Engineered Bacteria with Genetic Circuits Accumulating Nanomagnets as MRI Contrast Agents.

The demand for highly efficient cancer diagnostic tools increases alongside the high cancer incidence nowadays. Moreover, there is an imperative need for novel cancer treatment therapies that lack the side effects of conventional treatment options. Developments in this aspect employ magnetic nanoparticles (MNPs) for biomedical applications due to their stability, biocompatibility, and magnetic properties. Certain organisms, including many bacteria, can synthesize magnetic nanocrystals, which help their spatial orientation and survival by sensing the earth's geomagnetic field. This work aims to convert Escherichia coli to accumulate magnetite, which can further be coupled with drug delivery modules. The authors design magnetite accumulating bacterial machines using genetic circuitries hiring Mms6 with iron-binding activity and essential in magnetite crystal formation. The work demonstrates that the combinatorial effect of Mms6 with ferroxidase, iron transporter protein, and material binding peptide enhances the paramagnetic behavior of the cells in magnetic resonance imaging (MRI) measurements. Cellular machines are also engineered to display Mms6 peptide on the cell surface via an autotransporter protein that shows augmented MRI performance. The findings are promising for endowing a probiotic bacterium, able to accumulate magnetite intracellularly or extracellularly, serving as a theranostics agent for cancer diagnostics via MRI scanning and hyperthermia treatment.

SARS-CoV-2 Detection with De Novo-Designed Synthetic Riboregulators.

SARS-CoV-2 is a human pathogen and the main cause of COVID-19 disease, announced as a global pandemic by the World Health Organization. COVID-19 is characterized by severe conditions, and early diagnosis can make dramatic changes for both personal and public health. Low-cost, easy-to-use diagnostic capabilities can have a very critical role in controlling the transmission of the disease. Here, we are reporting a state-of-the-art diagnostic tool developed with an in vitro synthetic biology approach by employing engineered de novo riboregulators. Our design coupled with a home-made point-of-care device can detect and report the presence of SARS-CoV-2-specific genes. The presence of SARS-CoV-2-related genes triggers the translation of sfGFP mRNAs, resulting in a green fluorescence output. The approach proposed here has the potential of being a game changer in SARS-CoV-2 diagnostics by providing an easy-to-run, low-cost diagnostic capability.

Cellular Biocatalysts Using Synthetic Genetic Circuits for Prolonged and Durable Enzymatic Activity.

Cellular biocatalysts hold great promise for the synthesis of difficult to achieve compounds, such as complex active molecules. Whole-cell biocatalysts can be programmed through genetic circuits to be more efficient, but they suffer from low stability. The catalytic activity of whole cells decays under stressful conditions, such as prolonged incubation times or high temperatures. In nature, microbial communities cope with these conditions by forming biofilm structures. In this study, it is shown that the use of biofilm structures can enhance the stability of whole-cell biocatalysts. We employed two different strategies to increase the stability of whole-cell catalysts and decrease their susceptibility to high temperature. In the first approach, the formation of a biofilm structure is induced by controlling the expression of one of the curli component, CsgA. The alkaline phosphatase (ALP) enzyme was used to monitor the catalytic activity of cells in the biofilm structure. In the second approach, the ALP enzyme was fused to the CsgA curli fiber subunit to utilize the protective properties of the biofilm on enzyme biofilms. Furthermore, an AND logic gate is introduced between the expression of CsgA and ALP by toehold RNA switches and recombinases to enable logical programming of the whole-cell catalyst for biofilm formation and catalytic action with different tools. The study presents viable approaches to engineer a platform for biocatalysis processes.

Genetically-Tunable Mechanical Properties of Bacterial Functional Amyloid Nanofibers.

Bacterial biofilms are highly ordered, complex, dynamic material systems including cells, carbohydrates, and proteins. They are known to be resistant against chemical, physical, and biological disturbances. These superior properties make them promising candidates for next generation biomaterials. Here we investigated the morphological and mechanical properties (in terms of Young's modulus) of genetically-engineered bacterial amyloid nanofibers of Escherichia coli (E. coli) by imaging and force spectroscopy conducted via atomic force microscopy (AFM). In particular, we tuned the expression and biochemical properties of the major and minor biofilm proteins of E. coli (CsgA and CsgB, respectively). Using appropriate mutants, amyloid nanofibers constituting biofilm backbones are formed with different combinations of CsgA and CsgB, as well as the optional addition of tagging sequences. AFM imaging and force spectroscopy are used to probe the morphology and measure the Young's moduli of biofilm protein nanofibers as a function of protein composition. The obtained results reveal that genetically-controlled secretion of biofilm protein components may lead to the rational tuning of Young's moduli of biofilms as promising candidates at the bionano interface.

Engineered peptides for nanohybrid assemblies.

Inspired by biological material synthesis, synthetic biomineralization peptides have been screened through a laboratory evolution using biocombinatorial techniques. In this study, using the fine examples in nature, silica binding peptides and gold binding peptides were fused together to form a hybrid peptide. We designed fusion peptides with different gold binding and silica binding parts. First, we have tested the binding capability of the fusion peptides using quartz crystal microbalance on gold surface and silica surface. Second, S1G1 hybrid peptide enabled assembly of gold nanoparticles on a silica surface was achieved. Finally, nanomaterial synthesis ability of the S1G1 peptide was presented by the formation of a silica film on a gold surface. In this study, we are presenting a hybrid peptide tool for nanohybrid assembly as a promising route for nanotechnology applications.

Thermodynamics of engineered gold binding peptides: establishing the structure-activity relationships.

Adsorption behavior of a gold binding peptide was experimentally studied to achieve kinetics and thermodynamics parameters toward understanding of the binding of an engineered peptide onto a solid metal surface. The gold-binding peptide, GBP1, was originally selected using a cell surface display library and contains 14 amino acid residues. In this work, single- and three-repeats of GBP1 were used to assess the effects of two parameters: molecular architecture versus secondary structure on adsorption on to gold substrate. The adsorption measurements were carried out using surface plasmon resonance (SPR) spectroscopy at temperatures ranging from 10 to 55 °C. At all temperatures, two different regimes of peptide adsorption were observed, which, based on the model, correspond to two sets of thermodynamics values. The values of enthalpy, ΔH(ads), and entropy, ΔS(ads), in these two regimes were determined using the van't Hoff approach and Gibbs-Helmholtz relationship. In general, the values of enthalpy for both peptides are negative indicating GBP1 binding to gold is an exothermic phenomenon and that the binding of three repeat gold binding peptide (3l-GBP1) is almost 5 times tighter than that for the single repeat (l-GBP1). More intriguing result is that the entropy of adsorption for the 3l-GBP1 is negative (-43.4 ± 8.5 cal/(mol K)), while that for the l-GBP1 is positive (10.90 ± 1.3 cal/(mol K)). Among a number of factors that synergistically contribute to the decrease of entropy, long-range ordered self-assembly of the 3l-GBP1 on gold surface is the most effective, probably through both peptide-solid and peptide-peptide intermolecular interactions. Additional adsorption experiments were conducted in the presence of 2,2,2-trifluoroethanol (TFE) to determine how the conformational structures of the biomolecules responded to the environmental perturbation. We found that the peptides differ in their conformational responses to the change in solution conditions; while l-GBP does not fold in the presence of TFE, 3l-GBP1 adopted two types of secondary structure (ß-strand, α-helix) and that peptide's binding to the solid is enhanced by the presence of low percentages of TFE solvent. Not only do these kinetics and thermodynamics results provide adsorption behavior and binding of genetically engineered peptides for inorganics (GEPI), but they could also provide considerable insights into fundamental understanding peptide molecular recognition and their selective specificity for the solids. Moreover, comprehensive work described herein suggests that multiple repeat forms of the solid binding peptides possess a conformational component that can be exploited to further tailor affinity and binding of a given sequence to a solid material followed by ordered assembly as a convenient tool in future practical applications.

An Antibiotic-Degrading Engineered Biofilm Platform to Combat Environmental Antibiotic Resistance.

The presence of antibiotics in natural water bodies is a growing problem regarding the occurrence of antibiotic resistance among various species. This is mainly caused by the excessive use of medical and veterinary antibiotics as well as the lack of effective treatment processes for eliminating residual antibiotics from wastewaters. In this study, we introduce a genetically engineered biomaterial as a solution for the effective degradation of one of the dominantly found antibiotics in natural water bodies. Our biomaterial harnesses laccase-type enzymes, which are known to attack specific types of antibiotics, i.e., fluoroquinolone-type synthetic antibiotics, and as a result degradation occurs. The engineered biomaterial is built using Escherichia coli biofilm protein CsgA as a scaffold, which is fused separately to two different laccase enzymes with the SpyTag-SpyCatcher peptide-protein duo. The designed biofilm materials were successful in degrading ciprofloxacin, as demonstrated with the data obtained from mass spectrometry analysis and cell viability assays.

RNA-Based Sensor Systems for Affordable Diagnostics in the Age of Pandemics.

In the era of the COVID-19 pandemic, the significance of point-of-care (POC) diagnostic tools has become increasingly vital, driven by the need for quick and precise virus identification. RNA-based sensors, particularly toehold sensors, have emerged as promising candidates for POC detection systems due to their selectivity and sensitivity. Toehold sensors operate by employing an RNA switch that changes the conformation when it binds to a target RNA molecule, resulting in a detectable signal. This review focuses on the development and deployment of RNA-based sensors for POC viral RNA detection with a particular emphasis on toehold sensors. The benefits and limits of toehold sensors are explored, and obstacles and future directions for improving their performance within POC detection systems are presented. The use of RNA-based sensors as a technology for rapid and sensitive detection of viral RNA holds great potential for effectively managing (dealing/coping) with present and future pandemics in resource-constrained settings.

Screening Peptide Drug Candidates To Neutralize Whole Viral Agents: A Case Study with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

The COVID-19 pandemic revealed the need for therapeutic and pharmaceutical molecule development in a short time with different approaches. Although boosting immunological memory by vaccination was the quickest and robust strategy, still medication is required for the immediate treatment of a patient. A popular approach is the mining of new therapeutic molecules. Peptide-based drug candidates are also becoming a popular avenue. To target whole pathogenic viral agents, peptide libraries can be employed. With this motivation, we have used the 12mer M13 phage display library for selecting SARS-CoV-2 targeting peptides as potential neutralizing molecules to prevent viral infections. Panning was applied with four iterative cycles to select SARS-CoV-2 targeting phage particles displaying 12-amino acid-long peptides. Randomly selected peptide sequences were synthesized by a solid-state peptide synthesis method. Later, selected peptides were analyzed by the quartz crystal microbalance method to characterize their molecular interaction with SARS-CoV-2's S protein. Finally, the neutralization activity of the selected peptides was probed with an in-house enzyme-linked immunosorbent assay. The results showed that scpep3, scpep8, and scpep10 peptides have both binding and neutralizing capacity for S1 protein as a candidate for therapeutic molecule. The results of this study have a translational potential with future in vivo and human studies.

Making the Next Generation of Therapeutics: mRNA Meets Synthetic Biology.

The development of mRNA-based therapeutics centers around the natural functioning of mRNA molecules to provide the genetic information required for protein translation. To improve the efficacy of these therapeutics and minimize side effects, researchers can focus on the features of mRNA itself or the properties of the delivery agent to achieve the desired response. The tools considered for mRNA manipulation can be improved in terms of targetability, tunability, and translatability to medicine. While ongoing studies are dedicated to improving conventional approaches, innovative approaches can also be considered to unleash the full potential of mRNA-based therapeutics. Here, we discuss the opportunities that emerged from introducing synthetic biology to mRNA therapeutics. It includes a discussion of modular self-assembled mRNA nanoparticles, logic gates on a single mRNA molecule, and other possibilities.

Glycosylation Circuit Enables Improved Catalytic Properties for Recombinant Alkaline Phosphatase.

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins. Here, we engineered a Campylobacter jejuni N-linked glycosylation machinery by overexpressing one of the core glycosylation-related enzymes, PgIB, to increase the glycosylation rate. It has been previously shown that by utilizing N-linked glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. We utilized N-linked glycosylation using an engineered glycosylation pathway to glycosylate a model enzyme, the alkaline phosphatase (ALP) enzyme in Escherichia coli. We have investigated the effects of glycosylation on enzyme properties. Considering the glycosylation mechanism is highly dependent on accessibility of the glycosylation tag, ALP constructs carrying the glycosylation tag at different locations of the gene have been constructed, and glycosylation rates have been calculated. Our results showed that, upon glycosylation, ALP features in terms of thermostability, proteolytic stability, tolerance to suboptimal pH, and denaturing conditions are dramatically improved. The results indicated that the N-linked glycosylation mechanism can be employed for protein manipulation for industrial applications.

Synergistic Screening of Peptide-Based Biotechnological Drug Candidates for Neurodegenerative Diseases Using Yeast Display and Phage Display.

Peptide therapeutics are robust and promising molecules for treating diverse disease conditions. These molecules can be developed from naturally occurring or mimicking native peptides, through rational design and peptide libraries. We developed a new platform for the rapid screening of the peptide therapeutics for disease targets. In the course of the study, we aimed to employ our platform to screen a new generation of peptide therapeutic candidates against aggregation-prone protein targets. Two peptide drug candidates were screened for protein aggregation-prone diseases, namely, Parkinson's and Alzheimer's diseases. Currently, there are several therapeutic applications that are only effective in masking or slowing down symptom development. Nonetheless, different approaches are being developed for inhibiting amyloid aggregation in the secondary nucleation phase, which is critical for amyloid fibril formation. Instead of targeting secondary nucleated protein structures, we tried to inhibit the aggregation of monomeric amyloid units as a novel approach for halting the disease condition. To achieve this, we combined yeast surface display and phage display library platforms. We expressed α-synuclein, amyloid ß40, and amyloid ß42 on the yeast surface, and we selected peptides by using phage display library. After iterative biopanning cycles optimized for yeast cells, several peptides were selected for interaction studies. All of the peptides have been used for in vitro characterization methods, which are quartz crystal microbalance-dissipation (QCM-D) measurement, atomic force microscopy (AFM) imaging, dot-blotting, and ThT assay, and some of them have yielded promising results in blocking fibrillization. The rest of the peptides, although, interacted with amyloid units which made them usable as a sensor molecule candidate. Therefore, peptides selected by yeast surface display and phage display library combination are good choice for diverse disease-prone molecule inhibition, particularly those inhibiting fibrillization. Additionally, these selected peptides can be used as drugs and sensors to detect diseases quickly and halt disease progression.

Multiplexed cell-based diagnostic devices for detection of renal biomarkers.

The number of synthetic biology-based solutions employed in the medical industry is growing every year. The whole cell biosensors being one of them, have been proven valuable tools for developing low-cost, portable, personalized medicine alternatives to conventional techniques. Based on this concept, we targeted one of the major health problems in the world, Chronic Kidney Disease (CKD). To do so, we developed two novel biosensors for the detection of two important renal biomarkers: urea and uric acid. Using advanced gene expression control strategies, we improved the operational range and the response profiles of each biosensor to meet clinical specifications. We further engineered these systems to enable multiplexed detection as well as an AND-logic gate operating system. Finally, we tested the applicability of these systems and optimized their working dynamics inside complex medium human blood serum. This study could help the efforts to transition from labor-intensive and expensive laboratory techniques to widely available, portable, low-cost diagnostic options.

Highly Potent Peptide Therapeutics To Prevent Protein Aggregation in Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disorder resulting from a significant amplification of CAG repeats in exon 1 of the Huntingtin (Htt) gene. More than 36 CAG repeats result in the formation of a mutant Htt (mHtt) protein. These amino-terminal mHtt fragments lead to the formation of misfolded proteins, which then form aggregates in the relevant brain regions. Therapies that can delay the progression of the disease are imperative to halting the course of the disease. Peptide-based drug therapies provide such a platform. Inhibitory peptides were screened against monomeric units of both wild type (Htt(Q25)) and mHtt fragments, Htt(Q46) and Htt(Q103). Fibril kinetics was studied by utilizing the Thioflavin T (ThT) assay. Atomic force microscopy was also used to study the influence of the peptides on fibril formation. These experiments demonstrate that the chosen peptides suppress the formation of fibrils in mHtt proteins and can provide a therapeutic lead for further optimization and development.

Peptide-mediated constructs of quantum dot nanocomposites for enzymatic control of nonradiative energy transfer.

A bottom-up approach for constructing colloidal semiconductor quantum dot (QDot) nanocomposites that facilitate nonradiative Förster-type resonance energy transfer (FRET) using polyelectrolyte peptides was proposed and realized. The electrostatic interaction of these polypeptides with altering chain lengths was probed for thermodynamic, structural, and morphological aspects. The resulting nanocomposite film was successfully cut with the protease by digesting the biomimetic peptide layer upon which the QDot assembly was constructed. The ability to control photoluminescence decay lifetime was demonstrated by proteolytic enzyme activity, opening up new possibilities for biosensor applications.

Monitoring Molecular Assembly of Biofilms Using Quartz Crystal Microbalance with Dissipation (QCM-D).

The structure and the functionality of biofilm proteins, the main components of the extracellular matrix, can be tuned by protein engineering. The use of binding kinetics data has been demonstrated in the characterization of recombinantly produced biofilm proteins to control their behavior on certain surfaces or under certain conditions. Quartz crystal microbalance with dissipation monitoring (QCM-D) allows measuring the change in resonance frequency and the energy loss and distribution upon the interaction of molecules with the surface. The characterization of the molecular assembly of curli biofilm proteins on different surfaces using QCM-D is presented here as a detailed protocol. The experimental procedure detailed in this chapter can be applied and modified for other biofilm proteins or subunits to determine their surface adsorption and kinetic binding characteristics.

A Recombinase-Based Genetic Circuit for Heavy Metal Monitoring.

Rapid progress in the genetic circuit design enabled whole-cell biosensors (WCBs) to become prominent in detecting an extensive range of analytes with promise in many fields, from medical diagnostics to environmental toxicity assessment. However, several drawbacks, such as high background signal or low precision, limit WCBs to transfer from proof-of-concept studies to real-world applications, particularly for heavy metal toxicity monitoring. For an alternative WCB module design, we utilized Bxb1 recombinase that provides tight control as a switch to increase dose-response behavior concerning leakiness. The modularity of Bxb1 recombinase recognition elements allowed us to combine an engineered semi-specific heat shock response (HSR) promoter, sensitive to stress conditions including toxic ions such as cadmium, with cadmium resistance regulatory elements; a cadmium-responsive transcription factor and its cognitive promoter. We optimized the conditions for the recombinase-based cadmium biosensor to obtain increased fold change and shorter response time. This system can be expanded for various heavy metals to make an all-in-one type of WCB, even using semi-specific parts of a sensing system.

A Genetically Engineered Biofilm Material for SARS-CoV-2 Capturing and Isolation.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is continuously infecting people all around the world since its outbreak in 2019. Studies for numerous infection detection strategies are continuing. The sensitivity of detection methods is crucial to separate people with mild infections from people who are asymptomatic. In this sense, a strategy that would help to capture and isolate the SARS-CoV-2 virus prior to tests can be effective and beneficial. To this extent, genetically engineered biomaterials grounding from the biofilm protein of Escherichia coli are beneficial due to their robustness and adaptability to various application areas. Through functionalizing the E. coli biofilm protein, diverse properties can be attained such as enzyme display, nanoparticle production, and medical implant structures. Here, E. coli species are employed to express major curli protein CsgA and Griffithsin (GRFT) as fusion proteins, through a complex formation using SpyTag and SpyCatcher domains. In this study, a complex system with a CsgA scaffold harboring the affinity of GRFT against Spike protein to capture and isolate SARS-CoV-2 virus is successfully developed. It is shown that the hybrid recombinant protein can dramatically increase the sensitivity of currently available lateral flow assays for Sars-CoV-2 diagnostics.

A Highly Potent SARS-CoV-2 Blocking Lectin Protein.

The COVID-19 (coronavirus disease-19) pandemic affected more than 180 million people around the globe, causing more than five million deaths as of January 2022. SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), the new coronavirus, has been identified as the primary cause of the infection. The number of vaccinated people is increasing; however, prophylactic drugs are highly demanded to ensure secure social contact. A number of drug molecules have been repurposed to fight against SARS-CoV-2, and some of them have been proven to be effective in preventing hospitalization or ICU admissions. Here, we demonstrated griffithsin (GRFT), a lectin protein, to block the entry of SARS-CoV-2 and its variants, Delta and Omicron, into the Vero E6 cell lines and IFNAR-/- mouse models by attaching to the spike protein of SARS-CoV-2. Given the current mutation frequency of SARS-CoV-2, we believe that GRFT protein-based drugs will have a high impact in preventing the transmission of both the Wuhan strain as well as any other emerging variants, including Delta and Omicron variants, causing the high-speed spread of COVID-19.

In vitro labeling of hydroxyapatite minerals by an engineered protein.

Biological and biomimetic synthesis of inorganics have been a major focus in hard tissue engineering as well as in green processing of advanced materials. Among the minerals formed by organisms, calcium phosphate mineralization is studied extensively to understand the formation of mineral-rich tissues. Herein, we report an engineered fusion protein that not only targets calcium phosphate minerals but also allows monitoring of biomineralization. To produce the bi-functional fusion protein, nucleotide sequence encoding combinatorially selected hydroxyapatite-binding peptides (HABP) was genetically linked to the 3' end of the open reading frame of green fluorescence protein (GFPuv) and successfully expressed in Escherichia coli. The fluorescence and binding activities of the bi-functional proteins were characterized by, respectively, using fluorescence microscopy and quartz crystal microbalance spectroscopy. The utility of GFPuv-HABP fusion protein was assessed for both time-wise monitoring of mineralization and the visualization of the mineralized tissues. We used an alkaline phosphatase-based reaction to control phosphate release, thereby mimicking biological processes, to monitor calcium phosphate mineralization. The increase in mineral amount was observed using the fusion protein at different time points. GFPuv-HABP1 was also used for efficient fluorescence labeling of mineralized regions on the extracted human incisors. Our results demonstrate a simple and versatile application of inorganic-binding peptides conjugated with bioluminescence proteins as bi-functional bioimaging molecular probes that target mineralization, and which can be employed to a wide range of biomimetic processing and cell-free tissue engineering.