Real-time optogenetic control of intracellular protein concentration in microbial cell cultures

Abstract

Perturbations in the concentration of a specific protein are often used to study and control biological networks. The ability to "dial-in" and programmatically control the concentration of a desired protein in cultures of cells would be transformative for applications in research and biotechnology. We developed a culturing apparatus and feedback control scheme which, in combination with an optogenetic system, allows us to generate defined perturbations in the intracellular concentration of a specific protein in microbial cell culture. As light can be easily added and removed, we can control protein concentration in culture more dynamically than would be possible with long-lived chemical inducers. Control of protein concentration is achieved by sampling individual cells from the culture apparatus, imaging and quantifying protein concentration, and adjusting the inducing light appropriately. The culturing apparatus can be operated as a chemostat, allowing us to precisely control microbial growth and providing cell material for downstream assays. We illustrate the potential for this technology by generating fixed and time-varying concentrations of a specific protein in continuous steady-state cultures of the model organism Saccharomyces cerevisiae. We anticipate that this technology will allow for quantitative studies of biological networks as well as external tuning of synthetic gene circuits and bioprocesses.

An open-hardware platform for optogenetics and photobiology

“…Between 0.26 and 93.50 μmol m −2 s −1 , mCherry increases in a Hill-like manner with 5.6-fold dynamic range, n H of 1.3 and k 1/2 of 10.74 μmol m −2 s −1 (Fig. 5b), consistent with previous reports112452. Though mCherry increases slightly at higher intensities, we observe growth defects, likely due to heating or phototoxicity (Supplementary Fig.…”

“…All CRY2-CIB1 Y2H experiments were performed with the previously described S. cerevisiae strain yMM1081 (MAT α, trpΔ63, leu2Δ1, ura3Δ52, gal1ΔmCherry-caURA3) which carries plasmids pGal4AD-CIB1 and pGal4BD-CRY21152. A 3 mL SD (-Ura, -Trp, -Leu) medium starter culture was inoculated from a −80 °C stock and grown (30 °C, 250 r.p.m) overnight to a final density of approximately OD 600  = 2.…”

A photoconversion model for full spectral programming and multiplexing of optogenetic systems

“…However, if the nature of the alternative light signal is known, our approach can compensate for such perturbations (e.g., Figs and ). In silico feedback control has also been used to drive desired gene expression dynamics in optogenetic experiments (Milias‐Argeitis et al , , ; Melendez et al , ). The major benefit of this approach is that perturbations of unknown origin can be compensated by monitoring deviations in the output of an optogenetic tool relative to a reference.…”

How to train your microbe: methods for dynamically characterizing gene networks

“…Blue LEDs were recently combined with a custom chemostat and integrated fluorescence microscope to control a blue light activated promoter while measuring gene expression in single S. cerevisiae cells [57]. By combining the method of Olson and coworkers [55] (Fig.…”

“…However, recent studies have overcome this limitation by combining mathematical modeling and computational control with custom hardware and time-varying chemical inducers [24,52–54], or genetically-encoded light switchable proteins (optogenetic tools) [55–57]. In several cases, the resulting perturbations have been used to reveal new dynamical properties of promoters and gene circuits [24,54,55].…”