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Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets

Abstract

Understanding the dynamics of complex enzymatic reactions in highly crowded small volumes is crucial for the development of synthetic minimal cells. Compartmentalized biochemical reactions in cell-sized containers exhibit a degree of randomness due to the small number of molecules involved. However, it is unknown how the physical environment contributes to the stochastic nature of multistep enzymatic processes. Here, we present a robust method to quantify gene expression noise in vitro using droplet microfluidics. We study the changes in stochasticity in the cell-free gene expression of two genes compartmentalized within droplets as a function of DNA copy number and macromolecular crowding. We find that decreased diffusion caused by a crowded environment leads to the spontaneous formation of heterogeneous microenvironments of mRNA as local production rates exceed the diffusion rates of macromolecules. This heterogeneity leads to a higher probability of the molecular machinery staying in the same microenvironment, directly increasing the system's stochasticity.

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Figure 1: Correlated versus uncorrelated noise.
Figure 2: Effect of decreased copy number on inherent stochasticity of gene expression in pico-reactors.
Figure 3: Enhancement of uncorrelated noise in the presence of Ficoll.
Figure 4: Protein expression rates and ribosomal diffusion coefficients.
Figure 5: Inhomogeneous distribution of mRNA over one droplet at high Ficoll concentrations.
Figure 6: Theoretical modelling of gene expression noise.

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Acknowledgements

The authors thank R.Y. Tsien for kindly donating the genes encoding for YFP and CFP, F.H.T. Nelissen and D. Foschepoth for assisting with cloning work, E. Dubuc for designing the molecular beacon, and J. Thiele for designing the masters for the fluidic devices. This work was supported by a European Research Council (ERC) Advanced Grant (246812 Intercom), a VICI grant from the Netherlands Organization for Scientific Research (NWO), and funding from the Ministry of Education, Culture and Science (Gravity programme, 024.001.035).

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M.H., L.M. and W.H. conceived and designed the experiments. M.H., L.M., R.M. and M.V.R. performed the experiments. M.H. and L.M. analysed the data. E.S., J.G. and H.H. contributed materials/analysis tools. M.H. and W.H. co-wrote the paper.

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Correspondence to Wilhelm T. S. Huck.

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Hansen, M., Meijer, L., Spruijt, E. et al. Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets. Nature Nanotech 11, 191–197 (2016). https://doi.org/10.1038/nnano.2015.243

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