Screening putative polyester polyurethane degrading enzymes with semi-automated cell-free expression and nitrophenyl probes.

Cell-free expression (CFE) has shown recent utility in prototyping enzymes for discovery efforts. In this work, CFE is demonstrated as an effective tool to screen putative polyester polyurethane degrading enzyme sequences sourced from metagenomic analysis of biofilms prospected on aircraft and vehicles. An automated fluid handler with a controlled temperature block is used to assemble the numerous 30 µL CFE reactions to provide more consistent results over human assembly. In sum, 13 putative hydrolase enzymes from the biofilm organisms as well as a previously verified, polyester-degrading cutinase were expressed using in-house E. coli extract and minimal linear templates. The enzymes were then tested for esterase activity directly in extract using nitrophenyl conjugated substrates, showing highest sensitivity to shorter substrates (4-nitrophenyl hexanoate and 4-nNitrophenyl valerate). This screen identified 10 enzymes with statistically significant activities against these substrates; however, all were lower in measured relative activity, on a CFE volume basis, to the established cutinase control. This approach portends the use of CFE and reporter probes to rapidly prototype, screen and design for synthetic polymer degrading enzymes from environmental consortia. Graphical Abstract.

Lipid-Functionalized Single-Walled Carbon Nanotubes as Probes for Screening Cell Wall Disruptors.

Membrane-active molecules are of great importance to drug delivery and antimicrobials applications. While the ability to prototype new membrane-active molecules has improved greatly with the advent of automated chemistries and rapid biomolecule expression techniques, testing methods are still limited by throughput, cost, and modularity. Existing methods suffer from feasibility constraints of working with pathogenic living cells and by intrinsic limitations of model systems. Herein, we demonstrate an abiotic sensor that uses semiconducting single-walled carbon nanotubes (SWCNTs) as near-infrared fluorescent transducers to report membrane interactions. This sensor is composed of SWCNTs aqueously suspended in lipid, creating a cylindrical, bilayer corona; these SWCNT probes are very sensitive to solvent access (changes in permittivity) and thus report morphological changes to the lipid corona by modulation of fluorescent signals, where binding and disruption are reported as brightening and attenuation, respectively. This mechanism is first demonstrated with chemical and physical membrane-disruptive agents, including ethanol and sodium dodecyl sulfate, and application of electrical pulses. Known cell-penetrating and antimicrobial peptides are then used to demonstrate how the dynamic response of these sensors can be deconvoluted to evaluate different parallel mechanisms of interaction. Last, SWCNTs functionalized in several different bacterial lipopolysaccharides (Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli) are used to evaluate a panel of known membrane-disrupting antimicrobials to demonstrate that drug selectivity can be assessed by suspension of SWCNTs with different membrane materials.

Screening Enzymatic Degradation of Polyester Polyurethane with Fluorescent Single-walled Carbon Nanotube and Polymer Nanoparticle Conjugates.

Enzymatic biodegradation is a promising method to reclaim plastic materials. However, to date, a high-throughput method for screening potential enzyme candidates for biodegradation is still lacking. Here, we propose a single-walled carbon nanotube (SWCNT) fluorescence sensor for screening the enzymatic degradation of polyester polyurethane nanoparticles. Through wrapping the SWCNT with cationic chitosan, an electrostatic bond is formed between the SWCNT and Impranil, a widely applied model substrate of polyester polyurethane. As Impranil is being degraded by the enzymes, a characteristic quenching at a short reaction time followed by a brightening at a longer reaction time in the fluorescence signal is observed. The time-dependent fluorescence response is compared with turbidity measurement, and we conclude that the brightening in fluorescence results from the binding of the degradation product with the SWCNT. The proposed SWCNT sensor design has the potential to screen enzyme candidates for selective degradation of other plastic particles.

Characterizing the Interactions of Cell-Membrane-Disrupting Peptides with Lipid-Functionalized Single-Walled Carbon Nanotubes.

Lipid-functionalized single-walled carbon nanotubes (SWNTs) have garnered significant interest for their potential use in a wide range of biomedical applications. In this work, we used molecular dynamics simulations to study the equilibrium properties of SWNTs surrounded by the phosphatidylcholine (POPC) corona phase and their interactions with three cell membrane disruptor peptides: colistin, TAT peptide, and crotamine-derived peptide. Our results show that SWNTs favor asymmetrical positioning within the POPC corona, so that one side of the SWNT, covered by the thinnest part of the corona, comes in contact with charged and polar functional groups of POPC and water. We also observed that colistin and TAT insert deeply into the POPC corona, while crotamine-derived peptide only adsorbs to the corona surface. In separate simulations, we show that three examined peptides exhibit similar insertion and adsorption behaviors when interacting with POPC bilayers, confirming that peptide-induced perturbations to POPC in conjugates and bilayers are similar in nature and magnitude. Furthermore, we observed correlations between the peptide-induced structural perturbations and the near-infrared emission of the lipid-functionalized SWNTs, which suggest that the optical signal of the conjugates transduces the morphological changes in the lipid corona. Overall, our findings indicate that lipid-functionalized SWNTs could serve as simplified cell membrane model systems for prescreening of new antimicrobial compounds that disrupt cell membranes.

Contact-Free, Passive, Electromagnetic Resonant Sensors for Enclosed Biomedical Applications: A Perspective on Opportunities and Challenges.

Inexpensive and accurate tools for monitoring conditions in enclosed environments (through garments, bandages, tissue, etc.) have been a long-standing goal of medicine. Passive resonant sensors are a promising solution for such wearable health sensors as well as off-body diagnostics. They are simple circuits with inherent inductance and capacitance (LC tank) that have a measurable resonant frequency. Changes in local parameters, e.g., permittivity or geometry, effect inductance and capacitance which cause a resonant frequency shift response. This signal transduction has been applied to several biomedical applications such as intracranial pressure, hemodynamics, epidermal hydration, etc. Despite these many promising applications presented in the literature, resonant sensors still do not see widespread adoption in biomedical applications, especially as wearable or embedded sensing devices. This perspective highlights some of the current challenges facing LC resonant sensors in biomedical applications, such as positional sensitivity, and potential strategies that have been developed to overcome them. An outlook on adoption in medicine and health monitoring is presented, and a perspective is given on next steps for research in this field.

Characterizing the Interactions of Cell Membrane-Disrupting Peptides with Lipid-Functionalized Single-Walled Carbon Nanotube Systems for Antimicrobial Screening.

Lipid-functionalized single-walled carbon nanotubes (SWNTs) have garnered significant interest for their potential use in a wide range of biomedical applications. In this work, we used molecular dynamics simulations to study the equilibrium properties of SWNTs surrounded by the phosphatidylcholine (POPC) corona phase, and their interactions with three cell membrane disruptor peptides: colistin, TAT peptide, and crotamine-derived peptide. Our results show that SWNTs favor asymmetrical positioning within the POPC corona, so that one side of the SWNT, covered by the thinnest part of the corona, comes in contact with charged and polar functional groups of POPC and water. We also observed that colistin and TAT insert deeply into POPC corona, while crotamine-derived peptide only adsorbs to the corona surface. Compared to crotamine-derived peptide, colistin and TAT also induce larger perturbations in the thinnest region of the corona, by allowing more water molecules to directly contact the SWNT surface. In separate simulations, we show that three examined peptides exhibit similar insertion and adsorption behaviors when interacting with POPC bilayers, confirming that peptide-induced perturbations to POPC in conjugates and bilayers are similar in nature and magnitude. Furthermore, we observed correlations between the peptide-induced structural perturbations and the near-infrared emission of the lipid-functionalized SWNTs, which suggest that the optical signal of the conjugates transduces the morphological changes in the lipid corona. Overall, our findings indicate that lipid-functionalized SWNTs could serve as simplified cell membrane model systems for pre-screening of new antimicrobial compounds that disrupt cell membranes.

Rapid Characterization of Solid Tumors Using Resonant Sensors.

Cancer continues to be a significant cause of non-traumatic pediatric mortality. Diagnosis of pediatric solid tumors is paramount to prescribing the correct treatment regimen. Recent efforts have focused on non-invasive methods to obtain tumor tissues, but one of the challenges encountered is the ability to obtain an adequate amount of viable tissue. In this study, a wireless, inductor-capacitor (LC) sensor was employed to detect relative permittivity of pediatric tumor tissues. There is a comparison of resonant frequencies of tumor tissues between live versus dead tissues, the primary tumor tissue versus tissue from the organs of origin or metastasis, and treated versus untreated tumors. The results show significant shifts in resonant frequencies between the comparison groups. Dead tissues demonstrated a significant shift in resonant frequencies compared to alive tissues. There were significant differences between the resonant frequencies of normal tissues versus tumor tissues. Resonant frequencies were also significantly different between primary tumors compared to their respective metastases. These data indicate that there are potential clinical applications of LC technology in the detection and diagnosis of pediatric solid tumors.

Toward Mail-in-Sensors for SARS-CoV-2 Detection: Interfacing Gel Switch Resonators with Cell-Free Toehold Switches.

The COVID-19 pandemic has emphasized the importance of widespread testing to control the spread of infectious diseases. The rapid development, scale-up, and deployment of viral and antibody detection methods since the beginning of the pandemic have greatly increased testing capacity. Desirable attributes of detection methods are low product costs, self-administered protocols, and the ability to be mailed in sealed envelopes for the safe analysis and subsequent logging to public health databases. Herein, such a platform is demonstrated with a screen-printed, inductor-capacitor (LC) resonator as a transducer and a toehold switch coupled with cell-free expression as the biological selective recognition element. In the presence of the N-gene from SARS-CoV-2, the toehold switch relaxes, protease enzyme is expressed, and it degrades a gelatin switch that ultimately shifts the resonant frequency of the planar resonant sensor. The gelatin switch resonator (GSR) can be analyzed through a sealed envelope allowing for assessment without the need for careful sample handling with personal protective equipment or the need for workup with other reagents. The toehold switch used in this sensor demonstrated selectivity to SARS-CoV-2 virus over three seasonal coronaviruses and SARS-CoV-1, with a limit of detection of 100 copies/µL. The functionality of the platform and assessment in a sealed envelope with an automated scanner is shown with overnight shipment, and further improvements are discussed to increase signal stability and further simplify user protocols toward a mail-in platform.

Single-Walled Carbon Nanotube Probes for the Characterization of Biofilm-Degrading Enzymes Demonstrated against Pseudomonas aeruginosa Extracellular Matrices.

Hydrolase co-therapies that degrade biofilm extracellular polymeric substances (EPS) allow for a better diffusion of antibiotics and more effective treatment; current methods for quantitatively measuring the enzymatic degradation of EPS are not amendable to high-throughput screening. Herein, we present biofilm EPS-functionalized single-walled carbon nanotube (SWCNT) probes for rapid screening of hydrolytic enzyme selectivity and activity on EPS. The extent of biofilm EPS degradation is quantified by monitoring the quenching of the SWCNT fluorescence. We used this platform to screen 16 hydrolases with varying bond breaking selectivity against a panel of wild-type Pseudomonas aeruginosa and mutants deficient or altered in one or more EPS. Next, we performed concentration-dependent studies of six enzymes on two common strains found in cystic fibrosis (CF) environments and, for each enzyme, extracted three first-order rate constants and their relative contributions by fitting a parallel, multi-site degradation model, with a good model fit (R2 from 0.65 to 0.97). Reaction rates (turnover rates) are dependent on the enzyme concentration and range from 6.67 × 10-11 to 2.80 × 10-3 *s-1 per mg/mL of enzymes. Lastly, we confirmed findings from this new assay using an established crystal-violet staining assay for a subset of hydrolase panels. In summary, our work shows that this modular sensor is amendable to the high-throughput screening of EPS degradation, thereby improving the rate of discovery and development of novel hydrolases.

Effects of Sequence Features on Machine-Learned Enzyme Classification Fidelity.

Assigning enzyme commission (EC) numbers using sequence information alone has been the subject of recent classification algorithms where statistics, homology and machine-learning based methods are used. This work benchmarks performance of a few of these algorithms as a function of sequence features such as chain length and amino acid composition (AAC). This enables determination of optimal classification windows for de novo sequence generation and enzyme design. In this work we developed a parallelization workflow which efficiently processes >500,000 annotated sequences through each candidate algorithm and a visualization workflow to observe the performance of the classifier over changing enzyme length, main EC class and AAC. We applied these workflows to the entire SwissProt database to date (n = 565245) using two, locally installable classifiers, ECpred and DeepEC, and collecting results from two other webserver-based tools, Deepre and BENZ-ws. It is observed that all the classifiers exhibit peak performance in the range of 300 to 500 amino acids in length. In terms of main EC class, classifiers were most accurate at predicting translocases (EC-6) and were least accurate in determining hydrolases (EC-3) and oxidoreductases (EC-1). We also identified AAC ranges that are most common in the annotated enzymes and found that all classifiers work best in this common range. Among the four classifiers, ECpred showed the best consistency in changing feature space. These workflows can be used to benchmark new algorithms as they are developed and find optimum design spaces for the generation of new, synthetic enzymes.

Rapid, Enzymatic Methods for Amplification of Minimal, Linear Templates for Protein Prototyping using Cell-Free Systems.

This protocol describes the design of a minimal DNA template and the steps for enzymatic amplification, enabling rapid prototyping of assayable proteins in less than 24 h using cell-free expression. After receiving DNA from a vendor, the gene fragment is PCR-amplified, cut, circularized, and cryo-banked. A small amount of the banked DNA is then diluted and amplified significantly (up to 106x) using isothermal rolling circle amplification (RCA). RCA can yield microgram quantities of the minimal expression template from picogram levels of starting material (mg levels if all starting synthetic fragment is used). In this work, a starting amount of 20 pg resulted in 4 µg of the final product. The resulting RCA product (concatemer of the minimal template) can be added directly to a cell-free reaction with no purification steps. Due to this method being entirely PCR-based, it may enable future high-throughput screening efforts when coupled with automated liquid handling systems.

Inexpensive Near-Infrared Fluorimeters: Enabling Translation of nIR-Based Assays to the Field.

The practical impact of analytical probes that transduce in the near-infrared (nIR) has been dampened by the lack of cost-effective and portable nIR fluorimeters. Herein, we demonstrate straightforward designs for an inexpensive microplate reader and a portable fluorimeter. These instruments require minimally complex machining and fabrication and operate with an open-source programming language (Python). Complete wiring diagrams, assembly diagrams, and scripts are provided. To demonstrate the utility of these two instruments, we performed high-throughput and field-side measurements of soil samples to evaluate the effect of soil management strategies on extracellular proteolytic, cellulolytic, and lignin-modifying activities. This was accomplished with fluorescent enzyme probes that utilized uniquely sensitive transducers exclusive to the nIR spectrum, single-walled carbon nanotubes. We also used the portable fluorimeter to evaluate spatial variations of proteolytic activity within individual field plots, while minimizing the effects of soil storage and handling. These demonstrations indicate the utility of these fluorimeters for translating analytical probes that operate in the nIR beyond the laboratory and into actual use.

Anaerobic Conditioning of E. coli Cell Lysate for Enhanced In Vitro Protein Synthesis.

Cell-free protein expression (CFPS) from E. coli cell lysate is an established chemical biology technique. Common efforts to improve synthesis capacity, such as strain engineering and process improvements, have overlooked the opportunity to increase productivity by reducing the dependence on limited, dissolved oxygen. Here we demonstrate conditioning E. coli cells for anaerobic respiration which increases the initial protein expression rate up to 4-fold and increases titer by 50% as compared to traditional aerobic cell lysate when using sfGFP as a reporter protein in CFPS reactions run at atmospheric conditions. This enhancement is even more significant when run in an oxygen-depleted environment, where anaerobic respiration preconditioned cells increase yield when supplemented with nitrite as a terminal electron acceptor (TEA). Furthermore, we test knockout mutants to determine key proteins responsible for enhancing the anaerobically prepared CFPS lysate. Further improvements could be made in preconditioning cells by increasing expression levels of critical pathway enzymes or by screening other TEA.

Optimization of E. Coli Tip-Sonication for High-Yield Cell-Free Extract using Finite Element Modeling.

Optimal tip sonication settings, namely tip position, input power, and pulse durations, are necessary for temperature sensitive procedures like preparation of viable cell extract. In this paper, the optimum tip immersion depth (20-30% height below the liquid surface) is estimated which ensures maximum mixing thereby enhancing thermal dissipation of local cavitation hotspots. A finite element (FE) heat transfer model is presented, validated experimentally with (R2 > 97%) and used to observe the effect of temperature rise on cell extract performance of E. coli BL21 DE3 star strain and estimate the temperature threshold. Relative yields in the top 10% are observed for solution temperatures maintained below 32°C; this reduces below 50% relative yield at temperatures above 47°C. A generalized workflow for direct simulation using the COMSOL code as well as master plots for estimation of sonication parameters (power input and pulse settings) is also presented.

Monitoring Wound Health through Bandages with Passive LC Resonant Sensors.

This paper details a passive, inductor-capacitor (LC) resonant sensor embedded in a commercial dressing for low-cost, contact-free monitoring of a wound; this would enable tracking of the healing process while keeping the site closed and sterile. Spiral LC resonators were fabricated from flexible, copper-coated polyimide and interrogated using external reader antennas connected to a two-port vector network analyzer; the forward transmission scattering parameter (S21) magnitude was collected, and the resonant frequency (MHz) and the peak-to-peak amplitude of the resonant feature were identified. These increase during the healing process as the permittivity and conductivity of the tissue change. The sensor was first tested on gelatin-based tissue-mimicking phantoms that simulate layers of muscle, blood, fat, and skin at varying phases of wound healing. Finite element modeling was also used to verify the empirical results based on the expected variations in dielectric properties of the tissue. The performance of the resonant sensors for in vivo applications was investigated by conducting animal studies using canine patients that presented with a natural wound as well as a controlled cohort of rat models with surgically administered wounds. Finally, transfer functions are presented that relate the resonant frequency to wound size using an exponential model (R2 = 0.58-0.96). The next steps in sensor design and fabrication as well as the reading platform to achieve the goal of a universal calibration curve are then discussed.

Automated classification of bacterial cell sub-populations with convolutional neural networks.

Quantification of phenotypic heterogeneity present amongst bacterial cells can be a challenging task. Conventionally, classification and counting of bacteria sub-populations is achieved with manual microscopy, due to the lack of alternative, high-throughput, autonomous approaches. In this work, we apply classification-type convolutional neural networks (cCNN) to classify and enumerate bacterial cell sub-populations (B. subtilis clusters). Here, we demonstrate that the accuracy of the cCNN developed in this study can be as high as 86% when trained on a relatively small dataset (81 images). We also developed a new image preprocessing algorithm, specific to fluorescent microscope images, which increases the amount of training data available for the neural network by 72 times. By summing the classified cells together, the algorithm provides a total cell count which is on parity with manual counting, but is 10.2 times more consistent and 3.8 times faster. Finally, this work presents a complete solution framework for those wishing to learn and implement cCNN in their synthetic biology work.

Design and validation of a frugal, automated, solid-phase peptide synthesizer.

Solid phase peptide synthesis (SPPS) has enabled widespread use of synthetic peptides in applications ranging from pharmaceuticals to materials science. The demand for synthetic peptides has driven recent efforts to produce automated SPPS synthesizers which utilize fluid-handling components common to chemistry laboratories to drive costs down to several thousand dollars. Herein, we describe the design and validation of a more 'frugal' SPPS synthesizer that uses inexpensive, consumer-grade fluid-handling components to achieve a prototype price point between US$300 and $600. We demonstrated functionality by preparing and characterizing peptides with a variety of distinct properties including binding functionality, nanoscale self-assembly, and oxidation-induced fluorescence. This system yielded micromoles of peptide at a cost of approximately $1/residue, a cost which may be further reduced by optimization and bulk purchasing.

Sweat monitoring beneath garments using passive, wireless resonant sensors interfaced with laser-ablated microfluidics.

Sweat loss can help determine hydration status of individuals working in harsh conditions, which is especially relevant to those who wear thick personal protective equipment (PPE) such as firefighters. A wireless, passive, conformable sweat sensor sticker is described here that can be worn under and interrogated through thick clothing to simultaneously measure sweat loss volume and conductivity. The sticker consists of a laser-ablated, microfluidic channel and a resonant sensor transducer. The resonant sensor is wirelessly read with a handheld vector network analyzer coupled to two, co-planar, interrogation antennas that measure the transmission loss. A sweat proxy is used to fill the channels and it is determined that the sensor can orthogonally determine the sweat conductivity and volume filled in the channel via peak transmission loss magnitude and frequency respectively. A four-person study is then used to determine level of sensor variance caused by local tissue dielectric heterogeneity and sensor-reader orientation.

Methods to reduce variability in E. Coli-based cell-free protein expression experiments.

Cell-free protein synthesis (CFPS) is an established biotechnology tool that has shown great utility in many applications such as prototyping proteins, building genetic circuits, designing biosensors, and expressing cytotoxic proteins. Although CFPS has been widely deployed, the many, varied methods presented in the literature can be challenging for new users to adopt. From our experience and others who newly enter the field, one of the most frustrating aspects of applying CFPS as a laboratory can be the large levels of variability that are present within experimental replicates. Herein we provide a retrospective summary of CFPS methods that reduce variability significantly. These methods include optimized extract preparation, fully solubilizing the master mix components, and careful mixing of the reaction. These have reduced our coefficient of variation from 97.3% to 1.2%. Moreover, these methods allow complete novices (e.g. semester rotation undergraduate students) to provide data that is comparable to experienced users, thus allowing broader participation in this exciting research area.

Controlling Heterogeneity and Increasing Titer from Riboswitch-Regulated Bacillus subtilis Spores for Time-Delayed Protein Expression Applications.

Sporulated cells have potential as time-delayed expression chassis of proteins for applications such as "on-demand" biologics production, whole cell biosensors, or oral vaccines. However, the desired attributes of high expression rates and low product variances are difficult to maintain from germinated spores. In this work, we study the effect of an integrating vs theta-replicating plasmid in a wild-type Bacillus subtilis and two PolY mutants. The cells were engineered to produce a fluorescent reporter protein (RFP) under the control of a riboswitch activated by theophylline. This allowed for greater sensitivity to point mutations. The fluorescence and cell-growth curves were fit with a custom kinetic model, and a peak kinetic rate (LKPmax) was extracted for each clonal population (n = 30 for all cell, vector, and growth combinations). Plasmid-based expression yields higher (8.7×) expression rates because of an increased copy number of the expression cassette (10× over integrated). The variance of LKPmax values increased 2.1× after sporulation for the wild-type strain. This increase in variance from sporulation is very similar to what is observed with UV exposure. This effect can be partially mitigated by the use of PolY knockouts observed in suspended cell growths and adherent biofilms.