GreA and GreB Enhance Expression of Escherichia coil RNA Polymerase Promoters in a Reconstituted Transcription-Translation System

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate of Escherichia coli RNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of natural E. coli promoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and synthetic E. coli promoters and characterization of their regulation in minimal and defined reaction conditions, making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology.

Implementation and Characterization of Dynamic Genetic Networks in Vitro

Henrike Marie Niederholtmeyer

Transcription and translation (TX-TL) can be performed in vitro, outside of cells, allowing the assembly and analysis of genetic networks. This approach to engineering biological networks in a less complex and more controllable environment could one day allow rapid prototyping of network designs before implementing them in living cells. Furthermore, the in vitro approach provides insight into how natural biological systems are built and is instructive to define the rules for engineering biological systems from bottom up. Despite progress in engineering TX-TL mixes for higher yields and longer synthesis times it remains challenging to implement complex genetic networks, such as oscillators, in vitro. The reason is that the reactions are usually performed in a batch format, where reaction products accumulate and synthesis rates decline over time. We addressed the problems associated with batch reactions by developing a microfluidic chip with nanoliter-scale reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these nano-reactors we can run TX-TL reactions in continuous mode keeping synthesis rates at constant steady state levels for more than 30h. The setup allows close control over the reaction conditions such as dilution rates and DNA template concentration, and to monitor mRNA and reporter protein levels in real time. We can test any genetic program of our choice just by adding the DNA templates coding for the desired functions. We implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. As a proof of concept for this reactor-based approach to engineering genetic networks we designed and implemented a novel genetic oscillator. Its network architecture consists of a positive feedback loop coupled to delayed negative feedback. Varying dilution rates and DNA template concentrations we mapped its phase diagram showing that steady state conditions were necessary to produce oscillations. The period of oscillations could be tuned by dilution rate. To demonstrate that in vitro synthetic biology is useful for prototyping of dynamic genetic networks, we compared the behavior of biomolecular ring oscillators in a cell-free framework and Escherichia coli. We implemented and characterized the ârepressilatorâ, a three-node negative feedback oscillator in vitro. We then used our cell-free framework to engineer novel three-node, four-node, and five-node negative feedback architectures going from the characterization of circuit components to the rapid analysis of complete networks. We validated our cell-free approach by transferring the three-node and five-node oscillators to E. coli, resulting in robust and synchronized oscillations reflecting our in vitro observations. Our results demonstrate that comprehensive circuit characterization can be rapidly performed in a cell-free system and that these in vitro results have direct applicability in vivo. The reactor-based approach to in vitro synthetic biology, which was developed in this thesis, will thus enable a quantitative and more complete characterization of synthetic and natural genetic networks.

EPFL2015

Enhancer loops appear stable during development and are associated with paused polymerase

Daniël Noordermeer

Developmental enhancers initiate transcription and are fundamental to our understanding of developmental networks, evolution and disease. Despite their importance, the properties governing enhancer-promoter interactions and their dynamics during embryogenesis remain unclear. At the beta-globin locus, enhancer-promoter interactions appear dynamic and cell-type specific(1,2), whereas at the HoxD locus they are stable and ubiquitous, being present in tissues where the target genes are not expressed(3,4). The extent to which preformed enhancer-promoter conformations exist at other, more typical, loci and how transcription is eventually triggered is unclear. Here we generated a high-resolution map of enhancer three-dimensional contacts during Drosophila embryogenesis, covering two developmental stages and tissue contexts, at unprecedented resolution. Although local regulatory interactions are common, long-range interactions are highly prevalent within the compact Drosophila genome. Each enhancer contacts multiple enhancers, and promoters with similar expression, suggesting a role in their co-regulation. Notably, most interactions appear unchanged between tissue context and across development, arising before gene activation, and are frequently associated with paused RNA polymerase. Our results indicate that the general topology governing enhancer contacts is conserved from flies to humans and suggest that transcription initiates from preformed enhancer-promoter loops through release of paused polymerase.

Nature Publishing Group2014

Modeling of transcription mechanisms in mammals from kinetic measurements in single cells

Benjamin Zoller

In mammals, transcription was observed to occur predominantly in bursts, resulting from short and intense periods of gene transcription interspersed by longer silent periods. In order to elucidate the causes and the consequences of discontinuous transcription in mammals, we further extended a probabilistic and computational framework to model single-cell time-lapse recording of luminescent reporter based on stochastic gene expression models that describe the basic processes of gene activation, transcription, translation, and degradation of mRNA and proteins. Motivated by the recent finding of a refractory period in gene reactivation, we performed inference in promoter cycle models, where the gene activity is modeled by one active state and multiple sequential inactive states describing the promoter progression toward activation, and we characterized the rate limiting processes causing the refractory period. Our analysis demonstrated that the kinetic structure of the cycle is gene-specific. Several rate limiting steps (5 on average) are required to model the silent period of endogenous promoters while minimal synthetic promoters overall exhibit a simplified promoter cycle structure characterized by a reduced number of steps (3 on average) and longer silent period. Nevertheless, the promoter architecture might not solely be responsible for such an observed difference, as the synthetic promoters were all sharing similar chromatin context (same insertion locus). Based on the inferred kinetics, we investigate the implications on the transcriptional noise. It followed from the inferred bursting kinetics, that intrinsic noise is dominated by the fluctuations in the cycle, and globally maintained over the wide range of expression covered by the clones (100-fold). With minor modifications of the developed probabilistic framework, we investigated how the bursting kinetics of a mammalian gene (Ctgf) are modulated in response to two different induction pathways. Both stimuli resulted in a fast and acute increase in burst sizes. Whereas TGF-b1 induced a sustained increase of transcription rate and prolonged transcriptional activation, serum stimulation led to a large and temporally tight first transcriptional burst, followed by a refractory period in the range of hours. Those observations illustrated how different stimuli can result in kinetically distinct transcriptional responses of the same gene.