Characterization of translation levels of variants of synthetic bacterial RBSs

• Grant number: 18/11926-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: August 01, 2018
• End date: July 31, 2019
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: María Eugenia Guazzaroni
• Grantee: Caroline Moncaio Moda
• Host Institution: Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto (FFCLRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 15/04309-1 - Novel approaches to improve functional screening of biocatalysts in metagenomic libraries, AP.JP

Abstract

Ribosome Binding Sites (RBS) are small sequences that favor the interaction of the 3' region of the 16S ribosomal RNA, the minor subunit component of the prokaryotic ribosome, and are located upstream the initiation codon of the gene to be translated (SHULTZABERGER et al., 2001). Also known as "Shine-Dalgarno Sequences" in prokaryotic RNAs, such sequences influence mRNAs translation rate and have as fundamental characteristics the composition of nucleotides that form the between the region of ribosome recognition and the translation initiation region, which is fundamental to the recruitment of the cellular translational machinery. (NELSON; COX, 2008). Since the nucleotide sequence of a RBS is indicative of its ability to interact with the ribosome, in silico models of the interactions between ribosomes and mRNA have been successfully used to predict relative initiation forces between RBS, making it possible to anticipate potential sequences with desired forces (SALIS et al., 2009). This allows the study of the impact of different translation rates on the cellular machinery during heterologous protein production processes, an analysis that can optimize production rates, especially when considering the demands of the industrial strains (GOROCHOWSKI et al., 2014). A recent study shows that even a small set of RBSs can contemplate protein translation levels in variations of up to two orders of magnitude, concluding that combinatorial assembly of RBSs can significantly modulate translation levels of multiple genes in a synthetic operon (ZELCBUCH et al., 2013). In our research group, an ongoing project aims to combine several levels of translation of three metagenomic genes related to acidity resistance, in order to obtain an optimized cluster of genes that confers resistance to low pH in bacteria. In this work, we intend to investigate the effect of the RBSs selected in the mentioned proposal using the green fluorescent protein (GFP) as a measure of the translation strength in Escherichia coli. The data generated will be integrated with those obtained in the project that aims to expand the resistance to acidity in bacteria using synthetic circuits.