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Tesi etd-09042016-164400

Thesis type
Tesi di laurea magistrale
Engineering the methanogenic-type seryl-tRNA synthetase from Methanosarcina barkeri: a new methodological approach
Corso di studi
relatore Dott.ssa Moschini, Roberta
Parole chiave
  • Screening di librerie
  • Library Creation
  • Espansione del codige genetico
  • Genetic code expansion
  • Amminoacil-tRNA sintetasi
  • Aminoacyl-tRNA synthetase
  • Creazione di librerie
  • Library screening
  • Site-saturation mutagenesis
Data inizio appello
Data di rilascio
Riassunto analitico
Protein synthesis is a fundamental process that involves the transcription of the genetic information contained in the DNA sequence into an mRNA molecule, that will be used as a template for the condensation of amino acids into a polypeptide chain. Such process is guided by the macromolecular complex of the ribosome. Amino acids are delivered to the ribosome linked to an adaptor, a tRNA molecule, capable of base pairing with the mRNA codons, thus coupling the alphabet of DNA with the one of proteins. The fundamental role to link amino acids with the correct tRNAs, generating the decoding scheme, is provided by a class of enzymes known as aminoacyl-tRNA synthetases.
Recently, among synthetic biologists the interest grew to expand the genetic code, introducing unnatural amino acids into proteins in vivo engineering the translational apparatus of the cells. If properly chosen, non-canonical amino acids (UAAs) can, for example:mimic or reproduce the effect of post-translational modification on residues of biological interest;selectively label proteins in a non-invasive manner;
interfere with proteins' activity with high spatial/temporal resolution etc.
For this technology to work, it is essential that a tRNA/synthetase pair exists that does not interfere with the endogenous translational apparatus (namely, that is orthogonal), and that can link the UAA to the tRNA. There are at least two orthogonal tRNA/synthetase pairs for E. coli: the tRNATyr/tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii and the tRNAPyl/pyrrolysyl-tRNA synthetase from Methanosarcina barkeri or mazei, both of which were engineered to recognise several different UAAs. Recently, the tRNASer/seryl-tRNA synthetase from Methanosarcina barkeri was observed to be orthogonal in E. coli and to possess several others properties that make it a suitable candidate to become a new tool for the genetic code expansion. Nevertheless, this possibility has not been explored so far. In this thesis I will describe an attempt to engineer the active site of the enzyme in order to allow the incorporation of different amino acids. To do so, two different libraries of mutants were built using site-saturation mutagenesis and analysed to find mutants active on the desired substrates. While hits of interest were not experimentally observed, possible candidates were found bioinformatically from the data analysis of a high-throughput experiment using NGS and will be tested in the near future. Furthermore, I developed new tools to improve the library construction process and overcome some of the difficulties that I faced during the experimental work.