Macromolecular crowding creates heterogeneous environments of gene expression in picolitre droplets.
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.
Radboud University, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Maike M K
MM
Hansen
Eindhoven University of Technology, Institute for Complex Molecular Systems and Computational Biology Group, Eindhoven 5600 MB, The Netherlands.
Lenny H H
LH
Meijer
ESPCI ParisTech, Laboratoire de Physique et Mécanique des Milieux Hétérogènes, UMR 7636 du CNRS, Paris 75005, France.
Evan
E
Spruijt
Radboud University, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Roel J M
RJ
Maas
Radboud University, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Marta Ventosa
MV
Rosquelles
Radboud University, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Joost
J
Groen
Radboud University, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Hans A
HA
Heus
Radboud University, Institute for Molecules and Materials, Heyendaalseweg 135, 6525 AJ Nijmegen, The Netherlands.
Wilhelm T S
WT
Huck
D004926:Escherichia coli; D015870:Gene Expression / Q000502:physiology*; D046911:Macromolecular Substances / Q000737:chemistry*; D036103:Nanotechnology / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology
D046911:Macromolecular Substances; D012333:RNA, Messenger