Aptamers are often prone to nuclease digestion, which limits their utility in many biomedical applications. Here we describe a xeno-nucleic acid system based on α-L-threofuranosyl nucleic acid (TNA) that is completely refractory to nuclease digestion. The use of an engineered TNA polymerase permitted the isolation of functional TNA aptamers that bind to HIV reverse transcriptase (HIV RT) with K D 's of ∼0.4−4.0 nM. The aptamers were identified using a display strategy that provides a powerful genotype−phenotype linkage. The TNA aptamers remain active in the presence of nuclease and exhibit markedly higher thermal stability than monoclonal antibodies. The combined properties of biological stability, high binding affinity, and thermal stability make TNA aptamers a powerful system for the development of diagnostic and therapeutic agents.

Cell-free expression systems provide a suite of tools that are used in applications from sensing to biomanufacturing. One of these applications is genetic circuit prototyping, where the lack of cloning required and high degree of control over reaction components and conditions enables rapid testing of design candidates. Many studies have shown utility in the approach for characterizing genetic regulation elements, simple genetic circuit motifs, protein variants, or metabolic pathways. However, variability in cell-free expression systems is a known challenge, whether between individuals, labs, instruments, or batches of materials. While the issue of variability has begun to be quantified and explored, little effort has been put into understanding the implications of this variability. For genetic circuit prototyping, it is unclear when and how significantly variability in reaction activity will impact qualitative assessments of genetic components, e.g. relative activity between promoters. Here we explore this question by assessing DNA titrations of seven genetic circuits of increasing complexity using reaction conditions that ostensibly follow the same protocol but vary by person, instrument, and material batch. Though the raw activities vary widely between the conditions, by normalizing within each circuit across conditions, reasonably consistent qualitative performance emerges for the simpler circuits. For the most complex case involving expression of three proteins, we observe a departure from this qualitative consistency, offering a provisional cautionary line where normal variability may disrupt reliable reuse of prototyping results. Our results also suggest that a previously described closed loop controller circuit may help to mitigate such variability, encouraging further work to design systems that are robust to variability.

Lateral flow immunoassays (LFIs) are simple, point-of-care diagnostic devices used for detecting biological agents or other analytes of interest in a sample. LFIs are predominantly singleplex assays, interrogating one target analyte at a time. There is a need for multiplex LFI devices, e.g., a syndromic panel to differentiate pathogens causing diseases exhibiting similar symptoms. Multiplex LFI devices would be especially valuable in instances where sample quantity is limiting and reducing assay time and costs is critical. There are limitations to the design parameters and performance characteristics of a multiplex LFI assay with many horizontal test lines due to constraints in capillary flow dynamics. To address some of the performance issues, we have developed a spot array multiplex LFI using Braille format (hence called Blind Spot) and a sensor, MACAW (Modular Automated Colorimetric Analyses Widget), that can analyze and interpret the results. As a proof of concept, we created a multiplex toxin panel, for detecting three toxins, using two letter codes for each. The results indicated that the six-plex, triple toxin assay performs as well as singleplex assays. The sensor-based calls are better compared to human interpretation in discriminating and interpreting ambiguous test results correctly especially at lower antigen concentrations and from strips with blemishes.

No abstract

Cellular lysates capable of transcription and translation have become valuable tools for prototyping genetic circuits, screening engineered functional parts, and producing biological components. Here we report that lysates derived from Yersinia pestis CO92 − are functional and can utilize both the E. coli σ70 and the bacteriophage T7 promoter systems to produce green fluorescent protein (GFP). Because of the natural lifestyle of Y. pestis, lysates were produced from cultures grown at 21 °C, 26 °C, and 37 °C to mimic the infection cycle. Regardless of the promoter system the GFP production from 37 °C was the most productive and the 26 °C lysate was the least. When reactions are initiated with 5 nM of DNA, the GFP output of the 37 °C lysate is comparable with the productivity of other non-E. coli systems. The data we present demonstrate that, without genetic modification to enhance productivity, cell-free extracts from Y. pestis are functional and dependent on the temperature at which the bacterium was grown.

Recent advancements of engineered microbial systems capable of deployment in complex environments has enabled the creation of unique signatures for environmental forensics operations. These microbial systems must be robust, able to thrive in specific environments of interest and contain molecular signatures enabling the detection of the community across conditions. Furthermore, these systems must balance biocontainment concerns with the required stability and persistence required for environmental forensics. Here we evaluate the stability and persistence of a recently described microbial system comprised of germination-deficient Bacillus subtilis and Saccharomyces cerevisiae spores containing nonredundant DNA barcodes in a controlled simulated home environment. These spore-based microbial communities were found to be persistent in the simulated environment across 30-day periods and across multiple surface types. To improve the repeatability and reproducibility in detecting the DNA barcodes, we evaluated several spore lysis and sampling processes paired with CRISPR/Cas based detection (SHERLOCK). Finally, having optimized the detectability of the spores, we demonstrate that we can detect the spores transferring across multiple material types. Together we further demonstrate the utility of a recently described microbial forensics system and highlight the importance of independent validation and verification of synthetic biology tools and applications.