Characterizing bacterial gene circuit dynamics with optically programmed gene expression signals.

Gene circuits are dynamical systems that regulate cellular behaviors, often using protein signals as inputs and outputs. Here we have developed an optogenetic 'function generator' method for programming tailor-made gene expression signals in live bacterial cells. We designed precomputed light sequences based on experimentally calibrated mathematical models of light-switchable two-component systems and used them to drive intracellular protein levels to match user-defined reference time courses. We used this approach to generate accelerated and linearized dynamics, sinusoidal oscillations with desired amplitudes and periods, and a complex waveform, all with unprecedented accuracy and precision. We also combined the function generator with a dual fluorescent protein reporter system, analogous to a dual-channel oscilloscope, to reveal that a synthetic repressible promoter linearly transforms repressor signals with an approximate 7-min delay. Our approach will enable a new generation of dynamical analyses of synthetic and natural gene circuits, providing an essential step toward the predictive design and rigorous understanding of biological systems.

Optogenetic control of gut bacterial metabolism to promote longevity.

Gut microbial metabolism is associated with host longevity. However, because it requires direct manipulation of microbial metabolism in situ, establishing a causal link between these two processes remains challenging. We demonstrate an optogenetic method to control gene expression and metabolite production from bacteria residing in the host gut. We genetically engineer an Escherichia coli strain that secretes colanic acid (CA) under the quantitative control of light. Using this optogenetically-controlled strain to induce CA production directly in the Caenorhabditis elegans gut, we reveal the local effect of CA in protecting intestinal mitochondria from stress-induced hyper-fragmentation. We also demonstrate that the lifespan-extending effect of this strain is positively correlated with the intensity of green light, indicating a dose-dependent CA benefit on the host. Thus, optogenetics can be used to achieve quantitative and temporal control of gut bacterial metabolism in order to reveal its local and systemic effects on host health and aging.

Phosphatase activity tunes two-component system sensor detection threshold.

Two-component systems (TCSs) are the largest family of multi-step signal transduction pathways in biology, and a major source of sensors for biotechnology. However, the input concentrations to which biosensors respond are often mismatched with application requirements. Here, we utilize a mathematical model to show that TCS detection thresholds increase with the phosphatase activity of the sensor histidine kinase. We experimentally validate this result in engineered Bacillus subtilis nitrate and E. coli aspartate TCS sensors by tuning their detection threshold up to two orders of magnitude. We go on to apply our TCS tuning method to recently described tetrathionate and thiosulfate sensors by mutating a widely conserved residue previously shown to impact phosphatase activity. Finally, we apply TCS tuning to engineer B. subtilis to sense and report a wide range of fertilizer concentrations in soil. This work will enable the engineering of tailor-made biosensors for diverse synthetic biology applications.

An open-hardware platform for optogenetics and photobiology.

In optogenetics, researchers use light and genetically encoded photoreceptors to control biological processes with unmatched precision. However, outside of neuroscience, the impact of optogenetics has been limited by a lack of user-friendly, flexible, accessible hardware. Here, we engineer the Light Plate Apparatus (LPA), a device that can deliver two independent 310 to 1550 nm light signals to each well of a 24-well plate with intensity control over three orders of magnitude and millisecond resolution. Signals are programmed using an intuitive web tool named Iris. All components can be purchased for under $400 and the device can be assembled and calibrated by a non-expert in one day. We use the LPA to precisely control gene expression from blue, green, and red light responsive optogenetic tools in bacteria, yeast, and mammalian cells and simplify the entrainment of cyanobacterial circadian rhythm. The LPA dramatically reduces the entry barrier to optogenetics and photobiology experiments.