell fate selection; a recombinase attachment site is embedded within a repressor coding sequence, such that integration ...truncates a proteolysis domain, stabilizing the repressor and setting the switch.
The use of synthetic biological systems in research, healthcare, and manufacturing often requires autonomous history-dependent behavior and therefore some form of engineered biological memory. For ...example, the study or reprogramming of aging, cancer, or development would benefit from genetically encoded counters capable of recording up to several hundred cell division or differentiation events. Although genetic material itself provides a natural data storage medium, tools that allow researchers to reliably and reversibly write information to DNA in vivo are lacking. Here, we demonstrate a rewriteable recombinase addressable data (RAD) module that reliably stores digital information within a chromosome. RAD modules use serine integrase and excisionase functions adapted from bacteriophage to invert and restore specific DNA sequences. Our core RAD memory element is capable of passive information storage in the absence of heterologous gene expression for over 100 cell divisions and can be switched repeatedly without performance degradation, as is required to support combinatorial data storage. We also demonstrate how programmed stochasticity in RAD system performance arising from bidirectional recombination can be achieved and tuned by varying the synthesis and degradation rates of recombinase proteins. The serine recombinase functions used here do not require cell-specific cofactors and should be useful in extending computing and control methods to the study and engineering of many biological systems.
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