Synthetic biology approaches for deciphering the logic of signal integration in complex bacterial promoters

• Grant number: 12/22921-8
• Support Opportunities: Research Grants - Young Investigators Grants
• Duration: March 01, 2015 - February 29, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal Investigator: Rafael Silva Rocha
• Grantee: Rafael Silva Rocha
• Host Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
• Associated researchers: Stephen Busby ; Víctor de Lorenzo
• Associated scholarship(s): 19/14883-8 - Reconstruction of the gene regulatory network in Pseudomonas aeruginosa, BP.IC; 19/06672-7 - Genome assembly for identification of antibiotic resistance genes in clinical isolates of the Hospital das Clínicas de Ribeirão Preto, BP.IC; 19/04942-7 - Synthetic biology applied to Rhodosporidium toruloides for fine chemical production, BP.DR; + associated scholarships 19/04604-4 - Screening and characterization of hypoxia responsive promoters using functional genomics, BP.IC; 19/03212-5 - Standardization of a high-performance expression system for Escherichia coli based on benzoate inducer, BP.TT; 18/06509-6 - Design of novel synthetic yeast promoters based on in silico approaches, BP.MS; 18/04810-0 - Deconstructing complexity in the regulatory network for biofilm formation in gram-negative bacteria, BP.DD; 18/08607-5 - Quantifying the effect of space between -35/-10 boxes in synthetic bacterial promoters, BP.IC; 18/01820-5 - Standardization of a high-performance expression system for Escherichia coli based on benzoate inducer, BP.TT; 18/03857-3 - Deconstruction of regulatory complexity in Escherichia coli through systemic biology approaches, BP.IC; 18/03274-8 - Application of synthetic biology in the construction of genetic circuit to improve the antitumor effect of Salmonella spp., BP.MS; 17/17924-1 - Construction of synthetic promoters for the expression of cellulases using novel cis-regulatory elements in Trichoderma reesei, BP.DD; 17/04217-5 - Development of a computational tool for searching and strength prediction of bacterial promoters for applications in synthetic biology, BP.IC; 16/19179-9 - Deciphering the architecture/function relationship in complex bacterial promoters through synthetic biology approaches, BP.DR; 16/05472-6 - Synthetic biology approaches to engineer/mine novel regulatory elements in bacteria, BP.MS; 16/11093-8 - Construction of an ultrasensitive arsenic biosensor through synthetic biology approaches, BP.IC; 16/03763-3 - Unveiling the mechanisms of gene regulation at the single cell level in Trichoderma reesei, BP.MS; 16/01946-3 - Construction of synthetic promoters for the expression of cellulases using novel cis-regulatory elements in Trichoderma reesei, BP.MS; 15/22386-3 - Construction and characterization of constitutive mutant promoters and regulated by the global regulators CRP and Fis in Escherichia coli, BP.IC - associated scholarships

Abstract

The ability to coordinate expression of thousands of genes in response to different stimuli is an essential feature of any live organism. However, current knowledge on this matter is largely inferred from data on extant systems involving just a few regulatory components. In this context, this project propose the adoption of a genetically tractable experimental object (Escherichia coli) along with the conceptual and material tools of Synthetic Biology for examining the molecular mechanisms (hardware) and the underlying logic program (software) that account for the regulatory behavior of complex biological systems. The principal approach to this end will involve the generation of synthetic promoter libraries assembled adjacent to combinatorial arrays of upstream binding sequences for five known global regulators (IHF, Fis, Crp, NarL and Fnr). The behavior of the resulting promoter libraries will be thoroughly parameterized in single cells and in populations as a whole for quantification of their transcriptional capacity, kinetics and stochastic noise. The resulting data will be employed to feed computational models for identifying general rules that account for the regulation of the thereby generated promoters and predict the outcome of new ones. We expect that the results of this endeavor will contribute not only a fundamental understanding mechanisms of signal integration in prokaryotic promoters, but will also produce rules and methods for designing and building standardized, integrated biological systems to accomplish many particular tasks of biotechnological interest. (AU)

Scientific publications

(18)

(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)

NORA, LUISA CZAMANSKI; WESTMANN, CAUA ANTUNES; MARTINS-SANTANA, LEONARDO; ALVES, LUANA DE FATIMA; OLIVEIRA MONTEIRO, LUMMY MARIA; GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL. The art of vector engineering: towards the construction of next-generation genetic tools. MICROBIAL BIOTECHNOLOGY, v. 12, n. 1, p. 125-147, JAN 2019. (16/19179-9, 17/17924-1, 16/06323-4, 16/05472-6, 16/03763-3, 15/04309-1, 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)

AMORES, GERARDO RUIZ; GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL. Engineering Synthetic cis-Regulatory Elements for Simultaneous Recognition of Three Transcriptional Factors in Bacteria. ACS SYNTHETIC BIOLOGY, v. 4, n. 12, p. 1287-1294, DEC 2015. (12/22921-8)

OLIVEIRA MONTEIRO, LUMMY MARIA; SANCHES-MEDEIROS, ANANDA; WESTMANN, CAUA ANTUNES; SILVA-ROCHA, RAFAEL. Unraveling the Complex Interplay of Fis and IHF Through Synthetic Promoter Engineering. FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY, v. 8, JUN 18 2020. (18/04810-0, 12/22921-8, 16/05472-6, 16/19179-9, 17/50116-6)

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)

CAMPOS ANTONIETO, AMANDA CRISTINA; VIEIRA NOGUEIRA, KAROLINE MARIA; DE PAULA, RENATO GRACIANO; NORA, LUFSA CZAMANSKI; ANZOLINI CASSIANO, MURILO HENRIQUE; GUAZZARONI, MARIA-EUGENIA; ALMEIDA, FAUSTO; DA SILVA, THIAGO APARECIDO; RIES, LAURE NICOLAS ANNICK; DE ASSIS, LEANDRO JOSE; et al. A Novel Cys2His2 Zinc Finger Homolog of AZF1 Modulates Holocellulase Expression in Trichoderma reesei. MSYSTEMS, v. 4, n. 4, JUL-AUG 2019. (16/23233-9, 16/03322-7, 15/04309-1, 17/04217-5, 16/03763-3, 18/03766-8, 15/09553-8, 12/22921-8)

LUÍSA CZAMANSKI NORA; RELBER AGUIAR GONÇALES; LEONARDO MARTINS-SANTANA; BEATRIZ HENRIQUES FERREIRA; FERNANDO RODRIGUES; RAFAEL SILVA-ROCHA. Synthetic and minimalist vectors for Agrobacterium tumefaciens-mediated transformation of fungi. GENETICS AND MOLECULAR BIOLOGY, v. 42, n. 2, p. 395-398, 2019-06-13. (14/22561-7, 16/03763-3, 16/01946-3, 12/22921-8)

LEONARDO MARTINS-SANTANA; RENATO GRACIANO DE PAULA; ADRIANO GOMES SILVA; DOUGLAS CHRISTIAN BORGES LOPES; ROBERTO DO NASCIMENTO SILVA; RAFAEL SILVA-ROCHA. CRZ1 regulator and calcium cooperatively modulate holocellulases gene expression in Trichoderma reesei QM6a. GENETICS AND MOLECULAR BIOLOGY, v. 43, n. 2, 2020-05-08. (17/17924-1, 12/22921-8)

WESTMANN, CAUA ANTUNES; GUAZZARONI, MARA-EUGENIA; SILVA-ROCHA, RAFAEL. Engineering Complexity in Bacterial Regulatory Circuits for Biotechnological Applications. MSYSTEMS, v. 3, n. 2, SI, MAR-APR 2018. (15/04309-1, 16/05472-6, 12/22921-8)

DE FRIAS, ULYSSES AMANCIO; BONIFACIO PEREIRA, GREICY KELLY; GUAZZARONI, MARIA-EUGENIA; SILVA-ROCHA, RAFAEL. Boosting Secondary Metabolite Production and Discovery through the Engineering of Novel Microbial Biosensors. BIOMED RESEARCH INTERNATIONAL, 2018. (15/04309-1, 12/22921-8)

OLIVEIRA MONTEIRO, LUMMY MARIA; ARRUDA, LETICIA MAGALHAES; SILVA-ROCHA, RAFAEL. Emergent Properties in Complex Synthetic Bacterial Promoters. ACS SYNTHETIC BIOLOGY, v. 7, n. 2, p. 602-612, FEB 2018. (16/19179-9, 12/22921-8)

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)

NORA, LUISA CZAMANSKI; WEHRS, MAREN; KIM, JOONHOON; CHENG, JAN-FANG; TARVER, ANGELA; SIMMONS, BLAKE A.; MAGNUSON, JON; HARMON-SMITH, MIRANDA; SILVA-ROCHA, RAFAEL; GLADDEN, JOHN M.; et al. A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides. Microbial Cell Factories, v. 18, JUN 29 2019. (12/22921-8, 18/02227-6)

AMORES, GERARDO RUIZ; GUAZZARONI, MARIA-EUGENIA; ARRUDA, LETICIA MAGALHAES; SILVA-ROCHA, RAFAEL. Recent Progress on Systems and Synthetic Biology Approaches to Engineer Fungi As Microbial Cell Factories. CURRENT GENOMICS, v. 17, n. 2, p. 85-98, 2016. (15/04309-1, 12/22921-8)

ARRUDA, LETICIA M.; MONTEIRO, LUMMY M. O.; SILVA-ROCHA, RAFAEL. The Chromobacterium violaceum ArsR Arsenite Repressor Exerts Tighter Control on Its Cognate Promoter Than the Escherichia coil System. FRONTIERS IN MICROBIOLOGY, v. 7, NOV 21 2016. (12/22921-8)

NORA, LUISA CZAMANSKI; WESTMANN, CAUA ANTUNES; GUAZZARONI, MARIA-EUGENIA; SIDDAIAH, CHANDRANAYAKA; GUPTA, VIJAI KUMAR; SILVA-ROCHA, RAFAEL. Recent advances in plasmid-based tools for establishing novel microbial chassis. BIOTECHNOLOGY ADVANCES, v. 37, n. 8, DEC 2019. (16/03763-3, 15/04309-1, 16/05472-6, 12/22921-8)

GUPTA, VIJAI KUMAR; STEINDORFF, ANDREI STECCA; DE PAULA, RENATO GRACIANO; SILVA-ROCHA, RAFAEL; MACH-AIGNER, ASTRID R.; MACH, ROBERT L.; SILVA, ROBERTO N.. The Post-cenomic Era of Trichoderma reesei: What's Next?. TRENDS IN BIOTECHNOLOGY, v. 34, n. 12, p. 970-982, DEC 2016. (14/23653-2, 12/22921-8)

OLIVEIRA MONTEIRO, LUMMY MARIA; ARRUDA, LETICIA MAGALHAES; SANCHES-MEDEIROS, ANANDA; MARTINS-SANTANA, LEONARDO; DE FATIMA ALVES, LUANA; DEFELIPE, LUCAS; GUSTAVO TURJANSKI, ADRIAN; GUAZZARONI, MARIA-EUGENIA; DE LORENZO, VICTOR; SILVA-ROCHA, RAFAEL. Reverse Engineering of an Aspirin-Responsive Transcriptional Regulator in Escherichia coli. ACS SYNTHETIC BIOLOGY, v. 8, n. 8, p. 1890-1900, AUG 2019. (16/19179-9, 17/17924-1, 16/06323-4, 18/04810-0, 15/04309-1, 12/22921-8)