A tunable dual-input system for ‘on-demand’ dynamic gene expression regulation

Abstract

Cellular systems have evolved numerous mechanisms to finely control signalling pathway activation and properly respond to changing environmental stimuli. This is underpinned by dynamic spatiotemporal patterns of gene expression. Indeed, in addition to gene transcription and translation regulation, modulation of protein levels, dynamics and localization are also essential checkpoints that govern cell functions.The introduction of tetracycline-inducible promoters has allowed gene expression control using orthogonal small molecules, facilitating rapid and reversible manipulation to study gene function in biological systems. However, differing protein stabilities means this solely transcriptional regulation is insufficient to allow precise ON-OFF dynamics, thus hindering generation of temporal profiles of protein levels seen in vivo.We developed an improved Tet-On based system augmented with conditional destabilising elements at the post-translational level that permits simultaneous control of gene expression and protein stability. Integrating these properties to control expression of a fluorescent protein in mouse Embryonic Stem Cells (mESCs), we found that adding protein stability control allows faster response times to changes in small molecules, fully tunable and enhanced dynamic range, and vastly improved microfluidic-based in-silico feedback control of gene expression. Finally, we highlight the effectiveness of our dual-input system to finely modulate levels of signalling pathway components in stem cells.fusion protein of the herpes simplex virus VP16 activation domain and of the Escherichia coli Tet repressor protein (TetR) 9 . The presence of tetracycline or its derivative doxycycline prevents the interaction of the tTA to the tetO, blocking gene expression (Tet-Off system). The reverse-tTA (rtTA) is a tTA variant allowing gene expression activation in presence of an inducer; the resulting Tet-On system is generally preferred when rapid and dynamic gene induction is required 4,13 . A major limitation of inducible promoters is the significant time delay in switching proteins OFF and ON when using Tet-On and Tet-off systems, respectively 14 , diminishing the possibility of using these approaches to generate dynamic patterns of gene expression that faithfully recapitulate those observed natively 1 . Slow kinetic responses are also common to other techniques targeting precursor DNA or mRNA molecules (e.g. RNA interference 15 ), likely due to significantly different rates of innate protein degradation 15 .Recently, an alternative approach, relying on conditional protein destabilization to modulate turnover by the cellular degradation machinery, has been harnessed to probe complex biological functions. Engineered mutants of FKBP12 that are rapidly and constitutively degraded in mammalian cells can directly confer protein destabilization to the protein they are fused with. Addition of synthetic ligands, that bind the Destabilising Domain (DD) of FKBP12, prevent degradation and so can be used to alter levels of the f...

Cheetah: a computational toolkit for cybergenetic control

“…Experiments with mammalian cells used a previously generated mouse Embryonic Stem Cell (mESC) line 26 . Briefly, mESCs were subjected to two rounds of infection and drug-selection to stably express the transactivator (EF1a-rtTA, Neomycin) and the doxycycline-inducible vector (pLVX_TRE3GDDmCherry, Puromycin; Addgene plasmid #108679).…”

“…For mESCs, microfluidic chip loading and imaging were performed as reported previously 26 . The microfluidic device we used was designed in the laboratory of Prof Jeff Hasty at the During the open-loop experiment (Figures 2E, 2F) mESCs were exposed to plain media for the entire duration of the time-lapse, whereas dynamic switching between plain and Doxy/TMP media was automatically controlled during the closed-loop experiment (Figures 3C, 3D) to reach and maintain a desired reference red fluorescence level.…”

“…Having demonstrated the ability for Cheetah to robustly perform image analysis, we next attempted to validate its use for real-time external control of mammalian cells. Using the same engineered mESCs from the previous experiment, we employed an automated microscopy and fluidic control platform that allows for real-time live-cell imaging within microfluidic chips and the precise control of media and chemical inducers fed to the cells by the movement of motorised syringes ( Figure 3A) 28 . To allow for cells to be controlled by this system, mESCs carried a dual-input genetic construct where an mCherry fluorescent protein fused to a destabilisingdomain (DD) was under the control of a 'Tet-On' promoter ( Figure 3B, Methods) 28 .…”

ChipSeg: an automatic tool to segment bacteria and mammalian cells cultured in microfluidic devices

“…In the context of synthetic biology, the combination of control engineering algorithms with live-cell imaging has been shown to successfully enable the automatic regulation of gene expression across cellular chassis [1][2][3][4][5][6][7][8] . If employing microfluidics/microscopy platforms, the external feedback control action is implemented by measuring the relevant control output (e.g.…”

“…The same core functions are implemented for both cell types, making the code flexible for other chassis and applications. The algorithm showed robust segmentation results in external feedback control experiments with microfluidics/microscopy platforms [6][7][8][9] , and can be easily adapted for open-loop experiments, other cell types and experimental settings.…”