FLEET Velocimetry for Aerodynamics

Long-lasting emission from femtosecond excitation of nitrogen-based flows shows promise as a useful mechanism for a molecular tagging velocimetry instrument. The technique, known as femtosecond laser electronic excitation tagging (FLEET), was invented at Princeton a decade ago and has quickly been adopted and used in a variety of high-speed ground test flow facilities. The short temporal scales offered by femtosecond amplifiers permit nonresonant multiphoton excitation, dissociation, and weak ionization of a gaseous medium near the beam's focus without the generation of a laser spark observed with nanosecond systems. Gated, intensified imaging of the resulting emission enables the tracking of tagged molecules, thereby measuring one to three components of velocity. Effects of local heating and acoustic disturbances can be mitigated with the selection of a shorter-wavelength excitation source. This review surveys the development of FLEET over the decade since its inception, as it has been implemented in several test facilities to make accurate, precise, and seedless velocimetry measurements for studying complex high-speed flows.

Structural Analysis of the Ancestral Haloalkane Dehalogenase AncLinB-DmbA

Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.

Efficient linear dsDNA tagging using deoxyuridine excision

Site-specific strategies for exchanging segments of dsDNA are important for DNA library construction and molecular tagging. Deoxyuridine (dU) excision is an approach for generating 3′ ssDNA overhangs in gene assembly and molecular cloning procedures. Unlike approaches that use a multi-base pair motif to specify a DNA cut site, dU excision requires only a dT→dU substitution. Consequently, excision sites can be embedded in biologically active DNA sequences by placing dU substitutions at non-perturbative positions. In this work, I describe a molecular tagging method that uses dU excision to exchange a segment of a dsDNA strand with a long synthetic oligonucleotide. The core workflow of this method, called deoxyUridine eXcision-tagging (dUX-tagging), is an efficient one-pot reaction: strategically positioned dU nucleotides are excised from dsDNA to generate a 3′ overhang so that additional sequence can be appended by annealing and ligating a tagging oligonucleotide. The tagged DNA is then processed by one of two procedures to fill the 5′ overhang and remove excess tagging oligo. To facilitate its widespread use, all dUX-tagging procedures exclusively use commercially available reagents. As a result, dUX-tagging is a concise and easily implemented approach for high-efficiency linear dsDNA tagging.