Algorithms for optimizing cross-overs in DNA shuffling

DNA shuffling generates combinatorial libraries of chimeric genes by stochastically recombining parent genes. The resulting libraries are subjected to large-scale genetic selection or screening to identify those chimeras with favorable properties (e.g., enhanced stability or enzymatic activity). While DNA shuffling has been applied quite successfully, it is limited by its homology-dependent, stochastic nature. Consequently, it is used only with parents of sufficient overall sequence identity, and provides no control over the resulting chimeric library. Results: This paper presents efficient methods to extend the scope of DNA shuffling to handle significantly more diverse parents and to generate more predictable, optimized libraries. Our CODNS (cross-over optimization for DNA shuffling) approach employs polynomial-time dynamic programming algorithms to select codons for the parental amino acids, allowing for zero or a fixed number of conservative substitutions. We first present efficient algorithms to optimize the local sequence identity or the nearest-neighbor approximation of the change in free energy upon annealing, objectives that were previously optimized by computationally-expensive integer programming methods. We then present efficient algorithms for more powerful objectives that seek to localize and enhance the frequency of recombination by producing “runs” of common nucleotides either overall or according to the sequence diversity of the resulting chimeras. We demonstrate the effectiveness of CODNS in choosing codons and allocating substitutions to promote recombination between parents targeted in earlier studies: two GAR transformylases (41% amino acid sequence identity), two very distantly related DNA polymerases, Pol X and b (15%), and beta- lactamases of varying identity (26-47%). Conclusions: Our methods provide the protein engineer with a new approach to DNA shuffling that supports substantially more diverse parents, is more deterministic, and generates more predictable and more diverse chimeric libraries

Progress in strategies for sequence diversity library creation for directed evolution

Protein engineering has been the most attractive strategy for biologists to redesign enzymes. As the simplest technique of protein engineering, directed evolution has been applied to many fields, such as industry, agriculture and medicine. An experiment of directed evolution comprises mutant libraries creation and screening or selection for enzyme variants with desired properties. Therefore, a successful application of directed evolution depends on whether or not one can generate a quality library and perform effective screening to find the desired properties. Directed evolution is already increasingly used in many laboratories to improve protein stability and activity, alter enzyme substrate specificity, or design new activities. Meanwhile, many more effective novel strategies of mutant library generation and screening or selection have emerged in recent years, and will continue to be developed. Combining computational/rational design with directed evolution has been developed as more available means to redesign enzymes.Keywords: Protein engineering, directed evolution, sequence diversity creation, novel strategy, computational design, rational desig

Genetic Modification Approaches for Parasporins Bacillus thuringiensis Proteins with Anticancer Activity

Bacillus thuringiensis (Bt) is a bacterium capable of producing Cry toxins, which are recognized for their bio-controlling actions against insects. However, a few Bt strains encode proteins lacking insecticidal activity but showing cytotoxic activity against different cancer cell lines and low or no cytotoxicity toward normal human cells. A subset of Cry anticancer proteins, termed parasporins (PSs), has recently arisen as a potential alternative for cancer treatment. However, the molecular receptors that allow the binding of PSs to cells and their cytotoxic mechanisms of action have not been well established. Nonetheless, their selective cytotoxic activity against different types of cancer cell lines places PSs as a promising alternative treatment modality. In this review, we provide an overview of the classification, structures, mechanisms of action, and insights obtained from genetic modification approaches for PS proteins

Synthetic biology approaches to the metabolic engineering of Geobacillus thermoglucosidans for isobutanol production

Renewable green alternatives to fossil fuels need to be sought in order to address the challenges of environmental and energy crises. Up until now, ethanol has been the major biofuel. Geobacillus thermoglucosidans is a thermophilic bacterium that is capable of producing bioethanol in an industrial setting at high temperatures and is capable of metabolizing pentoses and hexoses commonly found in lignocellulosic biomass. Due to these attractive properties, the aim of this work has been to construct a toolbox of genetic components to develop G. thermoglucosidans as as the leading thermophile chassis for synthetic biology and metabolic engineering. The toolbox is composed of shuttle vectors that have higher transformation efficiencies than previous existing vectors and are modular, where the presence of restriction sites separating each of the components allows users to exchange parts easily and efficiently. Also included in the toolbox are the fluorescent reporters sfGFP, mCherry and BsFbFP that will permit the characterization of promoters. As a proof-of-principle application to demontrate the effectivity of the toolbox for the production of valuable compounds, this work explores the production of isobutanol by the thermophile bacteria Geobacillus thermoglucosidans. Isobutanol is a higher chain alcohol that is a significantly better fuel molecule than ethanol, both for energy content and infrastructure compatibility. The Geobacillus host was able to produce isobutanol in amounts of around 50 mg/L via the conversion of isobutyryl-CoA to isobutyraldehyde by an (ALDH) and from isobutyraldehyde to isobutanol by an alcohol dehydrogenase (ADH). It was observed that supplementing the growth medium with an intermediate of the valine biosynthesis pathway, 2-ketoisovalerate, resulted in the production of isobutanol and overexpressing ALDH increased the isobutanol titres.Open Acces

Protein Engineering of Escherichia coli β-glucuronidase

This thesis describes engineering studies with the Escherichia coli β-glucuronidase enzyme (E. coli β-GUS) that catalyzes the hydrolysis of D-glucuronic acids (glycone) that are conjugated through a β-O-glycosidic linkage to an aglycone. The enzyme is specific for the glucuronic acid component and will tolerate a variety of aglycones. A point mutation was known to convert β-GUS into a glucuronylsynthase, that is an enzyme capable of the synthesis of glucuronide conjugates. The long-term aim of the present research was to change the donor sugar specificity of the glucuronylsynthase from a glucuronyl donor to a glucosyl donor allowing the synthesis of glucosides. The approach taken to achieve the long-term aim was to first alter the specificity of β-GUS and to then convert the variants to a synthase. There were two approaches taken to altering the substrate specificity of β-GUS. The first approached was to use site saturation mutagenesis to alter key residues that are located at the active site. A more effective approach involved using directed evolution to generate variants with altered specificity. A selection of these variants were converted to (putative) synthetic enzymes and tested for activity. The structure-guided site saturation mutagenesis of 9 sites was carried out in an attempt to alter the substrate specificity of β-GUS. The choice of the residues to be altered was made with the aid of the structure of β-GUS with a bound substrate analogue. Nine codons in the β-GUS gene were randomised to create libraries that contained all possible amino acid of residues in and / or near the active site. Mutants were assayed using substrates presenting 5 different glycones. Of the positions randomised, most glycosyl binding residues were found not to tolerate amino acid substitutions, suggesting they are essential for β-GUS function while majority of non-glycosyl binding residues were found to tolerate amino acid substitutions – but not with good activity One of the common dogmas of directed evolution is the idea that evolvability is related to stability. We set out to test this idea while evolving substrate specificity. Other workers had generated a more thermostable variant of β-GUS. In parallel, we evolved 1) the native enzyme (GUS-WT) and 2) the thermostable (GUS-TR3337) variant of β-GUS. Mutant libraries of both GUS-WT and GUS-TR3337 were created under identical conditions and had the same distributions of mutations. After five rounds of evolution, the catalytic efficiency (kcat/Km) of the best mutant of the wild type parent for pNP-glucoside was increased ~307-fold while the best mutant of the thermophilic parent demonstrates a ~4-fold increased kcat/Km over the best mutant of wild type parent. Selected mutants from both libraries were characterised with regard to conformational properties and stability and these investigations, combined with kinetic data, provided valuable information about how thermostability promotes the ease of protein evolvability. The initial characterisation of β-GUS variants was done with crude lysates. It was observed that the GUS-WT and GUS-TR3337 variants lost their newly evolved activity after purification. It was eventually determined that the BugBusterTM reagent used to lyse cells prior to screening, had affected the directed evolution campaign. The n-octyl β-D-thioglucopyranoside (OTG) presents in the BugBusterTM reagent, was very similar in structure to pNP-glucoside and was identified as a competitive inhibitor that suppresses glucosidase activity of wild-type β-GUS, indicating that it can be bound in the active site. In response to the addition of OTG in the screening assay, OTG enhanced the glucosidase activity of selected mutants. This observation highlights the potential pitfall in the use of commercial reagents to lyse cells for enzymes with glycosidase activities. However, the evolution in the presence of OTG gives some insight into how an enzyme might evolve to be regulated by an effector molecule – OTG, in this case. Finally, improved cell lysis variants were converted to glucuronylsynthase variants by introducing the site specific mutation. Their glycosynthase ability was tested using a similar protocol developed by McLeod Group for assaying glucuronylsynthase activity. Unfortunately, glycosynthase activity was not observed with α-D-glucuronyl fluoride donor and steroid accepters. Time constraints did not allow other substrate to be tested

Understanding Quantum Technologies 2022

Understanding Quantum Technologies 2022 is a creative-commons ebook that provides a unique 360 degrees overview of quantum technologies from science and technology to geopolitical and societal issues. It covers quantum physics history, quantum physics 101, gate-based quantum computing, quantum computing engineering (including quantum error corrections and quantum computing energetics), quantum computing hardware (all qubit types, including quantum annealing and quantum simulation paradigms, history, science, research, implementation and vendors), quantum enabling technologies (cryogenics, control electronics, photonics, components fabs, raw materials), quantum computing algorithms, software development tools and use cases, unconventional computing (potential alternatives to quantum and classical computing), quantum telecommunications and cryptography, quantum sensing, quantum technologies around the world, quantum technologies societal impact and even quantum fake sciences. The main audience are computer science engineers, developers and IT specialists as well as quantum scientists and students who want to acquire a global view of how quantum technologies work, and particularly quantum computing. This version is an extensive update to the 2021 edition published in October 2021.Comment: 1132 pages, 920 figures, Letter forma

Analyses structurales et fonctionnelles de l'espace génique du chromosome 3B du blé tendre (Triticum aestivum L.)

Genome-wide studies of the bread wheat are a complicated task due to its large size (17 Gb), its allohexaploidy and its high content in repeat sequences (>80%). Using a chromosome-specific approach, the chromosome 3B (995 Mb) was successfully isolated and sequenced leading to the assembly of one pseudomolecule. The work presented in this thesis investigated the impact of the 3B chromosome size on the gene space organization. Production of transcriptomic data was achieved using RNA-Seq approach. The chromosome 3B was annotated and we predicted 7 264 features, including 5 326 full genes and 1 938 pseudogenes. We constructed RNA-Seq libraries for 15 developmental wheat conditions. Using this data we detected expression of 71.4% of the predictions, and 3 692 novel transcribed regions (NTR). We also detected alternative transcripts for 61% of the expressed genes, with 5.8 isoforms on average for one gene. Using these transcriptional data, we highlighted a partitioning of the chromosome 3B gene space. Indeed, transcription was found all along the chromosome, but genes were organized according to an increasing density gradient along the centromere-telomere axis. Based on recombination profile, we segmented the chromosome in 3 major regions: R1, R2 and R3. The region R2 was identified with low or no recombination rate corresponding to the centromeric and peri-centromeric regions (647 Mb). The regions R1 and R3 were associated with a higher recombination rate, both localized on the distal part of the short arm (58 Mb) and the long arm (69 Mb) respectively, where the recombination rate is higher. All three regions showed distinct level and specificity of gene expression as well as unique gene structure (variation size, exon number, intron size). Indeed, genes expressed in a specific condition and with a small number of alternatives transcripts were localized on regions R1 and R3. We showed that two evolutionary model could explain the link between gene structure and the level/specificity of expression : “selection for economy” and “genome design”. In conclusion, a transcriptomic studies was achieved along the 3B chromosome for the first time. This study demonstrated a relationship between gene characteristics (structure, expression level, expression specificity and evolution) and the chromosome 3B organization. Future pseudomolecule assemblies will help us to assess the structural organization of these chromosomes. In order to better understand the cellular mechanisms of gene expression, an epigenomic study of the 3B chromosome was started.De par sa taille (17 Gb), la complexité de son génome (allohexaploïde) ainsi que la forte proportion d’éléments répétés (>80%), l’étude du génome de blé tendre est une tâche particulièrement complexe et s’est souvent retrouvée confrontée aux limites technologies. Grâce une approche de tri de chromosomes, le chromosome 3B (995 Mb) a pu être isolé et séquencé. Ces données ont permis la construction d’une pseudomolécule. Mes travaux de thèse se sont basés sur des données de transcriptomique produites avec une approche RNA-Seq, afin d’investiguer l’impact de la taille de ce chromosome sur l’organisation de l’espace génique. L’annotation du chromosome 3B a permis de mettre en évidence : 5 326 gènes et 1 938 pseudogènes. L’analyse des librairies RNA-Seq pour 15 conditions de développement a permis de mettre en évidence l’expression de 71 % des gènes annotés, ainsi que 3 692 régions nouvellement transcrites (NTR). Nous avons aussi pu détecter des transcrits alternatifs pour 61% des gènes exprimés (en moyenne 6 isoformes). Nous avons donc pu mettre en évidence une structuration de l’espace génique pour le chromosome 3B. En effet, la transcription est répartie sur tout le chromosome, cependant les gènes sont organisés selon un gradient de densité croissant sur l’axe centromère-télomère. En nous basant sur le profil des données de recombinaison, nous avons divisé le chromosome en 3 régions : R1, R2 et R3. La région R2 correspondant à la région centrale du chromosome (647 Mb) où le taux de recombinaison est très faible voir absent. Les régions R1 (58 Mb) et R3 (69 Mb) correspondent respectivement aux parties distales du bras court et du bras long du chromosome, où le taux de recombinaison est le plus fort. Ces trois régions diffèrent par leur niveau et leur spécificité d'expression, ainsi que par leur structure génique (nombre d'exons, taille des introns …). En effet, les gènes ayant une expression tissu-spécifique, ainsi qu’un faible nombre de transcrits alternatifs sont retrouvés dans les régions R1 et R3. Deux modèles peuvent expliquer le lien observé entre la structure des gènes et leur niveau/spécificité d’expression : le modèle de la sélection pour l’économie et le modèle dessin génomique. En conclusion, ce travail a montré et ce, pour la première fois à l’échelle d’un chromosome entier de blé, l’impact de la taille du chromosome sur l’organisation ; mettant en relation la structure des gènes, leur niveau d’expression, leur spécificité d’expression, ainsi que leur nature évolutive. L’assemblage ainsi que l’annotation de pseudomolécules des autres chromosomes permettra de mettre en évidence si cette structure est conservée. Afin de mieux comprendre les mécanismes cellulaires impliqués dans la régulation de l’expression des gènes, une étude du paysage épigénomique a été engagée

Radiation Hybrid Fine Mapping of Two Fertility-Related Genes: Marking the Path to Wheat Hybrids

Over one billion people, more than 1/9th of the global population, are undernourished. Feeding the ever increasing population has to be the most important goal of plant sciences. Since cultivated areas are not likely to increase, I will need to produce more with what is available. This can be summarized in one word: yield. Unfortunately, wheat’s yield is expected to increase only 1.13% by 2019, a prediction that if converted into reality will likely indicate that I failed to cope with the world demographic increase. A new strategy to revolutionize wheat production is required, and some believe that this change might be represented by wheat hybrids. Achieving adequate commercial production of wheat hybrids has the potential to nearly double the yield of one of the world’s most important staple food. The first fundamental step toward this goal is to develop feasible methodologies to sterilize the male part of the complete wheat flowers. Two fertility-related genes are the primary target of this study, namely the species cytoplasm specific on chromosome 1D, and the desynaptic locus on chromosome 3B. This dissertation summarizes the important achievements obtained toward the cloning of the two loci by means of radiation hybrid functional analysis. Radiation hybrid is a technique that employs radiation to create genetic diversity along the targeted chromosome. Chapter 1 explains in details how this methodology can be applied to plants. The use of radiation hybrid mapping permitted creating a comprehensive map of wheat chromosome 3B, as discussed in Chapter 2, and then expanded the mapping information to identify the 2 Mb location of the desynaptic locus desw2, as discussed in Chapter 3. A similar approach on chromosome 1D allowed first to pinpoint the location of the species cytoplasm specific gene to a region of 2 Mb, as discussed in Chapter 4, and then ultimately to find a strong candidate for this locus, as discussed in Chapter 5. Now that the molecular locations of these genes have been unraveled by this study, their sequence can be streamlined into transformation to ultimately produce female wheat plants, and consequently hybrids