Carrera Ingeniería Bioquímica

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Author: search.filters.author.Herrera Aldaz, Bryan AlexanderSubject: search.filters.subject.IDEONELLA SAKAIENSIS

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    Diseño y validación in silico de primers para la construcción de las variantes mutantes S121E, S121D, D186H y R280A de la enzima PETasa de Ideonella sakaiensis mediante tres diferentes métodos de mutagénesis dirigida al sitio
    (Universidad Técnica de Ambato. Facultad de Ciencia e Ingeniería en Alimentos y Biotecnología. Carrera de Ingeniería Bioquímica, 2022-03) Herrera Aldaz, Bryan Alexander; Cerda Mejía, Liliana Alexandra
    The PETase enzyme from Ideonella sakaiensis (IsPETase) can be used to degrade PET, however, although it has been reported to date to have the highest enzymatic activity under normal conditions of all PET degrading enzymes, its low thermal stability limits its analysis and application. Therefore, by targeted mutagenesis, specific mutations can be introduced into the DNA to significantly increase its enzymatic activity. The purpose of this study was the design and in silico validation of primers for the construction of S121E, S121D, D186H and R280A mutant variants of IsPETase using three site-directed mutagenesis techniques: QuikChange, Q5 Site-Directed Mutagenesis and Phusion Site-Directed Mutagenesis. For the design of the primers, the parameters contained in the design guidelines for targeted mutagenesis methods were considered. The results showed that the 24 primers obtained meet the general criteria such as length, melting temperature, percentage of GC content and the position of the mutation in the primer. For the validation of the proposed primers, hairpin, autodimer and heterodimer analysis was performed with bioinformatics tools, the calculated values are within acceptable ranges. Finally, the specificity of the primers design was carried out by means of alignments, whose results indicate a high specificity for their respective targets. Likewise, an in silico PCR assay confirmed the specificity of the primers, which represent the expected amplification product, thus demonstrating the correct selection of the designed primers and the execution of the mutagenic PCR process.