Engineering input/output nodes in prokaryotic regulatory circuits

Abstract

A large number of prokaryotic regulatory elements have been interfaced artificially with biological circuits that execute specific expression programs. Engineering such circuits involves the association of input/output components that perform discrete signal-transfer steps in an autonomous fashion while connected to the rest of the network with a defined topology. Each of these nodes includes a signal-recognition component for the detection of the relevant physicochemical or biological stimulus, a molecular device able to translate the signal-sensing event into a defined output and a genetic module capable of understanding such an output as an input for the next component of the circuit. The final outcome of the process can be recorded by means of a reporter product. This review addresses three such aspects of forward engineering of signal-responding genetic parts. We first recap natural and non-natural regulatory assets for designing gene expression in response to predetermined signals - chemical or otherwise. These include transcriptional regulators developed by in vitro evolution (or designed from scratch), and synthetic riboswitches derived from in vitro selection of aptamers. Then we examine recent progress on reporter genes, whose expression allows the quantification and parametrization of signal-responding circuits in their entirety. Finally, we critically examine recent work on other reporters that confer bacteria with gross organoleptic properties (e.g. distinct odour) and the interfacing of signal-sensing devices with determinants of community behaviour.

The Logic of Decision Making in Environmental Bacteria

“…Alas, given the difficulty of the corresponding experiments, very little biochemical data on regulatory nodes is available from transcription experiments in vitro. Fortunately, the popularization of high-throughput methods for measuring promoter activity with optical reporter genes [6] helps us to remediate the shortage of such hard regulatory data. Determination of regulatory transfer functions in specific nodes of the network can in this way be easily deciphered in vivo.…”

“…Despite the various descriptive languages and modeling options just enumerated, the lack of benchmarking for measuring transfer functions in various laboratories [6] reduces the actual data that can be used to feed simulations. Typically, one group will employ lacZ as reporter to measure promoter activity, another will prefer GFP, and still others may rely on DNA array technology or RT-PCR.…”

“…This is because identification of general design principles should allow the correct integration of new regulatory circuits into preexisting systems [6], one of the key objectives of contemporary synthetic biology [19]. A growing approach to address the functions and properties of regulatory networks involves the formulation of models that translate the molecular interactions known for a given system into a set of equations that afford a simulation of the entire lot of physical and functional interplays [20].…”

Plants: biofactories for a sustainable future?

“…Such thinking has led to the assembly of collections of BioBricks as standardized components which can be readily assembled to generate new biological machines. Indeed, the concept of standardizing the toolkits used in synthetic biology based on long observed engineering principles has been central to the emergence of the science [20,21].…”

Whole-cell biochips for online water monitoring

“…4 An additional attractive characteristic of bacteria is their susceptibility to genetic manipulation, which allows one to engineer them to respond by a detectable dosedependent signal to pre-specified changes in their environment. 5,6 One well-established technique to achieve this is to transform bacteria with a plasmid that contains a fusion of a stress-or chemicalresponsive gene promoter to the bacterial bioluminescence genes luxCDABE. When the designated environmental conditions are met, the transcription of the lux operon is promoted and a light signal is produced that is proportional in intensity to the magnitude of the stimulus.…”