Biology subjects

Ng, J. T.

Publications and source records attributed to Ng, J. T..

3 recordsLinked to original sources

bioRxiv2020

Asymmetric second generation genomic incompatibility in interspecific crosses between Ciona robusta and Ciona intestinalis

Reproductive isolation is central to speciation, but interspecific crosses between two closely related species can produce viable and fertile hybrids. Two different species in the tunicate genus Ciona, Ciona robusta and Ciona intestinalis can produce hybrids. However, wild sympatric populations display limited gene flow, suggesting the existence of obstacles to interspecific reproduction that remain unknown. Here, we took advantage of a closed inland culture system to cross C. robusta with C. intestinalis and established F1 and F2 hybrids. We monitored post-embryonic development, survival, and sexual maturation to further probe the physiological mechanisms underlying reproductive isolation. Partial viability of first and second generation hybrids indicated that both pre- and postzygotic mechanisms contributed to genomic incompatibilities in hybrids. Asymmetrical second generation inviability and infertility suggested that interspecific genomic incompatibilities involved interactions between the maternal, zygotic and mitochondrial genomes during development. This study paves the way to quantitative genetic approaches to study the mechanisms underlying genomic incompatibilities and other complex traits in the genome-enabled Ciona model.

evolutionary biology↗

bioRxiv2019

The Low-Cost, Semi-Automated Shifter Microscope Stage Transforms Speed and Robustness of Manual Protein Crystal Harvesting

Despite the tremendous success of x-ray cryocrystallography over recent decades, the transfer of crystals from the drops where they grow to diffractometer sample mounts, remains a manual process in almost all laboratories. Here we describe the Shifter, a semi-automated microscope stage that offers an accessible and scalable approach to crystal mounting that exploits on the strengths of both humans and machines. The Shifter control software manoeuvres sample drops beneath a hole in a clear protective cover, for human mounting under a microscope. By allowing complete removal of film seals the tedium of cutting or removing the seal is eliminated. The control software also automatically captures experimental annotations for uploading to the users data repository, removing the overhead of manual documentation. The Shifter facilitates mounting rates of 100-240 crystals per hour, in a more controlled process than manual mounting, which greatly extends the lifetime of drops and thus allows for a dramatic increase in the number of crystals retrievable from any given drop, without loss of X-ray diffraction quality. In 2015 the first in a series of three Shifter devices was deployed as part of the XChem fragment screening facility at Diamond Light Source (DLS), where they have since facilitated the mounting of over 100,000 crystals. The Shifter was engineered to be simple, allowing for a low-cost device to be commercialised and thus potentially transformative as many research initiatives as possible. SynopsisA motorised X/Y microscope stage is presented that combines human fine motor control with machine automation and automated experiment documentation, to transform productivity in protein crystal harvesting.

biophysics↗

bioRxiv2016

Gentle, fast and effective crystal soaking by acoustic dispensing

SynopsisA high-throughput method is described for crystal soaking using acoustic droplet ejection, and its effectiveness demonstrated.\n\nAbstractBright light sources, agile robotics, and fast detectors are continually reducing the time it takes to perform an X-ray diffraction experiment, making high throughput experiments more feasible than ever. But this is also pushing the upstream bottleneck towards sample preparation, even for robust and well characterised crystal systems. Crystal soaking is routinely used to generate protein-ligand complex structures, yet protein crystals are often sensitive to changes in solvent composition, and frequently require gentle or careful stepwise soaking techniques, limiting overall throughput. Here, we describe the use of acoustic droplet ejection for soaking of protein crystals with small molecules, and show that it is both gentle on crystals and allows very high throughput, with 1000 unique soaks easily performed in under 10 minutes. In addition to having very low compound consumption (tens of nanolitres per sample), the positional precision of acoustic droplet ejection enables targeted placement of the compound/solvent away from crystals and towards drop edges, allowing for gradual diffusion of solvent across the drop. This ensures both an improvement in reproducibility of X-ray diffraction and an increased solvent tolerance of the crystals, thus enabling higher effective compound soaking concentrations. We detail the technique here with examples from the protein target JMJD2D, a histone lysine demethylase, having roles in cancer and the focus of active structure based drug design efforts.

biophysics↗