Modelling cell-free RNA and protein synthesis with minimal systems

Abstract

DNA-guided cell-free protein synthesis using a minimal set of purified components has emerged as a versatile platform in constructive biology. The E. coli-based PURE (Protein synthesis Using Recombinant Elements) system offers the basic protein synthesis factory in a prospective minimal cell relying on extant molecules. However, it becomes urgent to improve the system's performance, and to build a mechanistic computational model that can help interpret and predict gene expression dynamics. Herein, we utilized all three commercially available PURE system variants: PURExpress, PUREfrex and PUREfrex2.0. We monitored apparent kinetics of mRNA and protein synthesis by fluorescence spectroscopy at different concentrations of DNA template. Analysis of polysome distributions by atomic force microscopy, combined with a stochastic model of translation, revealed inefficient usage of ribosomes, consistent with the idea that translation initiation is a limiting step. This preliminary dataset was used to formulate hypotheses regarding possible mechanisms impeding robust gene expression. Next, we challenged these hypotheses by devising targeted experiments aimed to alleviate the current limitations of PUREfrex. We identified depletion of key initiation factors by translationally inactive mRNA as a possible inhibitory mechanism. This adverse process could partly be remedied by targeted mRNA degradation, whereas addition of more IFs and of the hrpA RNA helicase had no substantial effects. Moreover, depletion of tRNAs as peptidyl-tRNAs can become limiting in PUREfrex (but not in PURExpress), which can be alleviated by addition of peptidyl-tRNAhydrolase (PTH). We attempted to build a new model for PURE system dynamics integrating all experimental observations. Although a satisfying global fit can be obtained in specific conditions (with PTH), a unifying system's level model is still missing.

Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes

“…FtsA concentration does not significantly increase beyond the first 3 h of expression ( Supplementary Fig. 2), in agreement with the kinetic profiles of protein production with the PURE system 34 . Concentration of synthesized FtsA was compared after 3 and 6 h expression, yielding 4.5 ± 0.5 μM and 5.8 ± 1.1 μM, respectively.…”

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

“…Although highly popular, these systems are more expensive ($0.6-2/µL) than lysate systems ($0.3-0.5/µL). Moreover, despite the fact that the commercial systems are all based on the original PURE system, their exact composition is proprietary, and functional differences can be observed between them in terms of batch to batch variability, system yield, translation rate, lifespan of the reaction, and shelf-life (Doerr et al, 2019).…”

“…One direction for optimizing recombinant systems for protein synthesis yield is focused on finding optimal concentrations of the basic system components, such as proteins, energy sources, small molecules, and salts (Kazuta et al, 2014;Li et al, 2014Li et al, , 2017Doerr et al, 2019) (Figure 2C). Important work to improve our understanding of the system was done by Matsuura et al (2009), who performed titrations of all protein components.…”

“…To better understand the system behavior and to identify limiting factors, computational models of the PURE system have been developed. This includes coarse-grained ordinary differential equation (ODE) models containing effective lumped parameters and a small number of reactions (Mavelli et al, 2015;Carrara et al, 2018;Doerr et al, 2019), as well as more complex models based on modeling of a large number of elementary reactions, which can provide more detailed mechanistic insights but whose connection to experimental data as well as parameter inference is challenging (Matsuura et al, 2017(Matsuura et al, , 2018. These models show that a number of steps involving ribosomes could potentially become rate-limiting: these include slow elongation rates, peptide release, and ribosome dissociation; qualitatively similar results were observed experimentally (Kempf et al, 2017;Li et al, 2017;Doerr et al, 2019).…”

A self-regenerating synthetic cell model

“…Another key improvement was the supply of multiple solutions without the need for cooling. This was achieved by storing the energy and protein components separately, which when stored pre-mixed and without cooling resulted in non-productive resource consumption [35]. Secondly, reaction temperature was set to 34 • C, which decreased PURE degradation with only a minor decrease in protein synthesis rate ( Supplementary Fig.…”