Optimization of an E. coli L-rhamnose-inducible expression vector: test of various genetic module combinations

<p>Abstract</p> <p>Background</p> <p>A capable expression vector is mainly characterized by its production efficiency, stability and induction response. These features can be influenced by a variation of modifications and versatile genetic modules.</p> <p>Results</p> <p>We examined miscellaneous variations of a <it>rhaP</it>_{<it>BAD </it>}expression vector. The introduction of a stem loop into the translation initiation region of the <it>rhaP</it>_{<it>BAD </it>}promoter resulted in the most significant improvement of <it>eGFP </it>expression. Starting from this plasmid, we constructed a set of expression vectors bearing different genetic modules like <it>rop</it>, <it>ccdAB</it>, <it>cer </it>and combinations thereof, and tested the efficiency of expression and plasmid stability. The plasmid pWA21, containing the stem loop, one <it>cer </it>site and <it>rop</it>, attained high expression levels accompanied by a good stability, and on that score seems to be a well-balanced choice.</p> <p>Conclusion</p> <p>We report the generation of variations of the <it>rhaP</it>_{<it>BAD </it>}expression vector and characterization hereof. The genetic modules showed a complex interplay, therefore two positive effects combined sometimes resulted in a disadvantage.</p

Regulation of mtl operon promoter of Bacillus subtilis: requirements of its use in expression vectors

Several vector systems have been developed to express any gene desired to be studied in Bacillus subtilis. Among them, the transcriptionally regulated promoters involved in carbohydrate utilization are a research priority. Expression systems based on Bacillus promoters for xylose, maltose, and mannose utilization, as well as on the heterologous E. coli lactose promoter, have been successfully constructed. The promoter of the mtlAFD operon for utilization of mannitol is another promising candidate for its use in expression vectors. In this study, we investigated the regulation of the mtl genes in order to identify the elements needed to construct a strong mannitol inducible expression system in B. subtilis. Regulation of the promoters of Bacillus subtilis mtlAFD operon (PmtlA) and mtlR (PmtlR) encoding the activator were investigated by fusion to lacZ. Identification of the PmtlA and PmtlR transcription start sites revealed the sigma A like promoter structures. Also, the operator of PmtlA was determined by shortening, nucleotide exchange, and alignment of PmtlA and PmtlR operator regions. Deletion of the mannitol-specific PTS genes (mtlAF) resulted in PmtlA constitutive expression demonstrating the inhibitory effect of EIICBMtl and EIIAMtl on MtlR in the absence of mannitol. Disruption of mtlD made the cells sensitive to mannitol and glucitol. Both PmtlA and PmtlR were influenced by carbon catabolite repression (CCR). However, a CcpA deficient mutant showed only a slight reduction in PmtlR catabolite repression. Similarly, using PgroE as a constitutive promoter, putative cre sites of PmtlA and PmtlR slightly reduced the promoter activity in the presence of glucose. In contrast, glucose repression of PmtlA and PmtlR was completely abolished in a ptsG deletion mutant and significantly reduced in a MtlR (H342D) mutant

Towards inclusive GIS in the Congo Basin: an exploration of digital map creation and an evaluation of map understanding by non-literate hunter-gatherers

Sustainable and socially just natural resource management is one of the fundamental development challenges humanity is facing today. Communities living in remote areas possess unique insights about their natural resources. While this knowledge is critical to climate change, it is difficult for them to engage in environmental protection. Geographic Information Science (GIS) plays a central role in resource management, as it is utilised in spatial decision making processes. However, the literature argues that its use is too challenging for marginalised communities. Working with indigenous hunter-gatherers in the Congo Basin without prior exposure to technology or maps, this research moves towards enabling them to become active stakeholders in decision making so that they understand how to capture environmental knowledge and gain power through ownership. (Participatory) GIS, Human Computer Interaction, Action Research and Citizen Science concepts are adapted to the local context to address the lack of mapping of these areas, and the increased understanding of if and how digital, high resolution orthographic maps incorporated in digital mapping tools can be understood by people with no prior exposure to maps and technology. Different set-ups of low-cost Unmanned Aerial Vehicles and consumer grade cameras were tested and evaluated for suitability to generate high-resolution maps in-situ for previously unmapped and disconnected contexts. Applying a computer log analysis approach to overcome local obstacles, three experiments were carried out to test whether the resulting aerial orthophotos are understood as a representation of familiar geographical landscapes. For each of the experiments, a bespoke app functioning without an internet connection was developed. The research shows that the majority of the 136 participants could find as well as edit known features on the map and all participating groups were able to utilise a map for a Treasure Hunt game. Additionally, a number of methodological amendments are proposed to allow standardised research methods to be applied in a context where usability experiments are significantly challenged

FLEXIBLE MULTI-BODY DYNAMICS MODEL OF A BIO-INSPIRED ORNITHOPTER WITH EXPERIMENTAL VALIDATION

There is currently a large effort underway to understand the physics of avian-based flapping wing vehicles, known as ornithopters. There is a need for small aerial robots to conduct a variety of civilian and military missions. Efforts to model the flight physics of these vehicles have been complicated by a number of factors, including nonlinear elastic effects, multi-body characteristics, unsteady aerodynamics, and the strong coupling between fluid and structural dynamics. Experimental verification is crucial in order to achieve accurate simulation capabilities. A multi-disciplinary approach to modeling requires the use of tools representing individual disciplines, which must be combined to form a comprehensive model. In the framework of this research a five body flexible vehicle dynamics model and a novel experimental verification methodology is presented. For the model development and verification of the modeling assumptions, a data set providing refined wing kinematics of a test ornithopter research platform in free flight was used. Wing kinematics for the verification was obtained using a Vicon motion capture system. Lagrange equations of motion in terms of a generalized coordinate vector of the rigid and flexible bodies are formulated in order to model the flexible multi-body system. Model development and verification results are presented. The `luff region" and "thrust flap region" of the wing are modeled as flexible bodies. A floating reference frame formulation is used for the ornithopter. Flexible body constraints and modes are implemented using the Craig-Bampton method, which incorporates a semi-physical subspace method. A quasi-steady aerodynamic model using Blade Element Theory was correlated and verified for the problem using the experimental wing kinematics. The aerodynamic model was then formulated in terms of generalized coordinates of the five-body flexible multi-body system and is used in the resulting model in order to account for aero-elasticity. Modeling assumptions were verified and simulation results were compared with experimental free flight test data

Supporting collaboration with non-literate forest communities in the congo-basin

Providing indigenous communities with ICT tools and methods for collecting and sharing their Traditional Ecological Knowledge is increasingly recognised as an avenue for improvements in environmental governance and socialenvironmental justice. In this paper we show how we carried out a usability engineering effort in the “wild” context of the Congolese rainforest – designing, evaluating and iteratively improving novel collaborative data collection interfaces for non-literate forest communities that can subsequently be used to facilitate communication and information sharing with logging companies. Working in this context necessitates adopting a thoroughly flexible approach to the design, development, introduction and evaluation of technology and the modes of interaction it offers. We show that we have improved participant accuracy from about 75% towards 95% and provide a set of guidelines for designing and evaluating ICT solutions in “extreme circumstances” – which hold lessons for CSCW, HCI and ICT4D practitioners dealing with similar challenges

A timed off-switch for dynamic control of gene expression in Corynebacterium glutamicum

Dynamic control of gene expression mainly relies on inducible systems, which require supplementation of (costly) inducer molecules. In contrast, synthetic regulatory circuits, which allow the timed shutdown of gene expression, are rarely available and therefore represent highly attractive tools for metabolic engineering. To achieve this, we utilized the VanR/PvanABK* regulatory system of Corynebacterium glutamicum, which consists of the transcriptional repressor VanR and a modified promoter of the vanABK operon (PvanABK*). VanR activity is modulated by one of the phenolic compounds ferulic acid, vanillin or vanillic acid, which are co-metabolized with d-glucose. Thus, gene expression in the presence of d-glucose is turned off if one of the effector molecules is depleted from the medium. To dynamically control the expression of the aceE gene, encoding the E1 subunit of the pyruvate dehydrogenase complex that is essential for growth on d-glucose, we replaced the native promoter by vanR/PvanABK* yielding C. glutamicum ΔPaceE::vanR-PvanABK*. The biomass yield of this strain increased linearly with the supplemented amount of effector. After consumption of the phenolic compounds growth ceased, however, C. glutamicumΔPaceE::vanR-PvanABK* continued to utilize the residual d-glucose to produce significant amounts of pyruvate, l-alanine, and l-valine. Interestingly, equimolar concentrations of the three phenolic compounds resulted in different biomass yields; and with increasing effector concentration, the product spectrum shifted from pyruvate over l-alanine to l-valine. To further test the suitability of the VanR/PvanABK* system, we overexpressed the l-valine biosynthesis genes ilvBNCE in C. glutamicum ΔPaceE::vanR-PvanABK*, which resulted in efficient l-valine production with a yield of about 0.36 mol l-valine per mol d-glucose. These results demonstrate that the VanR/PvanABK* system is a valuable tool to control gene expression in C. glutamicum in a timed manner by the cheap and abundant phenolic compounds ferulic acid, vanillin, and vanillic acid

Crosslinking and Mechanical Properties Significantly Influence Cell Attachment, Proliferation, and Migration Within Collagen Glycosaminoglycan Scaffolds.

Crosslinking and the resultant changes in mechanical properties have been shown to influence cellular activity within collagen biomaterials. With this in mind, we sought to determine the effects of crosslinking on both the compressive modulus of collagen-glycosaminoglycan scaffolds and the activity of osteoblasts seeded within them. Dehydrothermal, 1-ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde crosslinking treatments were first investigated for their effect on the compressive modulus of the scaffolds. After this, the most promising treatments were used to study the effects of crosslinking on cellular attachment, proliferation, and infiltration. Our experiments have demonstrated that a wide range of scaffold compressive moduli can be attained by varying the parameters of the crosslinking treatments. 1-Ethyl-3-3-dimethyl aminopropyl carbodiimide and glutaraldehyde treatments produced the stiffest scaffolds (fourfold increase when compared to dehydrothermal crosslinking). When cells were seeded onto the scaffolds, the stiffest scaffolds also showed increased cell number and enhanced cellular distribution when compared to the other groups. Taken together, these results indicate that crosslinking can be used to produce collagen-glycosaminoglycan scaffolds with a range of compressive moduli, and that increased stiffness enhances cellular activity within the scaffolds

Ciência Cidadã Extrema: Uma Nova Abordagem

A conservação da biodiversidade é uma questão que tem preocupado o mundo todo. Nas últimas décadas, centenas de áreas protegidas foram criadas para assegurar a preservação da biodiversidade no planeta. Um grande número de áreas protegidas é habitado por comunidades que dependem do uso de seus recursos naturais não apenas para a sua sobrevivência, mas também para a sua reprodução social e cultural. Em muitos casos, as populações locais têm sido diretamente responsáveis pela gestão sustentável desses complexos ecossistemas por séculos. Iniciativas de Ciência Cidadã – entendida como a participação de amadores, voluntários e entusiastas em projetos científicos – têm envolvido o público na produção científica e em projetos de monitoramento da biodiversidade, mas têm limitado essa participação à coleta de dados, e têm normalmente ocorrido em locais afluentes, excluindo as populações não alfabetizadas ou letradas e que vivem em áreas remotas. Povos e comunidades tradicionais conhecem os aspectos ambientais das áreas por eles habitadas, o que pode ser benéfico para a gestão e o monitoramento bem-sucedidos da biodiversidade. Portanto, ao se tratar do monitoramento e da proteção da biodiversidade em áreas habitadas por populações humanas, o seu envolvimento é central e pode conduzir a um cenário onde todas as partes envolvidas se beneficiam. Extreme Citizen Science (ExCiteS) é um grupo de pesquisa interdisciplinar criado em 2011, na University College London, com a finalidade de avançar o atual conjunto de práticas da Ciência Cidadã. A ideia é permitir que qualquer comunidade, em qualquer lugar do mundo – desde grupos marginalizados que vivem nas periferias de áreas urbanas até grupos de caçadores e coletores da floresta amazônica –, comece um projeto de Ciência Cidadã para lidar com suas próprias questões. Este artigo apresenta os diversos aspectos que tornam a Ciência Cidadã “extrema” no trabalho do grupo ExCiteS, por meio da exposição de suas teorias, métodos e ferramentas, e dos estudos de caso atuais que envolvem comunidades tradicionais ao redor do mundo. Por fim, ressalta-se a maior preocupação do grupo, que é tornar a participação verdadeiramente efetiva, e sugere-se como iniciativas de monitoramento da biodiversidade podem ser realizadas de maneira colaborativa, trazendo benefícios a todos os atores envolvidos

Flight Testing of Novel Compliant Spines for Passive Wing Morphing on Ornithopters

Unmanned Aerial Vehicles (UAVs) are proliferating in both the civil and military markets. Flapping wing UAVs, or ornithopters, have the potential to combine the agility and maneuverability of rotary wing aircraft with excellent performance in low Reynolds number flight regimes. The purpose of this paper is to present new free flight experimental results for an ornithopter equipped with one degree of freedom (1DOF) compliant spines that were designed and optimized in terms of mass, maximum von-Mises stress, and desired wing bending deflections. The spines were inserted in an experimental ornithopter wing spar in order to achieve a set of desired kinematics during the up and down strokes of a flapping cycle. The ornithopter was flown at Wright Patterson Air Force Base in the Air Force Research Laboratory Small Unmanned Air Systems (SUAS) indoor flight facility. Vicon motion tracking cameras were used to track the motion of the vehicle for five different wing configurations. The effect of the presence of the compliant spine on wing kinematics and leading edge spar deflection during flight is presented. Results show that the ornithopter with the compliant spine inserted in its wing reduced the body acceleration during the upstroke which translates into overall lift gains