Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a D-alanine auxotrophy. Efficient DNA transfer (10 to 10 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-β-D-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.

We report on a new sensor strategy that integrates molecularly imprinted polymers (MIPs) with surface enhanced Raman scattering (SERS). The sensor was developed to detect the explosive, 2,4,6-trinitrotoluene (TNT). Micron thick films of sol gel-derived xerogels were deposited on a SERS-active surface as the sensing layer. Xerogels were molecularly imprinted for TNT using non-covalent interactions with the polymer matrix. Binding of the TNT within the polymer matrix results in unique SERS bands, which allow for detection and identification of the molecule in the MIP. This MIP-SERS sensor exhibits an apparent dissociation constant of (2.3 ± 0.3) × 10−5 M for TNT and a 3 μM detection limit. The response to TNT is reversible and the sensor is stable for at least 6 months. Key challenges, including developing a MIP formulation that is stable and integrated with the SERS substrate, and ensuring the MIP does not mask the spectral features of the target analyte through SERS polymer background, were successfully met. The results also suggest the MIP-SERS protocol can be extended to other target analytes of interest.

We describe a general synthetic strategy for developing high affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify a best binder. We describe epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies.

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The feasibility of using a novel detection scheme for the analysis of biological warfare agents is demonstrated using Bacillus globigii spores, a surrogate species for Bacillus anthracis. In this paper, a sensitive and selective enzyme-linked immunosorbent assay using a novel fluorogenic alkaline phosphatase substrate (dimethylacridinone phosphate) is combined with a compact biochip detection system, which includes a miniature diode laser for excitation. Detection of aerosolized spores was achieved by coupling the miniature system to a portable bioaerosol sampler, and the performance of the antibody-based recognition and enzyme amplification method was evaluated. The bioassay performance was found to be compatible with the air sampling device, and the enzymatic amplification was found to be an attractive amplification method for detection of low spore concentrations. The combined portable bioaerosol sampler and miniature biochip system detected 100 B. globigii spores, corresponding to 17 aerosolized spores/L of air. Moreover, the incorporation of the miniature diode laser with the self-contained biochip design allows for a compact system that is readily adaptable to field use. In addition, these studies have included investigations into the tradeoff between assay time and sensitivity.