Computer simulations have emerged as a cost efficient complement to laboratory experiments, as computers have become increasingly powerful. The aim of the present work is to explore the ideas of some state of the art software development practices, and ways in which these can be useful for developing high performance research codes. The introduction of these practices, and the modular designs that they give rise to, raises issues regarding a potential conflict between runtime efficiency on one hand and development efficiency on the other. Flexible software modules, based on mathematical abstractions, will provide support for convenient implementation and modification of numerical operators. Questions still remain about whether such modules will provide the efficiency which is required for high performance applications. To answer these questions, investigations were performed within two different problem domains. The first domain consisted of modular frameworks for the numerical solution of Partial Differential Equations. Such frameworks proved a suitable setting, since several of my research questions revolved around the issue of modularity. The second problem domain was that of symmetry exploiting algorithms. These algorithms are based on group theory, and make ample use of mathematical abstractions from that field. The domain of symmetry exploiting algorithms gave us opportunities to investigate difficulties in combining modularity based on high level abstractions with low level optimizations using data layout and parallelization. In conclusion, my investigation of software development practices for the area of high performance computing has proved very fruitful indeed. I have found that none of the concerns that were raised should lead us to refrain from the use of the practices that I have considered. On the contrary, in the two case studies presented here, these practices lead to designs that perform well in terms of usability as well as runtime efficiency.

This thesis reports the viability of E. coli cell extracts prepared using equipment that is both common to biotechnology laboratories and able to process small volume samples and expression of proteins containing unnatural amino acids (UAAs) at higher level using PCR amplified linear DNA templates (LETs) in cell-free protein synthesis (CFPS) system. E. coli-based cell extracts are a vital component of inexpensive and high-yielding CFPS reactions. However, effective preparation of E. coli cell extract is limited to high-pressure homogenizers (French press style or impinge-style) or bead mill homogenizers, which all require a significant capital investment. This work specifically assessed the following capital cost lysis techniques: (1) sonication, (2) bead vortex mixing, (3) freeze-thaw cycling, and (4) lysozyme incubation to prepare E. coli cell extract for CFPS. In this work, simple shake flask fermentation with a commercially available E. coli strain was used. Additionally, the RNA polymerase was over expressed in the E. coli cells prior to lysis which eliminated the need to add independently purified RNA polymerase to the CFPS reaction. As a result, high yielding E. coli-based cell extract was prepared using equipment requiring reduced capital investment and common to biotechnology laboratories. To our knowledge, this is the first successful prokaryote-based CFPS reaction to be carried out with extract prepared by sonication or bead vortex mixing. LETs are an attractive alternative to plasmids for site-specific incorporation of unnatural amino acids in proteins in the CFPS system because of their short preparation time and ease of production. However, major limitations associated with LETs are: (1) their degradation by RecBCD enzyme present in the cell-extract used for CFPS and (2) high CFPS energy costs. In this work, we report the optimization of LET-based CFPS for improved protein yield by inhibiting the RecBCD enzyme with small inhibitor molecules resulting in three fold increment in yield of protein containing UAA. We also assessed alternative energy sources such as glucose, fructose-1,6-bisphospate, creatine phosphate/creatine kinase, and high glutamate salt for cost reduction. This work could be important for high-throughput applications based on linear expression templates. This work demonstrates simple E. coli extract preparation and improved yield with linear expression templates for further advancements of cell-free protein synthesis system.

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