Implementation of cell-free biological networks at steady state

Living cells maintain a steady state of biochemical reaction rates by exchanging energy and matter with the environment. These exchanges usually do not occur in in vitro systems, which consequently go to chemical equilibrium. This in turn has severely constrained the complexity of biological networks that can be implemented in vitro. We developed nanoliter-scale microfluidic reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these reactors we achieved transcription and translation at steady state for 30 h and implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. We constructed and implemented an in vitro genetic oscillator and mapped its phase diagram showing that steady-state conditions were necessary to produce oscillations. This reactor-based approach will allow testing of whether fundamental limits exist to in vitro network complexity.

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

The contribution of the host stress response to the pathogenesis of Pseudomonas entomophila in the Drosophila intestine

Sveta Chakrabarti

Recently, several studies done in Drosophila have revealed that efficient and rapid recovery from bacterial infection in the gut is possible only when bacterial clearance by the immune system is coordinated with repair through renewal of the epithelium damaged by infection. In this thesis, I have analyzed how the entomopathogenic bacterium Pseudomonas entomophila affects the immune response and epithelium renewal. A microarray analysis first showed that P. entomophila is recognized by the Drosophila immune system since ingestion of this bacterium stimulated the expression of antimicrobial peptide genes by the Imd pathway. In addition, stress and damage related pathways were strongly induced by P. entomophila, which correlate with its capacity to inflict severe damage. While antibacterial genes were induced in the gut following infection with P. entomophila, the immune response was not productive due to a general inhibition of translation that affected all the newly synthesized transcripts. Furthermore, the blockage of translation also inhibited the repair program by blocking the production of JAK-STAT ligands (upd3, upd2) and growth factors, which stimulate the intestinal stem cells proliferation and differentiation. I next analyzed the pathways that link cellular damage to reduction of translation. Using a genetic approach, I showed that inhibition of translation was induced by two signaling pathways: i) the phosphorylation of elongation initiation factor 2α (eIF2α) by the stress kinase GCN2 and ii) the inhibition of the TOR pathway by the AMPK kinase. Both kinases sense metabolic deprivation, suggesting that cellular damages induced by P. entomophila induce a state of “starvation”. Inhibition of translation is usually an adaptive cellular response to adjust the metabolism to the energy status of the cells. The observation that GCN2-deficient flies survived better than wild-type flies to P. entomophila indicates that pathogenesis is linked to an over-activation of stress pathways that usually help to endure the consequence of an infection. In this in vivo model of infection, I also showed that the reduction of translation was a consequence of cellular damage to the intestine caused by host-derived reactive oxygen species (through the activity of Duox) and by the direct action of a pore-forming toxin produced by the pathogen. As a consequence of this translational arrest, flies succumbed P. entomophila infection because they were unable to repair gut damage. Finally, I showed that inhibition of translation also had a strong influence on innate immune responses observed upon P. entomophila infection. The specific activation of a systemic immune response (antimicrobial peptides produced by the fat body) observed upon P. entomophila infection could be recapitulated by feeding flies with a non-lethal pathogen and an inhibitor of translation. Hence, I showed translation inhibition could be an important feature that shapes the immune response. The p38 MAPK family is involved in stress and immunity in both mammals and Drosophila. In Drosophila, three p38-MAPK-encoding genes, p38a, p38b and p38c, have been identified but their function in the gut immune response is poorly characterized. In the second part of my thesis, I have analyzed the role of these three p38 MAPKs in Drosophila intestinal immunity, especially p38c, which is strongly enriched in the gut. I first confirmed that the three p38 are involved kinases are involved in the defense to oral infection with pathogenic (but non-lethal) bacteria such as Erwinia carotovora 15. Surprisingly, p38c mutant flies were more resistant to P. entomophila and did not show the reduction in translation observed in wild-type flies. Interestingly, the transcription of the ROS producing enzyme Duox was reduced in p38c raising the hypothesis that p38 contributes to P. entomophila pathogenicity by activating Duox. Furthermore, I have obtained preliminary data indicating that p38c and the transcription factor ATF-3 act in the same pathway contributing the host immune response and lipid metabolism. Together, my thesis reveals the complex cross-talks between stress and immune pathways during microbial infection. While, it shows that stress pathways usually contribute to endure the damage caused by infection, excessive activation of these pathways can contribute to pathogenesis.

EPFL2013

Establishment of polarized microglial cells derived from mouse embryonic stem cells

Céline Ruegsegger

Microglia are the resident immune cells of the central nervous system (CNS). Microglia undergo rapid activation in response to even minor pathological changes in the CNS. The activation state of microglia and tissue macrophages is characterized by two extremes that are known as M1 and M2. M1 is triggered upon pro-inflammatory stimuli and lead to the secretion of molecules involved in brain defense but also in neurotoxicity. M2 arises upon anti-inflammatory stimuli and is hallmarked by the secretion of molecules that mediate tolerance and neuroprotection. The aim of this study was to investigate the in vitro polarization capacity of microglia into M1 and M2 subtypes. Primary microglia are hard to obtain in sufficient number and therefore a recently developed protocol was applied to differentiate microglial precursors from mouse embryonic stem (ES) cells. The obtained ES cell derived microglial precursors (ESdM) were shown to be an appropriate substitute to primary microglia. Here we showed that ESdM stimulated with interferon-γ (IFN-gamma) and lipopolysaccharide (LPS) display typical M1 characteristics like higher transcription levels of pro-inflammatory cytokines as well as an increase in the expression level of M1 surface markers. They also secreted significantly higher levels of the chemokine CXCL10 and nitric oxide than untreated or interleukin-4 (IL-4) treated cells. These results indicate that ESdM can be polarized into a cytotoxic subtype in vitro using IFN-gamma and LPS. However, no evidence indicating that ESdM were polarized into a neuroprotective M2 subtype by IL-4 treatment has been found. Even though an increase in the transcription level of anti-inflammatory cytokines was observed upon IL-4 treatment, this increase was not significant. In addition, no increase in the expression levels of M2 markers or in the secretion of the anti-inflammatory cytokine IL-10 were found upon IL-4 treatment indicating that IL-4 might be insufficient to polarize ESdM into M2 subtype in vitro. Further experiments are required to better understand the mechanisms of microglial sub-differentiation and to determine the stability of the acquired M1 and M2 phenotypes over time

2010