Engineering Synthetic cis-Regulatory Elements for Simultaneous Recognition of Three Transcriptional Factors in Bacteria

Autor(es)

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

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo, FMRP, BR-05508020 Ribeirao Preto, SP - Brazil

^[2] Univ Sao Paulo, FFCLRP, BR-05508020 Ribeirao Preto, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: ACS SYNTHETIC BIOLOGY; v. 4, n. 12, p. 1287-1294, DEC 2015.
• Citações Web of Science: 8

Resumo

Recognition of cis-regulatory elements by transcription factors (TF) at target promoters is crucial to gene regulation in bacteria. In this process, binding of TFs to their cognate sequences depends on a set of physical interactions between these proteins and specific nucleotides in the operator region. Previously, we showed that in silico optimization algorithms are able to generate short sequences that are recognized by two different TFs of Escherichia coli, namely, CRP and IHF, thus generating an AND logic gate. Here, we expanded this approach in order to engineer DNA sequences that can be simultaneously recognized by three unrelated TFs (CRP, IHF, and Fis). Using in silica optimization and experimental validation strategies, we were able to obtain a candidate promoter (Plac-CFI1) regulated by only two TFs with an AND logic, thus demonstrating a limitation in the design. Subsequently, we modified the algorithm to allow the optimization of extended sequences, and were able to design two synthetic promoters (PCFI20-1 and PCFI22-5) that were functional in vivo. Expression assays in E. coli mutant strains for each TF revealed that while CRP positively regulates the promoter activities, IHF and Fis are strong repressors of both the promoter variants. Taken together, our results demonstrate the potential of in silica strategies in bacterial synthetic promoter engineering. Furthermore, the study also shows how small modifications in cis-regulatory elements can drastically affect the final logic of the resulting promoter. (AU)

• Processo FAPESP: 12/22921-8 - Abordagens de biologia sintética para decifrar os mecanismos de integração de sinais em promotores bacterianos complexos
• Beneficiário: Rafael Silva Rocha
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores