Rapid Characterization of Genetic Parts with Cell-free Systems.

Characterizing and cataloging genetic parts are critical to the design of useful genetic circuits. Having well-characterized parts allows for the fine-tuning of genetic circuits, such that their function results in predictable outcomes. With the growth of synthetic biology as a field, there has been an explosion of genetic circuits that have been implemented in microbes to execute functions pertaining to sensing, metabolic alteration, and cellular computing.

Rapid Visual Authentication Based on DNA Strand Displacement.

Novel ways to track and verify items of a high value or security is an ever-present need. Taggants made from deoxyribonucleic acid (DNA) have several advantageous properties, such as high information density and robust synthesis; however, existing methods require laboratory techniques to verify, limiting applications. Here, we leverage DNA nanotechnology to create DNA taggants that can be validated in the field in seconds to minutes with a simple equipment.

Impact of Porous Matrices and Concentration by Lyophilization on Cell-Free Expression.

Cell-free expression systems have drawn increasing attention as a tool to achieve complex biological functions outside of the cell. Several applications of the technology involve the delivery of functionality to challenging environments, such as field-forward diagnostics or point-of-need manufacturing of pharmaceuticals. To achieve these goals, cell-free reaction components are preserved using encapsulation or lyophilization methods, both of which often involve an embedding of components in porous matrices like paper or hydrogels.

Detection of Chemical Warfare Agents by Colorimetric Sensor Arrays.

We report the successful use of colorimetric arrays to identify chemical warfare agents (CWAs). Methods were developed to interpret and analyze a 73-indicator array with an entirely automated workflow. Using a cross-validated first-nearest-neighbor algorithm for assessing detection and identification performances on 632 exposures, at 30 min postexposure we report, on average, 78% correct chemical identification, 86% correct class-level identification, and 96% correct red light/green light (agent versus non-agent) detection.

Quantification of Interlaboratory Cell-Free Protein Synthesis Variability.

Cell-free protein synthesis (CFPS) platforms, once primarily a research tool to produce difficult to express proteins, are increasingly being pursued by the synthetic biology community for applications including biomanufacturing, rapid screening systems, and field-ready sensors. While consistency within individual studies is apparent in the literature, challenges with reproducing results between laboratories, or even between individuals within a laboratory, are discussed openly by practitioners. As the field continues to grow and move toward applications, a quantitative understanding of expected variability for CFPS and the relative contribution of underlying sources will become increasingly important.

A method for cost-effective and rapid characterization of engineered T7-based transcription factors by cell-free protein synthesis reveals insights into the regulation of T7 RNA polymerase-driven expression.

The T7 bacteriophage RNA polymerase (T7 RNAP) serves as a model for understanding RNA synthesis, as a tool for protein expression, and as an actuator for synthetic gene circuit design in bacterial cells and cell-free extract. T7 RNAP is an attractive tool for orthogonal protein expression in bacteria owing to its compact single subunit structure and orthogonal promoter specificity. Understanding the mechanisms underlying T7 RNAP regulation is important to the design of engineered T7-based transcription factors, which can be used in gene circuit design.

Detection and tracking of a novel genetically tagged biological simulant in the environment.

A variant of Bacillus thuringiensis subsp. kurstaki containing a single, stable copy of a uniquely amplifiable DNA oligomer integrated into the genome for tracking the fate of biological agents in the environment was developed. The use of genetically tagged spores overcomes the ambiguity of discerning the test material from pre-existing environmental microflora or from previously released background material.

Evaluation of handheld assays for the detection of ricin and staphylococcal enterotoxin B in disinfected waters.

Development of a rapid field test is needed capable of determining if field supplies of water are safe to drink by the warfighter during a military operation. The present study sought to assess the effectiveness of handheld assays (HHAs) in detecting ricin and Staphylococcal Enterotoxin B (SEB) in water. Performance of HHAs was evaluated in formulated tap water with and without chlorine, reverse osmosis water (RO) with chlorine, and RO with bromine.

An evaluation of suspicious powder screening tools for first responders.

Field screening tools are required which would allow first responders to quickly ascertain if a suspicious powder poses a potential threat necessitating additional testing for biological pathogens such as Bacillus anthracis. In this study, three commercially available generic screening technologies were evaluated for the effectiveness to accurately differentiate between a hoax powder and a true biological threat. The BioCheck Kit was able to detect the following biological agents 1 x 10(8)CFU of B.

Surface sampling of spores in dry-deposition aerosols.

The ability to reliably and reproducibly sample surfaces contaminated with a biological agent is a critical step in measuring the extent of contamination and determining if decontamination steps have been successful. The recovery operations following the 2001 attacks with Bacillus anthracis spores were complicated by the fact that no standard sample collection format or decontamination procedures were established. Recovery efficiencies traditionally have been calculated based upon biological agents which were applied to test surfaces in a liquid format and then allowed to dry prior to sampling tests, which may not be best suited for a real-world event with aerosolized biological agents.

Automated screening for biological weapons in homeland defense.

Biological threat detection programs that collect air samples and monitor for large-scale release of biowarfare agents generate large numbers of samples that must be quickly and accurately screened for the presence of biological agents. An impediment to the rapid analysis of large numbers of environmental biological samples is that manual laboratory processes are time-consuming and require resources to maintain infrastructure, trained personnel, and adequate supplies of testing reagents. An ideal screening system would be capable of processing multiple samples rapidly, cost-effectively, and with minimal personnel.

Evaluation of the Biological Sampling Kit (BiSKit) for large-area surface sampling.

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs.

Automated biological agent testing systems.

The events of September 11, 2001 revealed a need for regular monitoring of infrastructure systems. Efficient aerosol collection systems are currently in place in a variety of settings in order to sample the immediate surroundings. However, the consequence of regular monitoring is an exponential increase in the number of samples requiring rapid analysis.

Detection of Francisella tularensis within infected mouse tissues by using a hand-held PCR thermocycler.

The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen.