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Engineering 4D regulation toolbox to control spatiotemporal cell-free reconstitution
Engineering 4D regulation toolbox to control spatiotemporal cell-free reconstitution
Bottom-up reconstituting well-characterized functional molecular entities, parts and modules towards a synthetic cell will give new insights into the general mechanisms and molecular origins of life. However, a remaining central challenge is how to organize cellular processes spatiotemporally from their component parts in vitro. To this end, we developed a 4D regulation toolbox to facilitate a bottom-up reconstitution in both time and space. The spatiotemporal regulation of the 4D toolbox covers the aspects from dynamic gene transcription & translation, reversible protein interaction, spatially protein positioning, sequential protein assembly, extends to defining geometrical membrane boundaries and mimicking cellular anisotropic microenvironment. Firstly, we developed a thermo-genetic regulation toolbox based on synthetic RNA thermometers, for temporally controlling protein expression in vitro. We validated RNA thermometers from in vivo to in vitro and tuned RNA thermometers through utilizing cell free protein synthesis system. Then we generated the thermo-sensitive protocell by encapsulating thermo-regulated transcription and translation machine in water-in-oil droplets. With the temperature sensing devices, the protocells can be operated with logic AND gates, differentially processing temperature stimuli into biological signals. Secondly, we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on timescales of seconds. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. To develop an artificial anisotropic membrane environment, we introduced a 3D printed protein hydrogel device to induce pH-stimulated reversible shape changes in trapped vesicles. Deformations towards unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations. Moreover, the shape-tunable vesicle provides a spatially well-defined microenvironment for reconstituting shape-dependent protein systems, such as reaction-diffusion system that request explicitly non-spherical geometries. By taking advantages of the 3D printed hydrogel, we programmably engineered contractible scaffolds for actin-myosin motor reconstitution in 3D space. Nanoscale actomyosin motor as a bio-actuator could generate, transmit active contraction and then drive large-scale shape-morphing of complex 3D hydrogel scaffolds. In summary, by developing the spatiotemporal toolbox, this thesis introduces a promising step towards establishing bottom-up reconstitution in space and time, which could also guide future efforts in hierarchically building up the next level of complexity towards a minimal cell.
synthetic biology, cell free, 3D printing, bottom-up, RNA thermometer, membrane, optogenetics, actomyosin, micropatterning
Jia, Haiyang
2019
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Jia, Haiyang (2019): Engineering 4D regulation toolbox to control spatiotemporal cell-free reconstitution. Dissertation, LMU München: Faculty of Biology
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Abstract

Bottom-up reconstituting well-characterized functional molecular entities, parts and modules towards a synthetic cell will give new insights into the general mechanisms and molecular origins of life. However, a remaining central challenge is how to organize cellular processes spatiotemporally from their component parts in vitro. To this end, we developed a 4D regulation toolbox to facilitate a bottom-up reconstitution in both time and space. The spatiotemporal regulation of the 4D toolbox covers the aspects from dynamic gene transcription & translation, reversible protein interaction, spatially protein positioning, sequential protein assembly, extends to defining geometrical membrane boundaries and mimicking cellular anisotropic microenvironment. Firstly, we developed a thermo-genetic regulation toolbox based on synthetic RNA thermometers, for temporally controlling protein expression in vitro. We validated RNA thermometers from in vivo to in vitro and tuned RNA thermometers through utilizing cell free protein synthesis system. Then we generated the thermo-sensitive protocell by encapsulating thermo-regulated transcription and translation machine in water-in-oil droplets. With the temperature sensing devices, the protocells can be operated with logic AND gates, differentially processing temperature stimuli into biological signals. Secondly, we engineered the PhyB-PIF6 system to spatiotemporally target proteins by light onto model membranes and thus sequentially guide protein pattern formation and structural assembly in vitro from the bottom up. We show that complex micrometer-sized protein patterns can be printed on timescales of seconds. Moreover, when printing self-assembling proteins such as the bacterial cytoskeleton protein FtsZ, the targeted assembly into filaments and large-scale structures such as artificial rings can be accomplished. To develop an artificial anisotropic membrane environment, we introduced a 3D printed protein hydrogel device to induce pH-stimulated reversible shape changes in trapped vesicles. Deformations towards unusual quadratic or triangular shapes can be accomplished. Mechanical force induced by the cages to phase-separated membrane vesicles can lead to spontaneous shape deformations. Moreover, the shape-tunable vesicle provides a spatially well-defined microenvironment for reconstituting shape-dependent protein systems, such as reaction-diffusion system that request explicitly non-spherical geometries. By taking advantages of the 3D printed hydrogel, we programmably engineered contractible scaffolds for actin-myosin motor reconstitution in 3D space. Nanoscale actomyosin motor as a bio-actuator could generate, transmit active contraction and then drive large-scale shape-morphing of complex 3D hydrogel scaffolds. In summary, by developing the spatiotemporal toolbox, this thesis introduces a promising step towards establishing bottom-up reconstitution in space and time, which could also guide future efforts in hierarchically building up the next level of complexity towards a minimal cell.