Engineering orthogonal dual transcription factors for multi-input synthetic promoters

Synthetic biology has seen an explosive growth in the capability of engineering artificial gene circuits from transcription factors (TFs), particularly in bacteria. However, most artificial networks still employ the same core set of TFs (for example LacI, TetR and cI). The TFs mostly function via repression and it is difficult to integrate multiple inputs in promoter logic. Here we present to our knowledge the first set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants and multi-input promoter architectures. Our toolkit contains 12 TFs, flexibly operating as activators, repressors, dual activator–repressors or dual repressor–repressors, on up to 270 synthetic promoters. To engineer non cross-reacting cI variants, we design a new M13 phagemid-based system for the directed evolution of biomolecules. Because cI is used in so many synthetic biology projects, the new set of variants will easily slot into the existing projects of other groups, greatly expanding current engineering capacities

A unified design space of synthetic stripe-forming networks

Synthetic biology is a promising tool to study the function and properties of gene regulatory networks. Gene circuits with predefined behaviours have been successfully built and modelled, but largely on a case-by-case basis. Here we go beyond individual networks and explore both computationally and synthetically the design space of possible dynamical mechanisms for 3-node stripe-forming networks. First, we computationally test every possible 3-node network for stripe formation in a morphogen gradient. We discover four different dynamical mechanisms to form a stripe and identify the minimal network of each group. Next, with the help of newly established engineering criteria we build these four networks synthetically and show that they indeed operate with four fundamentally distinct mechanisms. Finally, this close match between theory and experiment allows us to infer and subsequently build a 2-node network that represents the archetype of the explored design space

Combining a Toggle Switch and a Repressilator within the AC-DC Circuit Generates Distinct Dynamical Behaviors.

Although the structure of a genetically encoded regulatory circuit is an important determinant of its function, the relationship between circuit topology and the dynamical behaviors it can exhibit is not well understood. Here, we explore the range of behaviors available to the AC-DC circuit. This circuit consists of three genes connected as a combination of a toggle switch and a repressilator. Using dynamical systems theory, we show that the AC-DC circuit exhibits both oscillations and bistability within the same region of parameter space; this generates emergent behaviors not available to either the toggle switch or the repressilator alone. The AC-DC circuit can switch on oscillations via two distinct mechanisms, one of which induces coherence into ensembles of oscillators. In addition, we show that in the presence of noise, the AC-DC circuit can behave as an excitable system capable of spatial signal propagation or coherence resonance. Together, these results demonstrate how combinations of simple motifs can exhibit multiple complex behaviors

Genetically encoded sender-receiver system in 3D mammalian cell culture

Engineering spatial patterning in mammalian cells, employing entirely genetically encoded components, requires solving several problems. These include how to code secreted activator or inhibitor molecules and how to send concentration-dependent signals to neighboring cells, to control gene expression. The Madin-Darby Canine Kidney (MDCK) cell line is a potential engineering scaffold as it forms hollow spheres (cysts) in 3D culture and tubulates in response to extracellular hepatocyte growth factor (HGF). We first aimed to graft a synthetic patterning system onto single developing MDCK cysts. We therefore developed a new localized transfection method to engineer distinct sender and receiver regions. A stable reporter line enabled reversible EGFP activation by HGF and modulation by a secreted repressor (a truncated HGF variant, NK4). By expanding the scale to wide fields of cysts, we generated morphogen diffusion gradients, controlling reporter gene expression. Together, these components provide a toolkit for engineering cell-cell communication networks in 3D cell culture.Facultad de Ciencias Exacta

Drug-inducible control of lethality genes: a low background destabilizing domain architecture applied to the Gal4-UAS system in Drosophila

Destabilizing domains (DDs) are genetic tags that conditionally control the level of abundance of proteins-of-interest (POI) with specific stabilizing small-molecule drugs, rapidly and reversibly, in a wide variety of organisms. The amount of the DD-tagged fusion protein directly impacts its molecular function. Hence, it is important that the background levels be tightly regulated in the absence of any drug. This is especially true for classes of proteins that function at extremely low levels, such as lethality genes involved in tissue development and certain transcriptional activator proteins. Here, we establish the uninduced background and induction levels for two widely used DDs (FKBP and DHFR) by developing an accurate quantification method. We show that both DDs exhibit functional background levels in the absence of a drug, but each to a different degree. To overcome this limitation, we systematically test a double architecture for these DDs (DD-POI-DD) that completely suppresses the protein’s function in an uninduced state, while allowing tunable functional levels upon adding a drug. As an example, we generate a drug-stabilizable Gal4 transcriptional activator with extremely low background levels. We show that this functions in vivo in the widely used Gal4-UAS bipartite expression system in Drosophila melanogaster. By regulating a cell death gene, we demonstrate that only the low background double architecture enables tight regulation of the lethal phenotype in vivo. These improved tools will enable applications requiring exceptionally tight control of protein function in living cells and organisms

Fluctuation induces evolutionary branching in a modeled microbial ecosystem

The impact of environmental fluctuation on species diversity is studied with a model of the evolutionary ecology of microorganisms. We show that environmental fluctuation induces evolutionary branching and assures the consequential coexistence of multiple species. Pairwise invasibility analysis is applied to illustrate the speciation process. We also discuss how fluctuation affects species diversity.Comment: 4 pages, 4 figures. Submitted to Physical Review Letter