title: Engineering Synthetic cis-Regulatory Elements for Simultaneous Recognition of Three Transcriptional Factors in Bacteria.
abstract: 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 silico 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 silico 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.
authors: FMRP, University of São Paulo , Ribeirão Preto, São Paulo 05508-020, Brazil.
authors: Gerardo Ruiz
authors: GR
authors: Amores
authors: FFCLRP, University of São Paulo , Ribeirão Preto, São Paulo 05508-020, Brazil.
authors: María-Eugenia
authors: ME
authors: Guazzaroni
authors: FMRP, University of São Paulo , Ribeirão Preto, São Paulo 05508-020, Brazil.
authors: Rafael
authors: R
authors: Silva-Rocha
mesh_terms: D000465:Algorithms; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D035021:Factor For Inversion Stimulation Protein / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering / Q000379:methods; D034982:Integration Host Factors / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D020218:Response Elements
chemical_list: D002373:Cyclic AMP Receptor Protein; D029968:Escherichia coli Proteins; D035021:Factor For Inversion Stimulation Protein; C501034:Fis protein, E coli; D034982:Integration Host Factors; C481662:crp protein, E coli; C440502:integration host factor, E coli
keywords: cis-regulatory elements; synthetic biology; synthetic promoters