Automated robotic liquid handling assembly of modular DNA devices

Recent advances in modular DNA assembly techniques have enabled synthetic biologists to test significantly more of the available "design space" represented by "devices" created as combinations of individual genetic components. However, manual assembly of such large numbers of devices is time-intensive, error-prone, and costly. The increasing sophistication and scale of synthetic biology research necessitates an efficient, reproducible way to accommodate large-scale, complex, and high throughput device construction. Here, a DNA assembly protocol using the Type-IIS restriction endonuclease based Modular Cloning (MoClo) technique is automated on two liquid-handling robotic platforms. Automated liquid-handling robots require careful, often times tedious optimization of pipetting parameters for liquids of different viscosities (e.g. enzymes, DNA, water, buffers), as well as explicit programming to ensure correct aspiration and dispensing of DNA parts and reagents. This makes manual script writing for complex assemblies just as problematic as manual DNA assembly, and necessitates a software tool that can automate script generation. To this end, we have developed a web-based software tool, http://mocloassembly.com, for generating combinatorial DNA device libraries from basic DNA parts uploaded as Genbank files. We provide access to the tool, and an export file from our liquid handler software which includes optimized liquid classes, labware parameters, and deck layout. All DNA parts used are available through Addgene, and their digital maps can be accessed via the Boston University BDC ICE Registry. Together, these elements provide a foundation for other organizations to automate modular cloning experiments and similar protocols. The automated DNA assembly workflow presented here enables the repeatable, automated, high-throughput production of DNA devices, and reduces the risk of human error arising from repetitive manual pipetting. Sequencing data show the automated DNA assembly reactions generated from this workflow are ~95% correct and require as little as 4% as much hands-on time, compared to manual reaction preparation

Extensive horizontal gene transfer during Staphylococcus aureus co-colonization in vivo.

Staphylococcus aureus is a commensal and major pathogen of humans and animals. Comparative genomics of S. aureus populations suggests that colonization of different host species is associated with carriage of mobile genetic elements (MGE), particularly bacteriophages and plasmids capable of encoding virulence, resistance, and immune evasion pathways. Antimicrobial-resistant S. aureus of livestock are a potential zoonotic threat to human health if they adapt to colonize humans efficiently. We utilized the technique of experimental evolution and co-colonized gnotobiotic piglets with both human- and pig-associated variants of the lineage clonal complex 398, and investigated growth and genetic changes over 16 days using whole genome sequencing. The human isolate survived co-colonization on piglets more efficiently than in vitro. During co-colonization, transfer of MGE from the pig to the human isolate was detected within 4 h. Extensive and repeated transfer of two bacteriophages and three plasmids resulted in colonization with isolates carrying a wide variety of mobilomes. Whole genome sequencing of progeny bacteria revealed no acquisition of core genome polymorphisms, highlighting the importance of MGE. Staphylococcus aureus bacteriophage recombination and integration into novel sites was detected experimentally for the first time. During colonization, clones coexisted and diversified rather than a single variant dominating. Unexpectedly, each piglet carried unique populations of bacterial variants, suggesting limited transmission of bacteria between piglets once colonized. Our data show that horizontal gene transfer occurs at very high frequency in vivo and significantly higher than that detectable in vitro

On-sky wide field adaptive optics correction using multiple laser guide stars at the MMT

We describe results from the first astronomical adaptive optics system to use multiple laser guide stars, located at the 6.5-m MMT telescope in Arizona. Its initial operational mode, ground-layer adaptive optics (GLAO), provides uniform stellar wavefront correction within the 2 arc minute diameter laser beacon constellation, reducing the stellar image widths by as much as 53%, from 0.70 to 0.33 arc seconds at lambda = 2.14 microns. GLAO is achieved by applying a correction to the telescope's adaptive secondary mirror that is an average of wavefront measurements from five laser beacons supplemented with image motion from a faint stellar source. Optimization of the adaptive optics system in subsequent commissioning runs will further improve correction performance where it is predicted to deliver 0.1 to 0.2 arc second resolution in the near-infrared during a majority of seeing conditions.Comment: 13 pages, 1 table, 7 figures. Accepted for publication in Astrophysical Journal. Expected March 200

Ship traffic connects Antarctica's fragile coasts to worldwide ecosystems.

Antarctica, an isolated and long considered pristine wilderness, is becoming increasingly exposed to the negative effects of ship-borne human activity, and especially the introduction of invasive species. Here, we provide a comprehensive quantitative analysis of ship movements into Antarctic waters and a spatially explicit assessment of introduction risk for nonnative marine species in all Antarctic waters. We show that vessels traverse Antarctica's isolating natural barriers, connecting it directly via an extensive network of ship activity to all global regions, especially South Atlantic and European ports. Ship visits are more than seven times higher to the Antarctic Peninsula (especially east of Anvers Island) and the South Shetland Islands than elsewhere around Antarctica, together accounting for 88% of visits to Southern Ocean ecoregions. Contrary to expectations, we show that while the five recognized "Antarctic Gateway cities" are important last ports of call, especially for research and tourism vessels, an additional 53 ports had vessels directly departing to Antarctica from 2014 to 2018. We identify ports outside Antarctica where biosecurity interventions could be most effectively implemented and the most vulnerable Antarctic locations where monitoring programs for high-risk invaders should be established

High-resolution spectroscopy of QY Sge -- An obscured RV Tauri variable?

The first high-resolution optical spectra of QY Sge are presented and discussed. Menzies & Whitelock (1988) on the basis of photometry and low-resolution spectra suggested that this G0I supergiant was obscured by dust and seen only by scattered light from a circumstellar reflection nebula. The new spectra confirm and extend this picture. Photospheric lines are unusually broad indicating scattering of photons from dust in the stellar wind. Presence of very broad Na D emission lines is confirmed. Sharp emission lines from low levels of abundant neutral metal atoms are reported for the first time. An abundance analysis of photospheric lines shows that the stellar atmosphere is of approximately solar composition but with highly condensible (e.g., Sc and Ti) elements depleted by factors of 5 to 10.Comment: 8 pages, 9 figures, accepted for publication in MNRA

Prevalence of true vein graft aneurysms: Implications for aneurysm pathogenesis

AbstractBackground: Circumstantial evidence suggests that arterial aneurysms have a different cause than atherosclerosis and may form part of a generalized dilating diathesis. The aim of this study was to compare the rates of spontaneous aneurysm formation in vein grafts performed either for popliteal aneurysms or for occlusive disease. The hypothesis was that if arterial aneurysms form a part of a systemic process, then the rates of vein graft aneurysms should be higher for patients with popliteal aneurysms than for patients with lower limb ischemia caused by atherosclerosis. Methods: Infrainguinal vein grafting procedures performed from 1990 to 1995 were entered into a prospective audit and graft surveillance program. Aneurysmal change was defined as a focal increase in the graft diameter of 1.5 cm or greater, excluding false aneurysms and dilatations after graft angioplasty. Results: During the study period, 221 grafting procedures were performed in 200 patients with occlusive disease and 24 grafting procedures were performed in 21 patients with popliteal aneurysms. Graft surveillance revealed spontaneous aneurysm formation in 10 of the 24 bypass grafts (42%) for popliteal aneurysms but in only 4 of the 221 grafting procedures (2%) that were performed for chronic lower limb ischemia. Conclusion:This study provides further evidence that aneurysmal disease is a systemic process, and this finding has clinical implications for the treatment of popliteal aneurysms. (J Vasc Surg 1999;29:403-8.

Anthropology and the Irish Encounter

In their discussion of ancestral versus contemporary anthropology in Ireland, Keith Egan and Fiona Murphy (this issue) do not draw a parallel distinction, quite probably deliberately, between “metropolitan” and “native” anthropologies. Positing a category of “native anthropology” opens up an explosive set of issues about the claim to be “native”—all the more combustible in a place that has known settler colonialism since the 12th century, tidalwaves of out-migration (and consequently a vast and tuned-in diaspora) due to famine in the 19th and economic stagnation in the 20th century, and a total demographic makeover through in-migration in the past two decades. Nonetheless, even though they do not resort to this distinction, Egan and Murphy are likely to agree that they are describing an Irish version of a quandary that is all too familiar to native anthropologists from marginal anthropological traditions, predominantly in the postcolonial world: namely, what is to be done when the acknowledged gold standard of metropolitan ethnographic writing renders your home place in a way that is unrecognizable to you

Commissioning the MMT ground-layer and laser tomography adaptive optics systems

A multi-laser adaptive optics system, at the 6.5 m MMT telescope, has been undergoing commissioning in preparation for wide-field, partially corrected as well as narrow-field, diffraction limited science observations in the thermal and near infrared. After several delays due to bad weather, we have successfully closed the full high order ground-layer adaptive optics (GLAO) control loop for the first time in February 2008 using five Rayleigh laser guide stars and a single tilt star. Characterization and automated correction of static aberrations such as non-common path errors were addressed in May 2008. Calibration measurements in preparation for laser tomography adaptive optics (LTAO) operation are planned for the fall of 2008 along with the start of shared-risk GLAO science observations. We present the results of GLAO observations with the PISCES imager, a 1 - 2.5 µm camera with a field of view of 110 arc seconds. The status of the remaining GLAO commissioning work is also reviewed. Finally, we present plans for commissioning work to implement the LTAO operating mode of the system

Fewer epistemological challenges for connectionism

Seventeen years ago, John McCarthy wrote the note Epistemological challenges for connectionism as a response to Paul Smolensky’s paper 'On the proper treatment of connectionism'. I will discuss the extent to which the four key challenges put forward by McCarthy have been solved, and what are the new challenges ahead. I argue that there are fewer epistemological challenges for connectionism, but progress has been slow. Nevertheless, there is now strong indication that neural-symbolic integration can provide effective systems of expressive reasoning and robust learning due to the recent developments in the field