Emergent Properties in Complex Synthetic Bacterial Promoters

Abstract

Regulation of gene expression in bacteria results from the interplay between hundreds of transcriptional factors (TFs) at target promoters. However, how the arrangement of binding sites for TFs generates the regulatory logic of promoters is not well-known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, in Escherichia coli, which are formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these two TFs. Using this approach, we found that while cis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as "UP" elements and stimulate transcription in several different architectures in the absence of this protein. However, the combination of CRP- and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.

Modulating Fis and IHF binding specificity, crosstalk and regulatory logic through the engineering of complex promoters

“…As can be seen in Fig. 3, most promoters assayed displayed low activity in wild type strain of E. coli and higher activity in the mutant strain lacking ihf, in agreement with previous data on complex IHF promoters (19).…”

“…2. 11 promoters variants previously described (19) were analyzed in wild type, Dfis and Dihf mutant strains of E. coli.…”

“…More recently, Synthetic Biology approaches have been used to construct artificial promoters through the combination of several cis-regulatory elements, and these have been characterized to decipher their architecture/dynamics relationship (15)(16)(17)(18). Yet, while most Synthetic Biology approaches have focused on cis-elements for local regulators (which not commonly regulate gene expression in a combinatorial way), we recently investigated this combinatorial regulation problem with global regulators (8,19,20). This is important since global regulators (such as IHF, Fis, CRP among others) have numerous binding sites along the genome of E. coli and very frequently co-occur at target promoters (8).…”

“…We previously explored how complex synthetic promoter harboring cis-regulatory elements for CRP and IHF can generate diverse regulatory logics depending on the final architecture of synthetic promoters, demonstrating that it is not possible to predict the regulatory logic of complex promoters from the known dynamics of their simple versions (19). Here, we further explore this approach to investigate the relationship of cis-regulatory elements for Fis and IHF.…”

The art of vector engineering: towards the construction of next‐generation genetic tools

“…The lack of biological information regarding the behaviour of simple genetic features such as promoters, terminators and origins of replication hinders the potential of engineering living organisms. Furthermore, we are currently experiencing the age of emergent phenomena in synthetic biology (P osfai et al, 2006;Kwok, 2010;Chen et al, 2015;Monteiro et al, 2017), an ubiquitous property of life, highlighted decades ago by systems biology (Bhalla and Iyengar, 1999;Kitano, 2002). In this context, the most well-characterized biological part might exhibit unpredictable behaviour when combined with other parts or when exposed to different compositional contexts such as the constraints imposed by vector biophysical structure/architecture or the combination of functional regulatory cis-elements (Cardinale and Arkin, 2012;Goñi-Moreno et al, 2017;Maria Oliveira Monteiro et al, 2017;Yeung et al, 2017).…”