Expanding the Logic of Bacterial Promoters Using Engineered Overlapping Operators for Global Regulators

Autor(es)

Guazzaroni, Maria-Eugenia ^[1] ; Silva-Rocha, Rafael ^[2]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, FFCLRP, Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, FMRP, Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: ACS SYNTHETIC BIOLOGY; v. 3, n. 9, p. 666-675, SEP 2014.
• Citações Web of Science: 9

Resumo

The understanding of how the architecture of cis-regulatory elements at bacterial promoters determines their final output is of central interest in modern biology. In this work, we attempt to gain insight into this process by analyzing complex promoter architectures in the model organism Escherichia coli. By focusing on the relationship between different TFs at the genomic scale in terms of their binding site arrangement and their effect on the target promoters, we found no strong constraint limiting the combinatorial assembly of TF pairs in E. coli. More strikingly, overlapping binding sites were found equally associated with both equivalent (both TFs have the same this information on hand, we set an in salt approach to design overlapping sites for three global regulators (GRs) of E. coli, specifically CRP, Fis, and IHF. Using random sequence assembly and an evolutionary algorithm, we were able to identify potential overlapping operators for all TF pairs. In order to validate our prediction, we constructed two lac promoter variants containing overlapping sites for CRP and IHF designed in silk. By assaying the synthetic promoters using a GFP reporter system, we demonstrated that these variants were functional and activated by CRP and IHF in vivo. Taken together, presented results add new information on the mechanisms of signal integration in bacterial promoters and provide new strategies for the engineering of synthetic regulatory circuits in bacteria. (AU)

• Processo FAPESP: 13/04125-2 - Novas ferramentas genéticas para Archaea: validação funcional e modelagem da rede regulatória de RNA não codificantes de Halobacterium salinarum
• Beneficiário: Rafael Silva Rocha
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado