A genetic time‐delay circuitry in mammalian cells

Abstract

Gene expression circuitries with time-delayed expression profiles regulate key events, such as oscillating systems, noise elimination, and coordinated multi-step processes, in all organisms from bacteria to mammalian cells. We present the rational synthesis of a genetic circuit displaying time-delayed expression in silico and in mammalian cells. The network is based on a time-delay circuit, where the tetracycline-responsive transactivator (tTA) induces expression of the pristinamycin-responsive repressor PIP-KRAB, which silences expression of the terminal human placental secreted alkaline phosphatase (SEAP). While the addition of pristinamycin I inactivates PIP-KRAB and results in the immediate resumption of SEAP expression, addition of tetracycline abolishes PIP-KRAB synthesis. Consequently, SEAP production remains repressed until the PIP-KRAB buffer in the cell is eliminated. We characterized in silico and in vivo the time-delayed expression properties and analyzed the impact of the size and stability of the PIP-KRAB buffer on fine-tuning of the response kinetics. This tunable time-delay circuitry represents a biologic building block for emulating a fundamental circuit topology in integrated artificial synthetic gene networks for the design of tailor-made cell types and organisms.

Recent advances in mammalian synthetic biology—design of synthetic transgene control networks

“…The synthetic time-delay circuit consists of modified tetracycline-dependent transactivator (tTA) whose individual domains TetR and VP16 have been fused to streptavidin (TetR-SA) and avitag (AT-VP16), respectively. Biotin, constitutively ligated to AT in the presence of the Escherichia coli biotin ligase BirA and able to non-covalently bind to SA, will heterodimerize TetR-SA and AT-VP16, thereby reconstituting a functional tetracycline-responsive transcription factor (TetR-SA-biotin-AT-VP16) [9,38] (Figure 2b). Owing to the irreversibility of the biotinylation reaction, heterodimerized transcription factors retain their functionality after biotin removal and continue to sustain target gene expression for some 30 h until the transcription factor is completely degraded.…”

“…Owing to the irreversibility of the biotinylation reaction, heterodimerized transcription factors retain their functionality after biotin removal and continue to sustain target gene expression for some 30 h until the transcription factor is completely degraded. This time-delayed shutdown of transgene expression is reminiscent of a capacitor whose discharge results in an immediate decrease in voltage [9].…”

“…Owing to their importance and success in basic and applied research a panoply of such heterologous transcription control modalities have been developed that are responsive to all kinds of small molecules. Since most mammalian transcription control systems have a generic circuit topology and a common genetic design they are functionally compatible and therefore seen by synthetic biologists as biological parts that could be assembled to higher order genetic control devices and regulatory networks that are approaching the complexity of electronic circuits [8,9,10 ]. Such synthetic networks reveal novel control dynamics [11] or provide prosthetic circuits for future therapeutic applications.…”

Taming Living Logic Using Formal Methods

“…The standard part libraries and toolboxes of well-characterized genetic components have been constructed through numerous laboratory experiments over the last decade [10][11][12][13][14][15][16][17][18]. These components have been extensively used to develop genetic circuits with different functionalities including oscillators [3], amplifiers [19,20], linearizer gene circuit [21], memory devices [22,23], switches [1,12,24], time-delay circuits [25,26], genetic logic gates [27][28][29][30] etc. …”

Coherent activation of a synthetic mammalian gene network

“…The recent development of inducible mammalian transgene control systems has allowed for the construction of synthetic gene circuits in mammalian cells and organisms (Greber and Fussenegger 2007). Mammalian transgene control elements have been used to build synthetic mammalian networks such as toggle switches (Kramer et al 2004), hysteretic switches (Kramer and Fussenegger 2005), and time-delay circuits (Weber et al 2007). Most of these engineered mammalian networks have been characterized using steady-state population level measurements hindering an examination of the dynamic network behavior in individual cells (Longo and Hasty 2006).…”