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Search Results: 1 - 10 of 95 matches for " Romy Kandzia "
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A One Pot, One Step, Precision Cloning Method with High Throughput Capability
Carola Engler, Romy Kandzia, Sylvestre Marillonnet
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003647
Abstract: Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.
Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes
Carola Engler, Ramona Gruetzner, Romy Kandzia, Sylvestre Marillonnet
PLOS ONE , 2009, DOI: 10.1371/journal.pone.0005553
Abstract: We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes
Frank Thieme, Carola Engler, Romy Kandzia, Sylvestre Marillonnet
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0020556
Abstract: We have developed an efficient strategy for cloning of PCR products that contain an unknown region flanked by a known sequence. As with ligation-independent cloning, the strategy is based on homology between sequences present in both the vector and the insert. However, in contrast to ligation-independent cloning, the cloning vector has homology with only one of the two primers used for amplification of the insert. The other side of the linearized cloning vector has homology with a sequence present in the insert, but nested and non-overlapping with the gene-specific primer used for amplification. Since only specific products contain this sequence, but none of the non-specific products, only specific products can be cloned. Cloning is performed using a one-step reaction that only requires incubation for 10 minutes at room temperature in the presence of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. The reaction mix is then directly transformed into E. coli where the annealed vector-insert complex is repaired and ligated. We have tested this method, which we call quick and clean cloning (QC cloning), for cloning of the variable regions of immunoglobulins expressed in non-Hodgkin lymphoma tumor samples. This method can also be applied to identify the flanking sequence of DNA elements such as T-DNA or transposon insertions, or be used for cloning of any PCR product with high specificity.
Resolution of night terrors after discontinuation of cetirizine  [PDF]
Lourdes DelRosso, Romy Hoque
Case Reports in Clinical Medicine (CRCM) , 2012, DOI: 10.4236/crcm.2012.11001
Abstract: We present a 4-year-old girl with allergies and asthma who developed night terrors after initia- tion of montelukast at 2 years of age. Montelu-kast was discontinued and cetirizine was started. Nigh terrors persisted. Sleep diaries were col- lected and diagnosticpolysomnogram (PSG) was ordered. Sleep diaries revealed an average sleep time of 10 hours with night terrors occur- ring three nights a week approximately two hours after sleep onset. The PSG did not show evidence of sleep disordered breathing or peri- odic leg movements. Cetirizine was discontin- ued and the night terrors ceased. Upon re-intro- duction of the medication, the sleep terrors re- curred.
Improving the Net Benefits from Tourism for People Living in Remote Northern Australia
Romy Greiner
Sustainability , 2010, DOI: 10.3390/su2072197
Abstract: Tourism can be an important source of livelihoods at a destination level. Yet, while there are economic benefits associated with more tourists, there can also be costs to destinations in the form of negative environmental and social impacts. This paper illustrates tourism-related dilemmas for two remote regions within Australia’s tropical savannas where increasing visitor numbers are straining not only the very environmental assets that attract tourist, but also the host communities. The paper draws on research conducted under the auspices of the Tropical Savannas Management Cooperative Research Centre. Tourism impacts on the regions are described and, where possible, quantified and distributional effects discussed. Evidence is provided that host populations in the remote of Australia’s tropical savannas are willing to trade off environmental and social costs for economic benefits, but that this situation may not be ecologically sustainable. The regions are parts of much larger destinations and consequently peripheral to their concerns. The onus for sustainable tourism and regional development strategies therefore falls on local decision makers. The research presented here provides a framework for local decision makers and stakeholders to ask questions, collect relevant data, and proceed with informed debates and choices.
De caza con el rey(1)
Hecht,Romy;
ARQ (Santiago) , 2010, DOI: 10.4067/S0717-69962010000100015
Abstract: the landscape is intervened to return to its natural state, with the objective of being utilized by the kings in hunting season. species of flora and fauna are forcibly introduced in a place to create gardens that we now recognize as parks. the different layers of history are superimposed and configure a particular terrain.
Trazado, paisaje y territorio: Cerro Sombrero y la arquitectura del petróleo en Magallanes
Hecht,Romy;
ARQ (Santiago) , 2002, DOI: 10.4067/S0717-69962002005100016
Abstract: this article seeks to highlight and analyse cerro sombrero as a foundation pattern in magallanes, not in the sense of occupying a territory by a repeated formal model. cerro sombrero shows an imposed and deliberated order in a territory without signals nor possibilities of occupation. at the end, the rescue of the southernmost modern architecture of the world
The importance of job characteristics in determining medical care-seeking in the Dutch working population, a longitudinal survey study
Steenbeek Romy
BMC Health Services Research , 2012, DOI: 10.1186/1472-6963-12-294
Abstract: Background The working population is ageing, which will increase the number of workers with chronic health complaints, and, as a consequence, the number of workers seeking health care. It is very important to understand factors that influence medical care-seeking in order to control the costs. I will investigate which work characteristics independently attribute to later care-seeking in order to find possibilities to prevent unnecessary or inefficient care-seeking. Methods Data were collected in a longitudinal two-wave study (n = 2305 workers). The outcome measures were visits (yes/no and frequency) to a general practitioner (GP), a physical therapist, a medical specialist and/or a mental health professional. Multivariate regression analyses were carried out separately for men and women for workers with health complaints. Results In the Dutch working population, personal, health, and work characteristics, but not sickness absence, were associated with later care-seeking. Work characteristics independently attributed to medical care-seeking but only for men and only for the frequency of visits to the GP. Women experience more health complaints and seek health care more often than men. For women, experiencing a work handicap (health complaints that impede work performance) was the only work characteristic associated with more care-seeking (GP). For men, work characteristics that led to less care-seeking were social support by colleagues (GP frequency), high levels of decision latitude (GP frequency) and high levels of social support by the supervisor (medical specialist). Other work characteristics led to more care-seeking: high levels of engagement (GP), full time work (GP frequency) and experiencing a work handicap (physical therapist). Conclusions We can conclude that personal and health characteristics are most important when explaining medical care-seeking in the Dutch working population. Work characteristics independently attributed to medical care-seeking but only for men and only for the frequency of visits to the GP. The association between work characteristics and later medical care-seeking differed between health care providers and between men and women. If we aim at reducing health care costs for workers by preventing unnecessary or inefficient care, it is important to reduce the number of workers that report that health complaints impede their work performance. The supervisor could provide more social support, closely monitor workload in combination with work pressure and decision latitude, and when possible help to adjust working co
Trazado, paisaje y territorio: Cerro Sombrero y la arquitectura del petróleo en Magallanes
Romy Hecht
ARQ , 2002,
Abstract: Este artículo presenta al campamento petrolero de Cerro Sombrero como un patrón fundacional en Magallanes; patrón no desde el punto de vista de ocupar el territorio en base a la repetición de un modelo formal, sino como un orden deliberado e impuesto a un territorio que, como veremos, no otorga se ales de ocupación. A la larga, un rescate patrimonial de la construcción de arquitectura moderna más al sur del mundo This article seeks to highlight and analyse Cerro Sombrero as a foundation pattern in Magallanes, not in the sense of occupying a territory by a repeated formal model. Cerro Sombrero shows an imposed and deliberated order in a territory without signals nor possibilities of occupation. At the end, the rescue of the southernmost modern architecture of the world
Neither home nor decoration
Romy Hecht
ARQ , 2002,
Abstract:
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