Year: 2023
Type: dissertation
Abstract: Streptococcus pneumoniae is a significant Gram-positive bacterial pathogen known for the global impact caused by the ability of its clinical strains to resist antibiotics and to cause disease. The understanding of how bacteria regulate protein expression can be improved through the design of synthetic RNA thermoswitches capable of distinguishing the temperature range found within an infected human host. However, achieving precise gene expression control is a complex task that requires an accurate prediction of the behavior of a particular RNA-based thermoswitch and a deep understanding of bacterial regulatory mechanisms and RNA thermodynamics. This work aimed to adapt a reported thermodynamic model to design pneumococcal thermoswitches. To determine the optimal aligned spacing in S. pneumoniae, which is required for the modeling process, several pneumococcal mutant strains were constructed carrying the gene encoding the reporter iCitrine protein with a variable spacing sequence between the ribosome binding site and the start codon. In addition, quantification of the fluorescence signal detected in each pneumococcal cell was facilitated by the development of an automated segmentation process that streamlined the analysis of many microscopy images. Despite ongoing work to determine the optimal spacing in S. pneumoniae, it was observed that maximum iCitrine expression can be achieved with a particular set of spacers. Moreover, it was confirmed that translation of fluorescent proteins can be influenced by the mRNA secondary structures present at the 5’ end of the transcribed molecules.
Author: Piteira, André Luís Caetano
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W6999606334