Cell-Free Expression System Derived from a Near-Minimal Synthetic Bacterium

Abstract

Cell-free expression (CFE) systems are fundamental to reconstituting metabolic pathways in vitro toward the construction of a synthetic cell. Although an Escherichia coli-based CFE system is well-established, simpler model organisms are necessary to understand the principles behind life-like behavior. Here, we report the successful creation of a CFE system derived from JCVI-syn3A (Syn3A), the minimal synthetic bacterium. Previously, high ribonuclease activity in Syn3A lysates impeded the establishment of functional CFE systems. Now, we describe how an unusual cell lysis method (nitrogen decompression) yielded Syn3A lysates with reduced ribonuclease activity that supported in vitro expression. To improve the protein yields in the Syn3A CFE system, we optimized the Syn3A CFE reaction mixture using an active machine learning tool. The optimized reaction mixture improved the CFE 3.2-fold compared to the preoptimized condition. This is the first report of a functional CFE system derived from a minimal synthetic bacterium, enabling further advances in bottom-up synthetic biology.

Keywords: JCVI-syn3A; Mycoplasma; active machine learning; cell-free expression system.

Figure 1

Syn3A ribosome activity and nitrogen decompression method. (a) Expression of GFP protein in PURE CFE platform using the original PURE ribosomes or Syn3A ribosomes. Error bars are standard deviation (n = 2). (b) Schematic illustration of the nitrogen decompression method from cell incubation (left), incubation at high nitrogen gas pressure (middle), to cell disruption (right). (c) Foamy layer formed after centrifugation was observed for every crude Syn3A cell lysate prepared (left). Air pockets can be observed under the brightfield microscope (right).

Figure 2

Building a functional Syn3A CFE system. (a) Total lipid analysis of foamy layer compared to other fractions of Syn3A and E. coli. Error bars are standard deviation (n = 4). (b) Analysis of rRNA (rRNA) stability in N₂-Syn3A lysate (left box) compared to rRNA from E. coli lysate (middle box) and digitonin-derived Syn3A lysate (right box) after incubation at 37 °C for 1h. Right lane is a diluted replicate of the left lane for each box. (c) Expression of mNG by a complete Syn3A CFE system (N₂-Syn3A lysate and Syn3A ribosomes) controlled by exogenous T7 RNA polymerase (gray line) or endogenous RNA polymerase (blue line). Error bars are standard deviation (n = 2). Results are representative of two biological replicates.

Figure 3

Optimization of feeding buffer components for the Syn3A CFE system. (a) Effect of tRNAs isolated from different organisms in the Syn3A CFE system. Only tRNAs from Mcap show a significant enhancement of Syn3A CFE. (b) Performance of different energy source molecules: 3-PGA, PEP, sucrose, maltose, glucose, and G6P. (c) Effect of CaCl₂ in the expression of mNG in the Syn3A CFE system. Error bars are relative standard deviation (n = 2); *one-way ANOVA, p ≤ 0.05.

Figure 4

Optimization of the entire reaction mixture for the Syn3A CFE system. (a) Boxplot of normalized protein yield progression per METIS iteration. Each dot is an independent experiment containing a distinct reaction mix composition suggested by METIS. Relative yield is a ratio of (mNG signal for each new reaction mix)/(mNG signal for the preoptimized reaction mix). (b) Expression of mNG using the optimized CFE reaction mix in the complete Syn3A CFE system (N₂-Syn3A lysate and Syn3A ribosomes). Error bars are standard deviation or relative standard deviation (n = 2).