Figure - available via license: Creative Commons Attribution 2.0 Generic
Content may be subject to copyright.
Schematic of positive-feedback amplifier. The basic design for the amplifier consists of GFP and LuxRΔ2-162 arranged in a bicistronic configuration under the control of the PluxI promoter. LuxRΔ2-162 functions in a positive feedback loop as it can bind to the PluxI promoter and activate its own transcription. In our design, LuxRΔ2-162 is also used as the input signal for the amplifier. LuxRΔ2-162, therefore, functions both as the input and positive feedback signal. GFP, the output signal, provides a measure of transcriptional activity.
Source publication
Positive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis. In the context of electrical circuit design, positive feedback is often considered in the design of amplifiers. Similar approaches, therefore, may be used for the design of...
Similar publications
We investigate the possibility of performing stochastic simulation of a synthetic gene circuit that includes a cell-to-cell communication system with an intracellular feedback control circuit. We propose an implementation of the CLE stochastic simulation method that makes possible to simulate gene synthetic circuits involving cell-to-cell communica...
Citations
... First, we designed a positive feedback loop in which pretroDNA_tetO under PLtetO-1 is induced by aTc and subsequently amplifies its own expression by decoying TetR (Fig. 3A). Positive feedback is useful for enhancing sensitivity with controlled leakage in biosensor development 40,41 , so we integrated the pretroDNA_tetO-based feedback with an optimized TetR-based aTc biosensor used in figure 2F and G 37 . This combined biosensor exhibited an order of magnitude improved sensitivity to ~0.14 ng/ml compared to the dRT control, with negligible background noise (Fig. 3B). ...
DNA-protein interactions are core components of myriad natural and synthetic gene networks. Despite the potential new design space, DNA-protein interactions remain underexploited in vivo due to challenges in controlling specific DNA segments, including protein-binding sequences, independently of the genome. Here we engineer retrons, prokaryotic retroelements, to intracellularly generate genome-independent programmable small DNA for sequence-specific protein-binding. Using reprogrammed retron-derived DNA for allosteric transcription factor, we demonstrated dynamic regulation of synthetic gene networks and construction of automated feedback circuits for signal amplification, adaptation, and memory. Furthermore, we developed a new class of stimuli-responsive molecular “bait and prey” that enable modular, rapid, and post-translational control of protein subcellular localization. This work substantially expands possible application area of DNA-protein interactions, laying the foundation for technical advances in synthetic biology.
One-Sentence Summary
We demonstrate new ways to control, design, and exploit DNA-protein interactions in living cells using engineered retron-generated small DNA.
... The application of artificial positive feedback loops in whole-cell biosensors can effectively complete the detection even when the concentration of a target substance is very low. In addition, Nistala et al. [46] designed an amplifier consisting of GFP and LuxR ∆2-162 arranged in series under the control of the PluxI promoter. The PluxI promoter activated transcription of LuxR ∆2-162 , and LuxR ∆2-162 in turn activated the PluxI promoter, thus amplifying the signal. ...
A whole-cell biosensor based on synthetic biology provides a promising new method for the on-site detection of food contaminants. The basic components of whole-cell biosensors include the sensing elements, such as transcription factors and riboswitches, and reporting elements, such as fluorescence, gas, etc. The sensing and reporting elements are coupled through gene expression regulation to form a simple gene circuit for the detection of target substances. Additionally, a more complex gene circuit can involve other functional elements or modules such as signal amplification, multiple detection, and delay reporting. With the help of synthetic biology, whole-cell biosensors are becoming more versatile and integrated, that is, integrating pre-detection sample processing, detection processes, and post-detection signal calculation and storage processes into cells. Due to the relative stability of the intracellular environment, whole-cell biosensors are highly resistant to interference without the need of complex sample preprocessing. Due to the reproduction of chassis cells, whole-cell biosensors replicate all elements automatically without the need for purification processing. Therefore, whole-cell biosensors are easy to operate and simple to produce. Based on the above advantages, whole-cell biosensors are more suitable for on-site detection than other rapid detection methods. Whole-cell biosensors have been applied in various forms such as test strips and kits, with the latest reported forms being wearable devices such as masks, hand rings, and clothing. This paper examines the composition, construction methods, and types of the fundamental components of synthetic biological whole-cell biosensors. We also introduce the prospect and development trend of whole-cell biosensors in commercial applications.
... Jia et al. incorporated a LuxR-based positive feedback into ArsRbased As biosensor to amplify output signal as well as to improve sensitivity (Jia et al., 2019). The LuxR protein lacked its N-terminal region (Δ2-162) and thus showed a constitutive transcription initiation activity even in the absence of its activator (Nistala et al., 2010). Two copies of the mutant luxR gene were incorporated into the biosensor. ...
Toxic heavy metal accumulation is one of anthropogenic environmental pollutions, which poses risks to human health and ecological systems. Conventional heavy metal remediation approaches rely on expensive chemical and physical processes leading to the formation and release of other toxic waste products. Instead, microbial bioremediation has gained interest as a promising and cost-effective alternative to conventional methods, but the genetic complexity of microorganisms and the lack of appropriate genetic engineering technologies have impeded the development of bioremediating microorganisms. Recently, the emerging synthetic biology opened a new avenue for microbial bioremediation research and development by addressing the challenges and providing novel tools for constructing bacteria with enhanced capabilities: rapid detection and degradation of heavy metals while enhanced tolerance to toxic heavy metals. Moreover, synthetic biology also offers new technologies to meet biosafety regulations since genetically modified microorganisms may disrupt natural ecosystems. In this review, we introduce the use of microorganisms developed based on synthetic biology technologies for the detection and detoxification of heavy metals. Additionally, this review explores the technical strategies developed to overcome the biosafety requirements associated with the use of genetically modified microorganisms.
... Examples of amplifiers include the activation of robust ligand-free TFs 42, 43 , a TF-free bacteriophage RNA polymerase or sigma factor-endogenous RNA polymerase pair 44 , an antirepressor RNA aptamer 35 , or by a tag-specific protease targeting a repressor 45 . The issue with adding an amplifier feedback loop or an amplification step of the analyte is that if either one is triggered falsely, the signal will be much higher than what it would have been without that extra step. ...
Cell-free protein synthesis-based biosensors have been developed as highly accurate, low-cost biosensors. However, since most biomarkers exist at low concentrations in various types of biopsies, the biosensor's dynamic range must be increased in the system to achieve the low limits of detection necessary while deciphering from higher background signals. Many attempts to increase the dynamic range have relied on amplifying the input signal from the analyte, which can lead to complications of false positives. In this study, we aimed to increase the protein synthesis capability of the cell-free protein synthesis system and the output signal of the reporter protein to achieve a lower limit of detection. We utilized a new fluorescent protein - mNeonGreen, which produces a higher output than those commonly used in cell-free biosensors. Optimizations of DNA sequence and the subsequent cell-free protein synthesis reaction conditions allowed characterizing protein expression variability by given DNA template types, reaction environment, and storage additives that cause the greatest time constraint on designing the cell-free biosensor. Finally, we characterized the fluorescence kinetics of mNeonGreen compared to the commonly used reporter protein, superfolder Green Fluorescent Protein. We expect that this finely tuned cell-free protein synthesis platform with the new reporter protein can be used with sophisticated synthetic gene circuitry networks to increase the dynamic range of a cell-free biosensor to reach lower detection limits and reduce false positives proportion.
... By fitting thermally regulated dynamic expression profiles with mechanistic and/or simple kinetic models, it can be highly valuable for bioproduction whereby one can derive the appropriate cooling/heating gradients and the desired duration and strength of thermal induction to achieve eventual performance. Although reasonable fold changes were observed in current systems, it can be further improved to suit specific applications potentially via the addition of positive feedbacks 49 and/or introducing temperature-sensitive proteolytic modules. 20 Different applications require specific temperature activation points. ...
... In order to improve the output signal and boost the sensitivity of WCBs, many studies incorporated genetic amplifier circuits (Nistala et al., 2010;Jia et al., 2018). However, negative feedback loops have a different role in enhancing loop sensitivity depending on the genetic context and regulatory factors present. ...
Although many whole-cell biosensors (WCBs) for the detection of Cd²⁺ have been developed over the years, most lack sensitivity and specificity. In this paper, we developed a Cd²⁺ WCB with a negative feedback amplifier in P. putida KT2440. Based on the slope of the linear detection curve as a measure of sensitivity, WCB with negative feedback amplifier greatly increased the output signal of the reporter mCherry, resulting in 33% greater sensitivity than in an equivalent WCB without the negative feedback circuit. Moreover, WCB with negative feedback amplifier exhibited increased Cd²⁺ tolerance and a lower detection limit of 0.1 nM, a remarkable 400-fold improvement compared to the WCB without the negative feedback circuit, which is significantly below the World Health Organization standard of 27 nM (0.003 mg/L) for cadmium in drinking water. Due to the superior amplification of the output signal, WCB with negative feedback amplifier can provide a detectable signal in a much shorter time, and a fast response is highly preferable for real field applications. In addition, the WCB with negative feedback amplifier showed an unusually high specificity for Cd²⁺ compared to other metal ions, giving signals with other metals that were between 17.6 and 41.4 times weaker than with Cd²⁺. In summary, the negative feedback amplifier WCB designed in this work meets the requirements of Cd²⁺ detection with very high sensitivity and specificity, which also demonstrates that genetic negative feedback amplifiers are excellent tools for improving the performance of WCBs.
... The minimum ratio of the fluorescence response of the sensor to Cd 2+ at a concentration of 0.1 μM to its fluorescence response to other heavy metal ions at a concentration of 10 μM was defined as the specificity of the sensor. WCBs have been extensively studied in order to detect toxic heavy metals in the environment, including cadmium, lead, mercury, and arsenic (Jain and Ali 2000;Jia et al. 2020;Nachman et al. 2005;Nistala et al. 2010;Wongsasuluk et al. 2014). Although many WCBs have been constructed for cadmium detection, most suffer from poor sensitivity or specificity (Bereza-Malcolm et al. 2017;Hou et al. 2015;Liao et al. 2006;Tao et al. 2013;Wu et al. 2009). ...
... Each experiment was repeated three times and standard deviations were shown as error bars) Appl Microbiol Biotechnol multi-copy reporting elements, and T7RNAP amplification module were 1 μM, 0.02 μM, and 0.01 μM, respectively (Fig. 3). Nistala et al. constructed a positive feedback-based amplifier to demonstrate the modular functionality of signal amplifiers (Nistala et al. 2010). Jia et al. coupled an arsenic WCB to a positive feedback circuit modulation module to construct a WCB that reduced the detection limit of As 3+ and increased the sensitivity by a factor of 20 (Jia et al. 2019). ...
Owing to the prevalence of cadmium contamination and its serious hazards, it is important to establish an efficient and low-cost monitoring technique for the detection of the heavy metal cadmium. In this study, we first designed 30 cadmium whole-cell biosensors (WCBs) using different combinations of detection elements, reporting elements, and the host. The best performing WCB KT-5-R with Pseudomonas putida KT2440 as the host and composed of CadR and mCherry was selected for further analysis and engineering. In order to enhance its sensitivity, a positive feedback amplifier was added or the gene dosage of the reporter gene was increased. The WCB with the T7RNAP amplification module, p2T7RNAPmut-68, had the best performance and improved tolerance to cadmium with a detection limit of 0.01 μM, which is the WHO standard. It also showed excellent specificity toward cadmium when assayed with mixed metal ions. This study demonstrated the power of circuit engineering in WCB design and provided valuable insights for the development of other WCBs.
Key points
• KT-5-R was selected after prescreening and engineered for better performance.
• Using multi-copy reporters and the T7RNAP amplifier greatly improved the performance.
• p2T7RNAPmut-68 had a detection limit of 0.01 μM and improved tolerance to cadmium.
... Xiaoqiang et al. (2018) created an improved lead biosensor using two strategies. Firstly, they changed the orientation of the regulatory elements and then incorporated positive feedback loops into the biosensor design [128]; based on a previous modified LuxR genetic amplifier [129]. Using this method they were able to improve both the sensitivity (K 1/2 in this case) and maximal output of their lead sensor. ...
It is becoming increasingly apparent that the microbiota has a profound effect on human health and disease. Modern synthetic biology provides tools that can be used to engineer new diagnostic and therapeutic circuits- facilitating microbiome engineering and the creation of engineered biotherapeutics. These engineered biotherapeutics have the potential to expand our knowledge of microbial communities, host-microbe interactions and human health. However, to achieve these ambitious goals several challenges remain to be solved. These involve the creation of novel model systems and design strategies that can be used to characterise and improve these engineered strains. The primary focus of this thesis are whole-cell biosensors, that can be used to monitor molecules relevant to human health. Within this work I develop a novel model system, based on the Caenorhabditis elegans nematode that can be used to characterise biosensor strains in vivo. Through the developed protocols I use the nematode model to show that ratiometric biosensors can detect and report on changes within the C. elegans digestive tract. This model could be used to improve engineered biosensor strains, while also expanding our understanding of nematode biology and host-microbe interactions. In addition, I engineer a range of new ratiometric plasmids that can be used in conjunction with the C. elegans model system in future. Finally, I develop a range of acetoacetate-inducible biosensors; while also exploring methods of rationally improving two-component system biosensors. Two component systems are a common sensing mechanism that can be used to create a range of biosensors, therefore methods of rationally improving these biosensors would be an invaluable tool. Overall, it is hoped that the tools developed within this thesis can be used to further engineer whole-cell biosensors, which may help expand our knowledge of host-microbe interactions and human health.
... Although approaching their attomolar sensitivity is still challenging for Cas protein itself or existing catalytic nucleic acid circuits, it is intriguing to see whether the integration of CRISPR-Cas system into catalytic nucleic acid circuits could provide simpler molecular mechanisms with comparable ultrasensitivity. Positive feedback circuits with exponential dynamics are a promising solution and are usually leveraged to achieve ultrasensitive response in biological regulation or artificial reaction networks (22)(23)(24). Considering that both inputs (DNA/RNA dual inputs) and outputs (cleaved nucleic acids) of Cas are nucleic acid-based, we envisioned that rational molecular programming of nucleic acid modules in the CRISPR-Cas system would wire up the inherent multifunctions of Cas to achieve the autocatalytic generation of active Cas proteins. This process could build a CRISPR-Cas-based positive feedback circuit with exponential amplification of catalysis. ...
Artificial nucleic acid circuits with precisely controllable dynamic and function have shown great promise in biosensing, but their utility in molecular diagnostics is still restrained by the inability to process genomic DNA directly and moderate sensitivity. To address this limitation, we present a CRISPR-Cas–powered catalytic nucleic acid circuit, namely, CRISPR-Cas–only amplification network (CONAN), for isothermally amplified detection of genomic DNA. By integrating the stringent target recognition, helicase activity, and trans-cleavage activity of Cas12a, a Cas12a autocatalysis-driven artificial reaction network is programmed to construct a positive feedback circuit with exponential dynamic in CONAN. Consequently, CONAN achieves one-enzyme, one-step, real-time detection of genomic DNA with attomolar sensitivity. Moreover, CONAN increases the intrinsic single-base specificity of Cas12a, and enables the effective detection of hepatitis B virus infection and human bladder cancer–associated single-nucleotide mutation in clinical samples, highlighting its potential as a powerful tool for disease diagnostics.
... Dynamic Range: Aside from performing random mutagenesis to improve the transcriptional output range, transcriptional amplifiers consisting of a transcriptional factor and its cognate promoter could also be adopted to extend the output range (Kim et al. 2016). The dynamic range can be further augmented by incorporating positive feedback loop (Nistala et al. 2010). ...
This chapter aims to review the underpinning process involved in gene circuit design, with the emphasis on applying it to cell-based biosensors. Accordingly, appropriate computer-aided design tools to be used during the design and construction phases, as well as modeling tools that facilitate the rational design-build-test-learn cycle, will be explored. Lastly, a compilation of the common failure modes as faced by typical users and recommended potential engineering solutions is presented.
