Characterisation of new metagenomics regulatory elements

• Grant number: 17/20818-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: January 01, 2018
• End date: June 30, 2018
• Field of knowledge: Biological Sciences - Microbiology
• Principal Investigator: María Eugenia Guazzaroni
• Grantee: Tiago Cabral Borelli
• 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

Comprehending gene expression in bacteria is fundamental to understand the logic of bacterial decisions, to create tools for Synthetic Biology and to elucidate gene expression profiles in complex metagenomics communities. In prokaryotes, there are two main levels of transcriptional regulation. The first one relies on sigma factors (Ã), which give specificity to RNA polymerases by guiding them to specific cis-regulatory sequences in the core promoter region; the second regulatory layer consists in the action of transcriptional factors (TF), whose role is to activate or repress gene expression in response to a myriad of environmental and intracellular stimuli. Together, these two layers comprise a fine-tuned mechanism of gene expression control. Thus, the current project aims to study both regulatory layers in Escherichia coli, by assessing the influence of sigma factors - Ã70 (RpoD), Ã38 (RpoS) and Ã32 (RpoH) and of two global TFs - CRP and Fis - on novel uncharacterized metagenomics promoters. For this purpose, we have used in silico methods for predicting the promoters' architecture and in vivo methods for validating both predictions and their expression profiles. Each promoter was transcriptionally fused to a GFP gene and assessed in vivo using time-lapse fluorescence assays under a broad range of E. coli mutant strains, RpoD, RpoS, RpoH, CRP, IHF, and their respective wild-type strains. Although our preliminary results were focused on establishing optimal experimental conditions for assessing each E. coli strain, we have already observed differences between the expression profiles of two metagenomics fragments. Further work will not only provide solid protocols for our experimental setup, but also essential information regarding the regulatory complexity of environmental promoters recovered in metagenomics libraries using E. coli as a host. (AU)

Scientific publications

(4)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

ALVES, LUANA DE FATIMA; BORELLI, TIAGO CABRAL; WESTMANN, CAUA ANTUNES; SILVA-ROCHA, RAFAEL; GUAZZARONI, MARIA-EUGENIA. Boundaries in metagenomic screenings using lacZ alpha-based vectors. GENETICS AND MOLECULAR BIOLOGY, v. 43, n. 1, 2020. (15/04309-1, 16/05472-6, 17/20818-9, 16/06323-4)

ALVES, LUANA DE FATIMA; WESTMANN, CAUA ANTUNES; LOVATE, GABRIEL LENCIONI; VIANA DE SIQUEIRA, GUILHERME MARCELINO; BORELLI, TIAGO CABRAL; GUAZZARONI, MARIA-EUGENIA. Metagenomic Approaches for Understanding New Concepts in Microbial Science. INTERNATIONAL JOURNAL OF GENOMICS, v. 2018, p. 15-pg., 2018-01-01. (17/20818-9, 16/05472-6, 16/06323-4, 15/04309-1)

ALVES, LUANA DE FATIMA; WESTMANN, CAUA ANTUNES; LOVATE, GABRIEL LENCIONI; VIANA DE SIQUEIRA, GUILHERME MARCELINO; BORELLI, TIAGO CABRAL; GUAZZARONI, MARIA-EUGENIA. Metagenomic Approaches for Understanding New Concepts in Microbial Science. INTERNATIONAL JOURNAL OF GENOMICS, 2018. (16/06323-4, 15/04309-1, 16/05472-6, 17/20818-9)

LUANA DE FÁTIMA ALVES; TIAGO CABRAL BORELLI; CAUÃ ANTUNES WESTMANN; RAFAEL SILVA-ROCHA; MARÍA-EUGENIA GUAZZARONI. Boundaries in metagenomic screenings using lacZα-based vectors. GENETICS AND MOLECULAR BIOLOGY, v. 43, n. 1, 2020-03-06. (16/06323-4, 17/20818-9, 16/05472-6, 15/04309-1)