Comparison of synthetic promoters expression activity in the chromosome and in plasmids

• Grant number: 17/04938-4
• Support Opportunities: Scholarships abroad - Research Internship - Scientific Initiation
• Start date: June 01, 2017
• End date: August 30, 2017
• Field of knowledge: Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
• Principal Investigator: Rafael Silva Rocha
• Grantee: Ananda Sanches Medeiros
• Supervisor: Victor de Lorenzo
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Institution abroad: Consejo Superior de Investigaciones Científicas (CSIC), Spain
• Associated to the scholarship: 15/22386-3 - Construction and characterization of constitutive mutant promoters and regulated by the global regulators CRP and Fis in Escherichia coli, BP.IC

Abstract

The study of gene expression logic in bacteria made in the field of Synthetic Biology is very dependent on the use of mutant strains for specific transcriptional factors since it allows characterizing each circuit component in isolation. Additionally, analysis of promoter activity in vivo is usually performed using fluorescent reporter genes (such as GFP) harbored in plasmids. However, this approach can cause cellular variations that could biases the analysis of gene expression, since it can burden the cellular metabolism or since plasmid copy number might vary from cell to cell. We have characterized the absence of pleiotropic effects on GFP expression regulated by Synthetic Constitutive Promoters inserted in mutant bacteria for Global Transcription Factors (CRP, IHF and Fis) using low copy plasmids. Here, we propose to implement a stable monocopy reporter system for chromosomal integration in order to characterize promoter activity in E. coli. Additionally, we will test novel synthetic promoters regulated by CRP and Fis from a library of promoter architectures in monocopy using chromosomal integration. This approach will be done using Transposons Vectors, largely described by Molecular Environmental Microbiology Laboratory, where this project will be developed. The approach proposed here will set a new methodology that will be later implemented in Ribeirão Preto with an important impact in the main project of the group. (AU)