Global ETD Search
Search theses and dissertations gathered from participating repositories worldwide. Every result links back to the library that holds it. No account is needed.
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Showing 1 to 18 of 18 for “"Saturation mutagenesis"”.
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Computational and experimental methods for CRISPR-based saturation mutagenesis screens
… The application of high-throughput CRISPR saturation mutagenesis screens for the functional interrogation of the coding and non-coding genome holds great promise in accelerating our understanding of how static DNA sequences encode and influence dynamic processes in human development and …
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A PERTURB-SEQ SATURATION MUTAGENESIS APPROACH TO DISSECT THE MOLECULAR BASES OF GENETIC DISEASES
… of patients. To address this point, MITEseq (Mutagenesis by Integrated TilEs) is a novel saturation mutagenesis technique that allows us to quickly test thousands of potentially pathogenic protein variants in a single high throughput biological assay. As proof of principle, we decided to apply …
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A technique to randomise consecutive codons in a sequence of DNA using MAX oligonucleotides
… the fact that consecutive codons cannot undergo saturation mutagenesis. This thesis describes the development of a method of saturation mutagenesis that can be used to randomise any number of consecutive codons in a DNA strand. The method makes use of “MAX” oligonucleotides coding for each of the …
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Engineering of Aniline Dioxygenase for Bioremediation and Industrial Applications
Another round of saturation mutagenesis on active site residues with 1-K31 as parent followed by random mutagenesis yielded the mutant 3-R21. Whole cell activity of 3-R21 for AN, 24DMA and 2IPA were 27.7, 9.8, and 12 nmol/min/mg protein respectively. The activities of 3-R21 for AN, 24DMA and 2IPA …
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Affinity Maturation of the D1.3 Antibody Using Yeast Surface Display and Flow Cytometry
… of selected mutations using site-directed mutagenesis produced a four-fold higher affinity mutant (MEC1). MEC1 was randomly mutagenized and screened by kinetic selection, identifying a large number of improved mutants. In both screens, library size was approximately 5 x 106, and mutagenesis …
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Genetic and biochemical analysis of a conserved DEAD-box helicase motif
… the role of the conserved motif Va. We combine saturation mutagenesis, in vivo selection, and next-generation sequencing as part of a novel, high-throughput approach to identify functional mutants of motif Va and individual amino acids that are crucial for Mss116p function. We use in vitro …
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Engineering Viruses for Gene Therapy: Isolating and Characterizing Murine Leukemia Virus With Improved Stability
… and infection at 37°C. After one round of mutagenesis and several rounds of selection, we isolated MLV variants with double the half-life of wild-type MLV. The improved stability of the mutant MLV leads to increased virus production, titer, and infection efficiency. Remarkably, a single …
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Investigation of the Evolutionary Aspects of Thiamin Diphosphate-Dependent Decarboxylases
… method to increase the efficiency of the site-saturation mutagenesis used to study ThDP-dependent enzyme evolution.
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Protein and Pathway Engineering for Biosynthesis or Aromatic Compounds
… specificity, which was successfully altered via saturation mutagenesis guided by a homology structural model of PhlD. To improve phloroglucinol production for industrial manufacturing, a two-prone approach was undertaken. Firstly, directed evolution was carried out to improve the poor properties …
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Molecular Simulation of Mutation Effects on Protein Folding and Function
… ensemble (EE) method to perform in silico site saturation mutagenesis on miniprotein binders to the highly conserved influenza A H1 hemagglutinin stem region (HA2) de novo designed by the Baker [66]. These miniproteins were selected through an exhaustive design process with iterations of …
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Engineering of the quorum quenching lactonase GcL for altered substrate specificity by rational design
… These positions were used as targets for site saturation mutagenesis, and several mutants with altered substrate preference were identified in a screening step and confirmed with purified enzyme and kinetic characterization. Several mutations were combined, and the best mutants obtained thus …
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Engineering Estrogen Receptor-Based Gene Switches and a Superoxide Dismutase for Therapeutic Applications
… properties. In the first example, stepwise saturation mutagenesis of functionally important protein sites and random point mutagenesis throughout all protein sites, accompanied by a yeast two-hybrid screening system, was used to engineer variants of the human estrogen receptor alpha …
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Lariat peptide inhibitors of Abl kinase
… at each position of the A1 lariat using site-saturation mutagenesis (SSM). In the second step, we designed specific mutations to the A1 lariat based on the SSM results and evolved higher affinity variants. Synthetic and recombinant higher affinity lariats exhibited a strong inhibition of Abl …
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Conformational engineering of human chemokine receptors and HIV-1 Env using deep mutational scanning
… evolution and deep sequencing with site- saturation mutagenesis (SSM) to create unbiased, diverse libraries, it is now possible to track the phenotypic fitness of thousands of sequence variants in a single experiment. This is known as deep mutational scanning, and it allows for the …
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Engineering nitrilases for enhanced thermostability
… at alkaline pH have been discovered by random mutagenesis and directed evolution; however, these methods are slow and limited in their explorable sequence space. The aim of this project is to investigate the structural and chemical determinants of thermostability in cyanide dihydratases. This …
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Escherichia coli 2-oxoglutarate dehydrogenase multienzyme complex: e1 and e2 substrate specificty, e1 carboligase activity, and e2 interchain succinyl transfer
… 2-oxovalerate (2-OV). E1o was subjected to saturation mutagenesis at H is260 and H is298. The H is298Asp E1 o variant was identified in the screen to accept the unnatural substrate. In addition, it was discovered that His260 is required for substrate recognition, but His298 could be replaced …
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Designed bifunctional proteins for induced degradation of androgen receptor in prostate cancer
… well-behaved soluble proteins. Computational saturation mutagenesis can potentially be used to screen for optimal interaction residues that do not abolish protein stability or solubility. The second approach targeted AR’s DNA-binding domain (DBD) by utilising a short DNA sequence corresponding …
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Probing PAX6-DNA interactions using high-throughput yeast one-hybrid assays and deep mutational scanning
PAX6 is a highly conserved transcription factor essential for the correct development of the central nervous system, the pancreas, and the eye. Heterozygous deletions, nonsense, and frameshift mutations are generally well characterised as causing aniridia, while most missense variants produce a …