Optimization of Recombination Methods and Expanding the Utility of Penicillin G Acylase

Protein engineering can be performed by combinatorial techniques (directed evolution) and data-driven methods using machine-learning algorithms. The main characteristic of directed evolution (DE) is the application of an effective and efficient screen or selection on a diverse mutant library. As it is important to have a diverse mutant library for the success of DE, we compared the performance of DNA-shuffling and recombination PCR on fluorescent proteins using sequence information as well as statistical methods. We found that the diversity of the libraries DNA-shuffling and recombination PCR generates were dependent on type of skew primers used and sensitive to nucleotide identity levels between genes. DNA-shuffling and recombination PCR produced libraries with different crossover tendencies, suggesting that the two protocols could be used in combination to produce better libraries. Data-driven protein engineering uses sequence, structure and function data along with analyzed empirical activity information to guide library design. Boolean Learning Support Vector Machines (BLSVM) to identify interacting residues in fluorescent proteins and the gene templates were modified to preserve interactions post recombination. By site-directed mutagenesis, recombination and expression experiments, we validated that BLSVM can be used to identify interacting residues and increase the fraction of active proteins in the library. As an extension to the above experiments, DE was applied on monomeric Red Fluorescent Proteins to improve its spectral characteristics and structure-guided protein engineering was performed on penicillin G acylase (PGA), an industrially relevant catalyst, to change its substrate specificity.Ph.D.Committee Chair: Bommarius, Andreas; Committee Member: Hu, Wei-Shou; Committee Member: Lee, Jay; Committee Member: Lutz, Stefan; Committee Member: Prausnitz, Mar

Considerations for determining optimal mouse caging density

At the 2006 National Meeting of the American Association of Laboratory Animal Science, a panel discussed the question of what constitutes optimal or acceptable housing density for mice. Though there is a consensus that present guidelines are somewhat arbitrarily defined, scientific research has not yet been able to provide clear recommendations for amending them. Speakers explored the many factors that influence decisions on mouse housing, including regulatory requirements, scientific data and their interpretation, financial considerations and ethical concerns. The panel largely agreed that animal well-being should be the measure of interest in evaluating housing density and that well-being includes not only physical health, but also animals\u27 behavior, productivity and preference

Globalisation, Entrepreneurship and the South Pacific: Reframing Australian Colonial Architecture, 1800-1850

In 1957, Clinton Hartley Grattan, one of Australia’s most important foreign observers, wrote of the shadow of the “urban” in legends of the Australian “bush”.1 He argued that the early frontiers of Australian settlement were frontiers of men with private capital, or entrepreneurs, and those frontiers thus carried more elements of the urban than is commonly realised. Such early colonial enterprises around Australia’s south and southeastern coasts, and across the Tasman included sealing, whaling, milling and pastoralism, as well as missionary, trading and finance ventures. In advance of official settlements in the late-eighteenth and early-nineteenth centuries, entrepreneurs mapped coastlines, pioneered trade routes and colonised lands. Backed by private capital they established colonial infrastructural architecture effecting urban expansion in the Australian colonies, New Zealand and beyond. Yet this architecture is rarely a subject of architectural histories

Revealing biases inherent in recombination protocols

<p>Abstract</p> <p>Background</p> <p>The recombination of homologous genes is an effective protein engineering tool to evolve proteins. DNA shuffling by gene fragmentation and reassembly has dominated the literature since its first publication, but this fragmentation-based method is labor intensive. Recently, a fragmentation-free PCR based protocol has been published, termed recombination-dependent PCR, which is easy to perform. However, a detailed comparison of both methods is still missing.</p> <p>Results</p> <p>We developed different test systems to compare and reveal biases from DNA shuffling and recombination-dependent PCR (RD-PCR), a StEP-like recombination protocol. An assay based on the reactivation of β-lactamase was developed to simulate the recombination of point mutations. Both protocols performed similarly here, with slight advantages for RD-PCR. However, clear differences in the performance of the recombination protocols were observed when applied to homologous genes of varying DNA identities. Most importantly, the recombination-dependent PCR showed a less pronounced bias of the crossovers in regions with high sequence identity. We discovered that template variations, including engineered terminal truncations, have significant influence on the position of the crossovers in the recombination-dependent PCR. In comparison, DNA shuffling can produce higher crossover numbers, while the recombination-dependent PCR frequently results in one crossover. Lastly, DNA shuffling and recombination-dependent PCR both produce counter-productive variants such as parental sequences and have chimeras that are over-represented in a library, respectively. Lastly, only RD-PCR yielded chimeras in the low homology situation of GFP/mRFP (45% DNA identity level).</p> <p>Conclusion</p> <p>By comparing different recombination scenarios, this study expands on existing recombination knowledge and sheds new light on known biases, which should improve library-creation efforts. It could be shown that the recombination-dependent PCR is an easy to perform alternative to DNA shuffling.</p

Collaboratively charting the gene-to-phenotype network of human congenital heart defects

Background How to efficiently integrate the daily practice of molecular biologists, geneticists, and clinicians with the emerging computational strategies from systems biology is still much of an open question. Description We built on the recent advances in Wiki-based technologies to develop a collaborative knowledge base and gene prioritization portal aimed at mapping genes and genomic regions, and untangling their relations with corresponding human phenotypes, congenital heart defects (CHDs). This portal is not only an evolving community repository of current knowledge on the genetic basis of CHDs, but also a collaborative environment for the study of candidate genes potentially implicated in CHDs - in particular by integrating recent strategies for the statistical prioritization of candidate genes. It thus serves and connects the broad community that is facing CHDs, ranging from the pediatric cardiologist and clinical geneticist to the basic investigator of cardiogenesis. Conclusions This study describes the first specialized portal to collaboratively annotate and analyze gene-phenotype networks. Of broad interest to the biological community, we argue that such portals will play a significant role in systems biology studies of numerous complex biological processes. CHDWiki is accessible at http://www.esat.kuleuven.be/~bioiuser/chdwikistatus: publishe

Automorphism Modular Invariants of Current Algebras

We consider those two-dimensional rational conformal field theories (RCFTs) whose chiral algebras, when maximally extended, are isomorphic to the current algebra formed from some affine non-twisted Kac--Moody algebra at fixed level. In this case the partition function is specified by an automorphism of the fusion ring and corresponding symmetry of the Kac--Peterson modular matrices. We classify all such partition functions when the underlying finite-dimensional Lie algebra is simple. This gives all possible spectra for this class of RCFTs. While accomplishing this, we also find the primary fields with second smallest quantum dimension.Comment: 32 pages, plain Te

Opportunities for improving animal welfare in rodent models of epilepsy and seizures

Animal models of epilepsy and seizures, mostly involving mice and rats, are used to understand the pathophysiology of the different forms of epilepsy and their comorbidities, to identify biomarkers, and to discover new antiepileptic drugs and treatments for comorbidities. Such models represent an important area for application of the 3Rs (replacement, reduction and refinement of animal use). This report provides background information and recommendations aimed at minimising pain, suffering and distress in rodent models of epilepsy and seizures in order to improve animal welfare and optimise the quality of studies in this area. The report includes practical guidance on principles of choosing a model, induction procedures, in vivo recordings, perioperative care, welfare assessment, humane endpoints, social housing, environmental enrichment, reporting of studies and data sharing. In addition, some model-specific welfare considerations are discussed, and data gaps and areas for further research are identified. The guidance is based upon a systematic review of the scientific literature, survey of the international epilepsy research community, consultation with veterinarians and animal care and welfare officers, and the expert opinion and practical experience of the members of a Working Group convened by the United Kingdom's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)

Considerations for determining optimal mouse caging density

At the 2006 National Meeting of the American Association of Laboratory Animal Science, a panel discussed the question of what constitutes optimal or acceptable housing density for mice. Though there is a consensus that present guidelines are somewhat arbitrarily defined, scientific research has not yet been able to provide clear recommendations for amending them. Speakers explored the many factors that influence decisions on mouse housing, including regulatory requirements, scientific data and their interpretation, financial considerations and ethical concerns. The panel largely agreed that animal well-being should be the measure of interest in evaluating housing density and that well-being includes not only physical health, but also animals\u27 behavior, productivity and preference