A Miniaturized Escherichia coli Green Light Sensor with High Dynamic Range

Abstract

Genetically engineered photoreceptors enable unrivaled control over gene expression. Previously, we ported the Synechocystis PCC 6803 CcaSR two-component system, which is activated by green light and deactivated by red, into Escherichia coli, resulting in a sensor with a sixfold dynamic range. Later, we optimized pathway protein expression levels and the output promoter sequence to decrease transcriptional leakiness and to increase the dynamic range to approximately 120-fold. These CcaSR v 1.0 and v 2.0 systems have been used for precise quantitative, temporal, and spatial control of gene expression for a variety of applications. Recently, other workers deleted two PAS domains of unknown function from the CcaS sensor histidine kinase in a system similar to CcaSR v 1.0. Here we apply these deletions to CcaSR v 2.0, resulting in a v 3.0 light sensor with an output four times less leaky and a dynamic range of nearly 600-fold. We demonstrate that the PAS domain deletions have no deleterious effect on CcaSR green light sensitivity or response dynamics. CcaSR v 3.0 is the best-performing engineered bacterial green light sensor available, and should have broad applications in fundamental and synthetic biology studies.

An inducible AraC that responds to blue light instead of arabinose

“…Moreover, since we constructed pBLADE using pBAD33 as template ( Supplementary Fig. 2a While many other light-inducible TFs have been developed to date 61,[68][69][70][71][72] , some of which featuring extremely high dark/light fold changes 61, 72 , we explicitly aimed to engineer a system based on a well-known and pervasive TF (namely AraC) that is particularly suited for microbiological applications, thus stimulating the use of optogenetics in microbiology. We took special care to engineer BLADE with minimal leakiness.…”

Light‐inducible flux control of triosephosphate isomerase on glycolysis in Escherichia coli

“…A third generation E . coli CcaSR system with lower leakiness and 600‐fold dynamic range has been developed by removing its two PAS domains (Ong & Tabor, ). Implementation of this new system can be expected to eliminate the leaky effect on growth rate in red light condition.…”

“…Though this CcaS/CcaR system was evolved in cyanobacteria, it was previously ported into and optimized for function in E . coli (Levskaya et al, ; Schmidl, Sheth, Wu, & Tabor, ; Tabor et al, ; Ong & Tabor, ). The CcaS and CcaR are a light sensor and its response regulator, respectively.…”

In situ characterisation and manipulation of biological systems with Chi.Bio

“…Real-time interfacing between in silico computational elements and biological systems can facilitate novel studies [31] and optimise experimental schemes [32]. We implemented such a scheme as a 'custom program' in the platform, which uses the red (623 nm) and green (523 nm) LEDs as actuating inputs for the CcaS-CcaR optogenetic system coupled to green fluorescent protein (GFP) expression [33]. In Fig 4A and 4B, this system is probed with square wave inputs of varying frequency, which yield smoothed output signals (due to the dynamics of protein expression/maturation).…”