Transcription and Translation in Cytomimetic Protocells Perform Most Efficiently at Distinct Macromolecular Crowding Conditions

Abstract

The formation of cytomimetic protocells that capture the physicochemical aspects of living cells is an important goal in bottom-up synthetic biology. Here, we recreated the crowded cytoplasm in liposome-based protocells and studied the kinetics of cell-free gene expression in these crowded containers. We found that diffusion of key components is affected not only by macromolecular crowding but also by enzymatic activity in the protocell. Surprisingly, size-dependent diffusion in crowded conditions yielded two distinct maxima for protein synthesis, reflecting the differential impact of crowding on transcription and translation. Our experimental data show, for the first time, that macromolecular crowding induces a switch from reaction to diffusion control and that this switch depends on the sizes of the macromolecules involved. These results highlight the need to control the physical environment in the design of synthetic cells.

A Relationship between NTP and Cell Extract Concentration for Cell-Free Protein Expression

“…The first possibility we tested was that inadequate small molecule environments such as magnesium concentration reduced CFPS activity. A previous study showed a high concentration of magnesium can restore the decrease of CFPS activity using HCCE [ 34 ]. To test the possibility, CFPS activity of HCCE under various magnesium concentrations was investigated.…”

Macromolecular crowding effects on the kinetics of opposing reactions catalyzed by alcohol dehydrogenase

“…This observation is consistent with a previous study showing that crowding had distinct effects on isoenzymes of β-galactosidase with different rate-limiting steps [ 99 ]. Furthermore, there is precedent gene regulation studies that crowding can alter the rate-limiting step of a reaction [ 10 ].…”

“…For transition-state-limited reactions, though, crowding effects are less predictable due to the complex, often opposing, interplay of excluded volume, viscosity, soft interactions, and hydration effects [ 9 ]. These trends are further complicated by the fact that crowding has differing effects on steps within an enzymatic process and can thereby alter the rate-limiting step of a reaction [ 10 ]. Excluded volume can increase enzyme activity by improving substrate binding [ 11 ], stabilizing the enzyme [ 12 ], or promoting the proper oligomerization state [ 13 ]; yet, crowding can also decrease enzyme activity by enhancing product inhibition [ 14 ], altering thermodynamic activities [ 15 ], impeding a critical conformational change [ 16 ], aggregating the enzyme [ 17 ], interacting with the substrate [ 18 ], or dehydrating the protein [ 19 , 20 ].…”

“…As such, the kinetics of diffusion-limited reactions are slower in heterogeneous environments than corresponding homogeneous ones due to crowding-induced anisotropic transport [ 27 ]. Furthermore, molecules are directed towards and then remain in less densely packed regions of this heterogeneous environment [ 30 ], thereby promoting co-localization of necessary machinery (mRNA and ribosomes) [ 10 , 26 ]. Taken together, this information suggests that cells may use crowding a means to regulate gene expression and to direct molecules to specific cellular locations [ 25 ].…”

Macromolecular Crowding: a Hidden Link Between Cell Volume and Everything Else

“…However, the classical MC effects are supposed to be largely independent of the nature of "crowder" (which is not always observed in reality), and the typical in vitro crowding experiment often uses inert polymers (dextran, Ficoll, or polyethylene glycol) to mimic the environment inside the cell [9]. The next approximation to nature can be achieved by encapsulating the reagents and crowders in liposomes [10][11][12]. In this paper, we will look at the crowding effects from the CV regulation perspective, including their possible roles in the regulatory volume responses, volume sensing, and apoptosis.…”