Construction and characterization of constitutive mutant promoters and regulated by the global regulators CRP and Fis in Escherichia coli

• Grant number: 15/22386-3
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2016
• End date: December 31, 2017
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Rafael Silva Rocha
• Grantee: Ananda Sanches Medeiros
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated research grant: 12/22921-8 - Synthetic biology approaches for deciphering the logic of signal integration in complex bacterial promoters, AP.JP
• Associated scholarship(s): 17/04938-4 - Comparison of synthetic promoters expression activity in the chromosome and in plasmids, BE.EP.IC

Abstract

Bacteria have global regulators responsible for the expression of multiple genes. For example, the transcriptional factors CRP, Fis and IHF are very important to the bacteria. In fact, mutant strain lacking these regulators have strong alteration in the metabolism and growth, leading to the modification of gene expression and cell physiology. In this way, this proposition aims to analyze the behavior of some previously reported promoters in E. coli wild type and mutant for IHF and Fis in order to standardize gene expression in these strains. Additionally, we aim to analyze the interaction between these factors through the construction of complex promoter for CRP and Fis. This library will be constructed through the ligation of consensus binding sites for CRP and Fis, generating synthetic promoters. These synthetic promoters will be cloned into pMR1 vector, allowing the quantification of promoter activity using GFP reporter gene. These assays will be performed in a plate reader, as well as by flow cytometry and fluorescence microscopy.

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)

AMORES, GERARDO RUIZ; DE LAS HERAS, AITOR; SANCHES-MEDEIROS, ANANDA; ELFICK, ALISTAIR; SILVA-ROCHA, RAFAEL. Systematic identification of novel regulatory interactions controlling biofilm formation in the bacterium Escherichia coli. SCIENTIFIC REPORTS, v. 7, DEC 1 2017. (15/22386-3, 12/22921-8)

SANCHES-MEDEIROS, ANANDA; OLIVEIRA MONTEIRO, LUMMY MARIA; SILVA-ROCHA, RAFAEL. Calibrating Transcriptional Activity Using Constitutive Synthetic Promoters in Mutants for Global Regulators in Escherichia coli. INTERNATIONAL JOURNAL OF GENOMICS, v. 2018, p. 10-pg., 2018-01-01. (16/19179-9, 12/22921-8, 15/22386-3)

SANCHES-MEDEIROS, ANANDA; OLIVEIRA MONTEIRO, LUMMY MARIA; SILVA-ROCHA, RAFAEL. Calibrating Transcriptional Activity Using Constitutive Synthetic Promoters in Mutants for Global Regulators in Escherichia coli. INTERNATIONAL JOURNAL OF GENOMICS, 2018. (16/19179-9, 15/22386-3, 12/22921-8)

MARTINS-SANTANA, LEONARDO; NORA, LUISA C.; SANCHES-MEDEIROS, ANANDA; LOVATE, GABRIEL L.; CASSIANO, MURILO H. A.; SILVA-ROCHA, RAFAEL. Systems and Synthetic Biology Approaches to Engineer Fungi for Fine Chemical Production. FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, v. 6, OCT 3 2018. (15/22386-3, 17/17924-1, 16/11093-8, 17/04217-5, 16/03763-3, 12/22921-8)