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A high-throughput approach to understand biofilm promoter architecture under different growth conditions

Grant number: 21/07728-6
Support type:Scholarships abroad - Research Internship - Doctorate (Direct)
Effective date (Start): October 01, 2021
Effective date (End): September 30, 2022
Field of knowledge:Biological Sciences - Microbiology - Biology and Physiology of Microorganisms
Principal researcher:Rafael Silva Rocha
Grantee:Ananda Sanches Medeiros
Supervisor abroad: Victor Sourjik
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Research place: Max Planck Society, Marburg, Germany  
Associated to the scholarship:18/04810-0 - Deconstructing complexity in the regulatory network for biofilm formation in gram-negative bacteria, BP.DD

Abstract

Biofilms are bacterial structures influenced by environmental changes like pH variation, temperature differences, carbon source availability, and bacterial growth phase. These influences are processed in different layers from transcriptional gene regulation to post-translational modifications. In transcriptional gene regulation, different transcription factors can bind and/or unbind from a vast variety of promoter binding sites according to their signal processing, comprising a complex biofilm development network. Thus, promoter binding site regions could vary according to environmental signals and bacterial growth phases. Nowadays, SortSeq is one of the most modern approaches to study gene regulation functionally, supporting a dynamic promoter binding site inference through a promoter library (containing different promoters and its random mutated variants) sorting by fluorescence intensity and sequencing. Additionally, it allows a high throughput analysis of several different promoters and their mutated variations in a few experiments. This method is particularly useful for biofilm promoter investigation since we can analyze a number of promoters under different conditions, allowing the evaluation of promoter architecture dynamics. Therefore, the aim of this project is applying and standardizing the SortSeq assay in promoter libraries from static submerged biofilms cultivated under different conditions in the strain Escherichia coli BW25113. These results will then be compared to the results from the SortSeq of the same library under the same conditions in the planktonic lifestyle, standardized at Ribeirao Preto Medical School (FMRP). We aim to analyze 21 different promoters with 349 mutated variants each, totaling 7350 promoters. The project will be done at Microbial Networks Lab directed by Prof. Dr. Victor Sourjik at Max Planck Institute for Terrestrial Microbiology (Marburg, Germany), considering that the receptor laboratory has developed the mentioned procedure and presents the expertise and wide experience in biofilm network studies.

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