Amplification of small molecule-inducible gene expression via tuning of intracellular receptor densities

Nucleic Acids Research Amplification of small molecule-inducible gene expression via tuning of intracellular receptor densities Baojun Wang 2 Mauricio Barahona 1 Martin Buck 0 0 Department of Life Sciences, Faculty of Natural Sciences, Imperial College London , London, SW7 2AZ , UK 1 Department of Mathematics, Faculty of Natural Sciences, Imperial College London , London, SW7 2AZ , UK 2 Centre for Synthetic and Systems Biology, School of Biological Sciences, University of Edinburgh , Edinburgh, EH9 3JR , UK - Ligand-responsive transcription factors in prokaryotes found simple small molecule-inducible gene expression systems. These have been extensively used for regulated protein production and associated biosynthesis of fine chemicals. However, the promoter and protein engineering approaches traditionally used often pose significant restrictions to predictably and rapidly tune the expression profiles of inducible expression systems. Here, we present a new unified and rational tuning method to amplify the sensitivity and dynamic ranges of versatile small molecule-inducible expression systems. We employ a systematic variation of the concentration of intracellular receptors for transcriptional control. We show that a low density of the repressor receptor (e.g. TetR and ArsR) in the cell can significantly increase the sensitivity and dynamic range, whereas a high activator receptor (e.g. LuxR) density achieves the same outcome. The intracellular concentration of receptors can be tuned in both discrete and continuous modes by adjusting the strength of their cognate driving promoters. We exemplified this approach in several synthetic receptor-mediated sensing circuits, including a tunable cell-based arsenic sensor. The approach offers a new paradigm to predictably tune and amplify ligand-responsive gene expression with potential applications in synthetic biology and industrial biotechnology. INTRODUCTION Ligand responsive transcription factors (TFs) in bacteria regulates cellular functions in response to environmental and metabolic cues (1). They play a central and classical role in the induction of gene expression systems by small molecules, such as tetracycline (tet), lactose (lac) and arabinose (ara) (2). Inducible expression systems have been widely utilized for recombinant protein production (3), gene function studies and, more recently, in synthetic biology for the control of gene networks (46) and metabolic pathways to produce fine chemicals and biofuels (79). However, the native effector-responsive TF-controlled systems have evolved to respond to their cognate ligands with a particular sensitivity, selectivity and dynamic ranges, and are therefore not optimized for direct reuse in, e.g. industrial biotechnology, to express various target genes. As a result, several approaches have been proposed to re-engineer the wild-type small molecule-mediated expression systems in order to generate modified systems that can achieve matched expression of various genes in synthetic biochemical pathways (1012). Typically, these approaches have focused on re-engineering directly the underlying promoters by either randomly mutating flanking sequences of the consensus DNA motifs (5,13), or by placing different numbers of enhancers or operator sites within different locations of the target promoter (2,1417). Despite notable successes, the somewhat unpredictable and labour-intensive nature of these promoter-engineering approaches poses significant restrictions to their use to predictably and rapidly tune the sensitivity and dynamic range of inducible expression systems. Hence, there is a need for new methods that will enable the systematic and controlled variation of the expression profiles of versatile small molecule-inducible expression systems. Here we present a new methodology to flexibly tune the sensitivity and dynamic range of small molecule-inducible gene expression systems in a predictable manner via variation of the concentrations of intracellular receptors in onecomponent signal transduction pathways. Bacterial ligandresponsive TF regulatory systems typically comprises an intracellular receptor protein, which acts as either a transcriptional activator or repressor and dynamically switches between two states through allostery upon binding to its target ligand in the cytoplasm, and a cognate target promoter, which is regulated by the receptor to control the transcription of a downstream gene (Figure 1) (1). Since the receptor protein both detects the target ligand and acts to activate/repress the target promoter, we expected that the concentration of receptor in the cytoplasm would determine the sensing sensitivity for the ligand as well as the transcriptional dynamic range of the cognate-inducible promoter. Thus, the density of the intracellular receptor could function as a dial to tune continuously the sensitivity and dynamic range of the gene expression system (Figure 1). In this study, we demonstrate that lowering the intra-cellular density of a repressor receptor protein (e.g. TetR and ArsR) increases significantly the sensitivity and dynamic range of these small molecule-inducible systems. Increasing the intra-cellular density of an activator receptor (e.g. LuxR) achieves a similar outcome. This approach establishes a new way to tune and amplify ligand-responsive gene expression systems in living cells and has the potential for applications in synthetic biology and industrial biotechnology. MATERIALS AND METHODS Strain, media and growth conditions Plasmid cloning work and circuit construct characterization were all performed in Escherichia coli TOP10 strain. Cells were cultured in LB (LuriaBertani Broth) media (10 g/l peptone, 5 g/l NaCl, 5 g/l yeast extract). The kanamycin used was 50 g/ml. Cells inoculated from single colonies on freshly streaked plates were grown overnight in 5 ml LB in sterile 20 ml universal tubes at 37C with shaking (200 revolutions per minute (rpm)). Overnight cultures were diluted into pre-warmed LB media at OD600 = 0.025 for the day cultures, which were induced (see below) and grown for 6 h at 37C prior to analysis. For fluorescence assay by fluorometry, diluted cultures were loaded into a 96-well microplate (Bio-Greiner, chimney black, flat clear bottom) and induced with 5 l (for single input induction) or 10 l (for double input induction) volumes of specified inducers to yield a final volume of 200 l per well. For the 2D characterization, cell cultures in the microplate were first induced with arabinose and grown for 3 h (to produce sufficient receptor proteins in the cytoplasm) before being induced by the second inducer chemical (aTc or NaAsO2 or 3OC6HSL) and grown for another 3 h. The microplate was covered by a ultraviolet transparent lid to counteract evaporation and incubated in the fluorometer (BMG FLUOstar) with continuous shaking (200 rpm, linear mode, 37C) between each cycle of repetitive measurements. Chemical reagents and ind (...truncated)