rapid-enzymatic-methods-for-amplification-minimal-linear-templates

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 ...

Iowa State University

Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.

Cell-free protein synthesis (CFPS) has become an established biotechnology tool for rapid protein expression. Over the past few decades many advances have elevated CFPS from a niche, low-efficiency system to one now capable of biomanufacturing custom proteins. Many research papers and reviews exist on the advances made in CFPS genetic template and cell extract preparation for use in E. coli systems. What is currently missing from the literature is a comprehensive review on the myriad of supplement recipes added to the CFPS reaction to support metabolism, transcription, and translation. This list of supplements has changed over the years, with a general drive towards greater simplification. Herein we provide a comprehensive list of the supplements used in CFPS, tracing major recipe classes as the field has evolved. We also provide an in-depth analysis of the proposed biochemical purpose each supplement has in the reaction. This review reveals the significance of correct supplements on overall CFPS productivity; however, the large range of supplements accommodated by CFPS also shows an inherent flexibility in the CFPS reaction as well as additional room for optimization and recipe simplification.

Cell-free supplement mixtures: Elucidating the history and biochemical utility of additives used to support in vitro protein synthesis in E. coli extract.

Noncontact methods to measure animal activity and physiology are necessary to monitor undisturbed states such as hibernation. Although some noncontact measurement systems are commercially available, they are often incompatible with realistic habitats, which feature freely moving animals in small, cluttered environments. A growing market of single-board computers, microcontrollers, and inexpensive sensors has made it possible to assemble bespoke integrated sensor systems at significantly lower price points. Herein, we describe a custom-built nesting box imager (NBI) that uses a single-board computer (Raspberry Pi) with a passive infrared (IR) motion sensor, silicon charge-coupled device (CCD), and IR camera CCD to monitor the activity, surface body temperature, and respiratory rate of the meadow jumping mouse during hibernation cycles. The data are logged up to 12 samples per minute and postprocessed using custom Matlab scripts. The entire unit can be built at a price point below US$400, which will be drastically reduced as IR (thermal) arrays are integrated into more consumer electronics and become less expensive.

Nesting box imager: Contact-free, real-time measurement of activity, surface body temperature, and respiratory rate applied to hibernating mouse models.

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  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 (LKP) was extracted for each clonal population ( = 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 LKP 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.

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

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.

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

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.

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

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.

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

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 (S) 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  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 ( = 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.

Monitoring Wound Health through Bandages with Passive LC Resonant Sensors.

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.

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

Cell-free protein expression (CFPS) from  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  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.

Nigel F. Reuel

Chemical and Biological Engineering

Iowa State University

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