Materials Synthetic Biology

The rapid development of synthetic biology is a paradigm of how the molecular diversity of naturally occurring gene control components can be used to design synthetic control devices and gene networks that provide precisely programmed transgene expression dynamics in space and time. Here we offer an overview on recent advances in the modular design of trigger-inducible mammalian expression devices that are either responsive by exogenous stimuli such as chemicals and physical cues or controlled by endogenous metabolites driving prosthetic circuits to treat metabolic disorders in a self-sufficient manner. Compatible genetic switches can also be assembled to synthetic gene networks that show highly complex expression dynamics such as temporally resolved band-detect functions or oscillating transgene expression profiles. The ongoing metagenomic discovery and characterization of the unexplored sequence space is constantly increasing the molecular diversity in fundamental control components that fuels the further development of synthetic biology. © 2011 Elsevier Ltd.

Synthetic quorum-sensing systems in mammalian cells has enabled the implementation of time- and distance-dependent bioprocesses, as well as the design of synthetic ecosystems emulating clinically important host–parasite interactions. In this chapter, we provide a detailed protocol of the design of a mammalian cell-to-cell signaling device and its integration into a mammalian quorum-sensing system for cell density-induced expression product genes. Cell-to-cell signaling is based on a sender cell, metabolically engineered for expression of alcohol dehydrogenase converting ethanol into acetaldehyde, and a receiver cell line for the dose-dependent translation of the acetaldehyde concentration into transgene expression by an acetaldehyde-responsive promoter. This protocol can be adapted easily to various cell types and transgenes for the design of versatile mammalian cell-based quorum-sensing systems. © 2011, Springer Science+Business Media, LLC.

Synthetic Biology aims at the design and construction of biologic systems with desired features by applying a modular strategy. This approach was used to investigate the interaction of multiple organisms in synthetic ecosystems.

Gene therapy scientists have developed expression systems for therapeutic transgenes within patients, which must be seamlessly integrated into the patient's physiology by developing sophisticated control mechanisms to titrate expression levels of the transgenes into the therapeutic window. However, despite these efforts, gene-based medicine still faces security concerns related to the administration of the therapeutic transgene vector. Here, molecular tools developed for therapeutic transgene expression can readily be transferred to materials science to design a humanized drug depot that can be implanted into mice and enables the trigger-inducible release of a therapeutic protein in response to a small-molecule inducer. The drug depot is constructed by embedding the vascular endothelial growth factor (VEGF121) as model therapeutic protein into a hydrogel consisting of linear Polyacrylamide crosslinked with a homodimeric variant of the human FKbinding protein 12 (F M), originally developed for gene therapeutic applications, as well as with dimethylsuberimidate. Administrating increasing concentrations of the inducer molecule FK506 triggers the dissociation of FM thereby loosening the hydrogel structure and releasing the VEGF121 payload in a dose-adjustable manner. Subcutaneous implantation of the drug depot into mice and subsequent administration of the inducer by injection or by oral intake triggers the release of VEGF121 as monitored in the mouse serum. This study is the first demonstration of a stimuli-responsive hydrogel that can be used in mammals to release a therapeutic protein on demand by the application of a small-molecule stimulus. This trigger-inducible release is a starting point for the further development of externally controlled drug depots for patient-compliant administration of biopharmaceuticals. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Synthetic biology as the discipline of reconstructing natural and designing novel biological systems is gaining increasing impact in signaling science. This review article provides insight into synthetic approaches for analyzing and synthesizing signaling processes starting with strategies into how natural and pathological signaling pathways can be reconstructed in an evolutionary distant host to study their topology and function while avoiding interference with the original host background. In the second part we integrate synthetic strategies in the rewiring of signaling systems at the nucleic acid and protein level to reprogram cellular functions for biotechnological applications. The last part focuses on synthetic inter-cell and inter-species signaling devices and their integration into synthetic ecosystems to study fundamental mechanisms governing the co-existence of species. We finally address current bottlenecks in the (re-)design of signaling pathways and discuss future directions in signaling-related synthetic biology. © 2010 The Royal Society of Chemistry.

The design and construction of synthetic gene circuits with complex spatiotemporal dynamics was pioneered in bacteria, but it took almost a decade until synthetic biologists were able to construct genetic circuits with complex spatiotemporal dynamics in mammalian cells. This review highlights the most recent advances in mammalian synthetic biology, and it describes metabolite, hormone, and light-triggered genetic switches as well as the design and construction of synthetic networks that feature tunable oscillations. We conclude by discussing not only the current limitations but also possible ways to transform the construction of synthetic mammalian systems from an art into a predictive engineering discipline. © 2010 Elsevier Ltd.

Inducer-dependent prokaryotic transcriptional repressor proteins that originally evolved to orchestrate the transcriptome with intracellular and extracellular metabolite pools, have become universal tools in synthetic biology, drug discovery, diagnostics and functional genomics. Production of the repressor proteins is often limited due to inhibiting effects on the production host and requires iterative process optimization for each individual repressor. At the example of the Streptomyces pristinaespiralis-derived streptogramin-dependent repressor PIP, the expression of which was shown to inhibit growth of Escherichia coli BL21*, we demonstrate that the addition of the PIP-specific streptogramin antibiotic pristinamycin I neutralizes the growth-inhibiting effect and results in >100-fold increased PIP titers. The yield of PIP was further increased 2.5-fold by the engineering of a new E. coli host suitable for the production of growth-inhibiting proteins encoded by an unfavorable codon usage. PIP produced in the presence of pristinamycin I was purified and was shown to retain the antibiotic-dependent binding to its operator pir as demonstrated by a fluorescence resonance energy transfer (FRET)-based approach. At the example of the macrolide-, tetracycline- and arsenic-dependent repressors MphR(A), TetR and ArsR, we further demonstrate that the production yields can be increased 2- to 3-fold by the addition of the cognate inducer molecules erythromycin, tetracycline and As3+, respectively. Therefore, the addition of inducer molecules specific to the target repressor protein seems to be a general strategy to increase the yield of this interesting protein class. © 2009 Elsevier Inc. All rights reserved.

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida-mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.

Synthetic biology, the science of engineering complex biological systems with novel functions, is increasingly fascinating researchers across disciplines who gather to design functional biological assemblies in a rational and systematic manner. Although initial success stories were based on reprogramming prokaryotic and lower eukaryotic cells, the design of synthetic mammalian gene circuits is becoming increasingly popular because it promises to foster novel therapeutic opportunities in the not-so-distant future. Here, we discuss the latest generation of mammalian synthetic biology devices assembled to form complex synthetic gene networks, such as regulatory cascades, logic evaluators, hysteretic circuits, epigenetic toggle switches, time-keeping components, drug discovery tools, and "cell phone" units. We further highlight how such circuits could be interconnected to achieve higher-order control networks such as synthetic hormone-like communication systems in animals or synthetic ecosystems with dynamic interspecies crosstalk. © 2009 Elsevier Ltd. All rights reserved.