Development of novel orthogonal genetic circuits, based on extracytoplasmic function (ECF) σ factors

The synthetic biology field aims to apply the engineering 'design-build-test-learn' cycle for the implementation of synthetic genetic circuits modifying the behavior of biological systems. In order to reach this goal, synthetic biology projects use a set of fully characterized biological parts that subsequently are assembled into complex synthetic circuits following a rational, model-driven design. However, even though the bottom-up design approach represents an optimal starting point to assay the behavior of the synthetic circuits under defined conditions, the rational design of such circuits is often restricted by the limited number of available DNA building blocks. These usually consist only of a handful of transcriptional regulators that additionally are often borrowed from natural biological systems. This, in turn, can lead to cross-reactions between the synthetic circuit and the host cell and eventually to loss of the original circuit function. Thus, one of the challenges in synthetic biology is to design synthetic circuits that perform the designated functions with minor cross-reactions (orthogonality). To overcome the restrictions of the widely used transcriptional regulators, this project aims to apply extracytoplasmic function (ECF) σ factors in the design novel orthogonal synthetic circuits. ECFs are the smallest and simplest alternative σ factors that recognize highly specific promoters. ECFs represent one of the most important mechanisms of signal transduction in bacteria, indeed, their activity is often controlled by anti-σ factors. Even though it was shown that the overexpression of heterologous anti-σ factors can generate an adverse effect on cell growth, they represent an attractive solution to control ECF activity. Finally, to date, we know thousands of ECF σ factors, widespread among different bacterial phyla, that are identifiable together with the cognate promoters and anti-σ factors, using bioinformatic approaches. All the above-mentioned features make ECF σ factors optimal candidates as core orthogonal regulators for the design of novel synthetic circuits. In this project, in order to establish ECF σ factors as standard building blocks in the synthetic biology field, we first established a high throughput experimental setup. This relies on microplate reader experiments performed using a highly sensitive luminescent reporter system. Luminescent reporters have a superior signal-to-noise ratio when compared to fluorescent reporters since they do not suffer from the high auto-fluorescence background of the bacterial cell. However, they also have a drawback represented by the constant light emission that can generate undesired cross-talk between neighboring wells on a microplate. To overcome this limitation, we developed a computational algorithm that corrects for luminescence bleed-through and estimates the “true” luminescence activity for each well of a microplate. We show that the correcting algorithm preserves low-level signals close to the background and that it is universally applicable to different experimental conditions. In order to simplify the assembly of large ECF-based synthetic circuits, we designed an ECF toolbox in E. coli. The toolbox allows for the combinatorial assembly of circuits into expression vectors, using a library of reusable genetic parts. Moreover, it also offers the possibility of integrating the newly generated synthetic circuits into four different phage attachment (att) sites present in the genome of E. coli. This allows for a flawless transition between plasmid-encoded and chromosomally integrated genetic circuits, expanding the possible genetic configurations of a given synthetic construct. Moreover, our results demonstrate that the four att sites are orthogonal in terms of the gene expression levels of the synthetic circuits. With the purpose of rationally design ECF-based synthetic circuits and taking advantage of the ECF toolbox, we characterized the dynamic behavior of a set of 15 ECF σ factors, their cognate promoters, and relative anti-σs. Overall, we found that ECFs are non-toxic and functional and that they display different binding affinities for the cognate target promoters. Moreover, our results show that it is possible to optimize the output dynamic range of the ECF-based switches by changing the copy number of the ECFs and target promoters, thus, tuning the input/output signal ratio. Next, by combining up to three ECF-switches, we generated a set of “genetic-timer circuits”, the first synthetic circuits harboring more than one ECF. ECF-based timer circuits sequentially activate a series of target genes with increasing time delays, moreover, the behavior of the circuits can be predicted by a set of mathematical models. In order to improve the dynamic response of the ECF-based constructs, we introduced anti-σ factors in our synthetic circuits. By doing so we first confirmed that anti-σ factors can exert an adverse effect on the growth of E. coli, thus we explored possible solutions. Our results demonstrate that anti-σ factors toxicity can be partially alleviated by generating truncated, soluble variants of the anti-σ factors and, eventually, completely abolished via chromosomal integration of the anti-σ factor-based circuits. Finally, after demonstrating that anti-σ factors can be used to generate a tunable time delay among ECF expression and target promoter activation, we designed ECF/AS-suicide circuits. Such circuits allow for the time-delayed cell-death of E. coli and will serve as a prototype for the further development of ECF/AS-based lysis circuits

EU rural policy: proposal and application of an agricultural sustainability index

In this paper I propose an Agricultural Sustainability Index (ASI) starting from a ‘political’ perspective: European legislation in the rural sector. I try to answer these questions. How can we measure sustainability in agriculture? How do we measure the enhancement (if any) of the European policy for sustainability in agriculture? Why do some geographical areas perform better than others? Considering these questions, the paper suggests a model for measuring sustainability in agriculture and an approach to compare performances among different geographical contexts. The model puts together different dimensions of sustainability in agriculture, combining Geographical Information System (GIS) analysis and Multi-Criteria Analysis (MCA). Using eighteen agricultural indicators divided into three dimensions, social, economic and environmental, the model incorporates the following stages: (i) indicator specification and definition of the decisional framework; (ii) indicators' normalisation by means of transformation functions based on the fuzzy logic approach; (iii) indicators weighted by Analytic Hierarchy Process (AHP) techniques; (iv) indicators aggregated to obtain the ASI. The model is tested on a specific area: Alta Val d’Agri, a rural area in the southern Basilicata Region. Final results show that ASI consistently synthesises the evolution of thirty years of rural development policy.Agricultural sustainability, Indicators, GIS-MCA

Economic growth, electricity consumption and foreign dependence in Italy between 1963 and 2007

The energy sector is assuming an increasing importance in the global economy. As a consequence, there is a vast literature on the causal relation between energy use and others economic variables. In this paper, I investigate the relationship between electricity consumption and economic growth for Italy using yearly data covering the period 1963–2007. Unlike previous works, this paper specifically concerns the causal link between the dynamics of GDP and the different sources of electricity production. Regarding the dependence from foreign suppliers, the paper tests the hypothesis of a causal relationship between economic growth and electricity imports. The results show a unidirectional causality from economic activity to other variables. More specifically, economic growth Granger cause total electricity consumption, industrial consumption and electricity import. For the others source of generation, any specific causal relationship has been found.: Energy and Growth, Italy, Vector Error Correction Model, Granger Causality

microRNA. Diagnostic Perspective

Biomarkers are biological measures of a biological state. An ideal marker should be safe and easy to measure, cost efficient, modifiable with treatment, and consistent across gender and ethnic groups. To date, none of the available biomarkers satisfy all of these criteria. In addition, the major limitations of these markers are low specificity, sensitivity, and false positive results. Recently identified, microRNAs (miRNAs) are endogenous, evolutionarily conserved small non-coding RNA (about 22-25 nt long), also known as micro-coordinators of gene expression, which have been shown to be an effective tools to study the biology of diseases and to have great potential as novel diagnostic and prognostic biomarkers with high specificity and sensitivity. In fact, it has been demonstrated that miRNAs play a pivotal role in the regulation of a wide range of developmental and physiological processes and their deficiencies have been related to a number of disease. In addition, miRNAs are stable and can be easily isolated and measured from tissues and body fluids. In this review, we provide a perspective on emerging concepts and potential usefulness of miRNAs as diagnostic markers, emphasizing the involvement of specific miRNAs in particular tumor types, subtypes, cardiovascular diseases, diabetes, infectious diseases, and forensic test

Marine flora and fauna of the eastern United States Mollusca: Cephalopoda

The cephalopods found in neritic waters of the northeastern United States include myopsid and oegopsid squids, sepiolid squids, and octopods. A key with diagnostic illustrations is provided to aid in identification of the eleven species common in the neritic waters between Cape Hatteras and Nova Scotia; included also is information on two oceanic species that occur over the continental shelf in this area and that can be confused with similar-looking neritic species. Other sections comprise a glossary of taxonomic characters used for identification of these species, an annotated systematic checklist, and checklists of the 89 other oceanic species and 18 Carolinian and subtropical neritic species that might occur occasionally off the northeastern United States. (PDF file contains 30 pages.

Open Content and Public Broadcasting Conference Report

On September 19-21, 2006, the WGBH Educational Foundation hosted a conference on "Open Content and Public Broadcasting." With an initial grant from the William and Flora Hewlett Foundation and additional funding from the Corporation for Public Broadcasting, WGBH proposed the conference as a way to explore public broadcasting issues related to producing and distributing open access content across multiple platforms.The conference grew out of the recognition that public broadcasting executives were interested in the Open Content movement, but were struggling to understand how it might both further public broadcasting's public service mission and support efforts to pursue strategic business models and sustainable solutions.The Open Content conference was designed to bring together key stakeholders in the public broadcasting system, academics who promoted open content in higher education, industry leaders in new media and technology, experts in intellectual property rights, and leaders in philanthropy. Together they examined existing models and efforts that might guide public broadcasting's entry into open content, possible barriers to adoption of open content models in public broadcasting, and opportunities to pursue.This report looks at the events of the conference and the key points discussed. It summarizes the group's conclusions and presents recommendations from the WGBH planners. Audio from the conference presentations and further information is available at http://opencontent.wgbh.org

Aspects of the early life history of Loligo pealei (Cephalopoda:Myopsida)

The long-finned squid Loligo pealei was the most common squid collected in 2 years of zooplankton sampling over the Middle Atlantic Bight off New Jersey and Virginia. Planktonic specimens of L. pealei were found in that area during spring, summer, and fa ll; there were no indications of multiple stocks. This species was captured in waters with a salinity range of 31.5 to 34.0 ppt, and was confined to coastal waters except when current conditions, such as the passage of a Gulf Stream eddy, resulted in strong, offshore surface transport. While abundances were greater in night surface samples, larger specimens occurred in night subsurface samples indicating ontogenetic descent. Tentacle length was closely correlated with dorsal mantle length (DML) in preserved specimens of less than 4.5 mm DML, indicating that tentacles are noncontractile in newly hatched specimens. This may be part of a major discontinuity in the development of L. pealei which separates hatchlings from juvenile

Notable and Notorious Idaho Women: An Annotated Bibliography

In the early days of Idaho’s penitentiary there was a common belief that women could not be held responsible for murder. Juries were comprised of only men at this time, and it was generally thought that women didn’t have the intellectual capability to be so plotting. Often women were not tried for first degree murder; they were characterized as crazy rather than manipulative. “Anecdotally, they [juries] didn’t have it in their hearts to hang a woman,” according to Amber Beierle, Site Manager of the Old Idaho Penitentiary. This is often reflected in the short amounts of time that they served for crimes such as murder and assault, but also reflected in the size of the women’s ward at the Old Penitentiary. When the Penitentiary was built there was no space for women at all

MakerLab Annual Report 2018

Albertsons Library’s MakerLab is available to Boise State University students, staff, and faculty to boost their creative confidence. Use of the equipment is free for all, and creates an environment where students can obtain job skills, invent, proto-type, explore, and build their ideas

A Multispecies Aggregation of Cirrate Octopods Trawled from North of the Bahamas

Two cruises in the western North Atlantic collected 38 trawl samples between the Bahamas and New England. Of the 22 cirrate octopods taken in these samples, 17 came from the area north of the Bahamas. Pooled catch rate (specimens per hour of bottom trawling time) was significantly higher north of the Bahamas than in any other area sampled. Although the taxonomy of these gelatinous benthopelagic cephalopods is not yet settled, morphological characters from these specimens indicate that this aggregation includes at least four species. Only one species (Cirrothauma murrayi) was widely distributed in these samples