Targeted mutagenesis and high-throughput screening of diversified gene and promoter libraries for isolating gain-of-function mutations

Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6–9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3–5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments

Towards a Deleuzoguattarian methodology for urban design

© 2018, © 2018 Informa UK Limited, trading as Taylor & Francis Group. The philosophy of Gilles Deleuze and Felix Guattari (Deleuzoguattarian) is now widely thought to be apropos for today’s world. This paper proposes Deleuzoguattarian philosophy as a new methodology for urban design research and practice. First, existing methodologies in the field of urban design are examined and their strengths and limitations outlined in relation to current urban problems. A reading of Deleuzoguattarian philosophy from an urban design perspective is then provided in order to propose a new methodology for research and practice. This reading mainly concentrates on the ontology and epistemology offered by the philosophy, aspects that have been neglected in the literature. The concept of territorialization as a complementary concept to assemblage theory is highlighted in order to illustrate the characteristics of the ontology and epistemology. Finally, normative goals are discussed. It is concluded that Deleuzoguattarian philosophy is not only an ethical response to modernism but is an epistemological need

Liveable Open Public Space - From Flaneur to Cyborg

Open public spaces have always been key elements of the city. Now they are also crucial for mixed reality. It is the main carrier of urban life, place for socialization, where users rest, have fun and talk. Moreover, “Seeing others and being seen” is a condition of socialization. Intensity of life in public spaces provides qualities like safety, comfort and attractiveness. Furthermore, open public spaces represent a spatial framework for meetings and multileveled interactions, and should include virtual flows, stimulating merging of physical and digital reality. Aim of the chapter is to present a critical analysis of public open spaces, aspects of their social role and liveability. It will also suggest how new technologies, in a mixed reality world, may enhance design approaches and upgrade the relationship between a user and his surroundings. New technologies are necessary for obtaining physical/digital spaces, becoming playable and liveable which will encourage walking, cycling, standing and interacting. Hence, they will attract more citizens and visitors, assure a healthy environment, quality of life and sociability. Public space, acting as an open book of the history of the city and of its future, should play a new role, being a place of reference for the flaneur/cyborg citizen personal and social life. The key result is a framework for understanding the particular importance of cyberparks in contemporary urban life in order to better adapt technologies in the modern urban life needs

Sugar-fermenting yeast as an organic source of carbon dioxide to attract the malaria mosquito Anopheles gambiae s.s.

<p>Abstract</p> <p>Background</p> <p>Carbon dioxide (CO₂) plays an important role in the host-seeking process of opportunistic, zoophilic and anthropophilic mosquito species and is, therefore, commonly added to mosquito sampling tools. The African malaria vector <it>Anopheles gambiae sensu stricto </it>is attracted to human volatiles augmented by CO₂. This study investigated whether CO₂, usually supplied from gas cylinders acquired from commercial industry, could be replaced by CO₂derived from fermenting yeast (yeast-produced CO₂).</p> <p>Methods</p> <p>Trapping experiments were conducted in the laboratory, semi-field and field, with <it>An. gambiae s.s</it>. as the target species. MM-X traps were baited with volatiles produced by mixtures of yeast, sugar and water, prepared in 1.5, 5 or 25 L bottles. Catches were compared with traps baited with industrial CO₂. The additional effect of human odours was also examined. In the laboratory and semi-field facility dual-choice experiments were conducted. The effect of traps baited with yeast-produced CO₂on the number of mosquitoes entering an African house was studied in the MalariaSphere. Carbon dioxide baited traps, placed outside human dwellings, were also tested in an African village setting. The laboratory and semi-field data were analysed by a χ²-test, the field data by GLM. In addition, CO₂concentrations produced by yeast-sugar solutions were measured over time.</p> <p>Results</p> <p>Traps baited with yeast-produced CO₂caught significantly more mosquitoes than unbaited traps (up to 34 h post mixing the ingredients) and also significantly more than traps baited with industrial CO₂, both in the laboratory and semi-field. Adding yeast-produced CO₂to traps baited with human odour significantly increased trap catches. In the MalariaSphere, outdoor traps baited with yeast-produced or industrial CO₂+ human odour reduced house entry of mosquitoes with a human host sleeping under a bed net indoors. <it>Anopheles gambiae s.s</it>. was not caught during the field trials. However, traps baited with yeast-produced CO₂caught similar numbers of <it>Anopheles arabiensis </it>as traps baited with industrial CO₂. Addition of human odour increased trap catches.</p> <p>Conclusions</p> <p>Yeast-produced CO₂can effectively replace industrial CO₂for sampling of <it>An. gambiae s.s</it>.. This will significantly reduce costs and allow sustainable mass-application of odour-baited devices for mosquito sampling in remote areas.</p

From rhetoric to reality: which resilience, why resilience, and whose resilience in spatial planning?

This paper analyses contrasting academic understandings of ‘equilibrium resilience’ and ‘evolutionary resilience’ and investigates how these nuances are reflected within both policy and practice. We reveal that there is a lack of clarity in policy, where these differences are not acknowledged with resilience mainly discussed as a singular, vague, but optimistic aim. This opaque political treatment of the term and the lack of guidance has affected practice by privileging an equilibrist interpretation over more transformative, evolutionary measures. In short, resilience within spatial planning is characterised by a simple return to normality that is more analogous with planning norms, engineered responses, dominant interests, and technomanagerial trends. The paper argues that, although presented as a possible paradigm shift, resilience policy and practice underpin existing behaviour and normalise risk. It leaves unaddressed wider sociocultural concerns and instead emerges as a narrow, regressive, technorational frame centred on reactive measures at the building scale