Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform

Abstract

Cell-free expression (CFE) systems are one of the main platforms for building synthetic cells. A major drawback is the orthogonality of cell-free systems across species. To generate a CFE system compatible with recently established minimal cell constructs, we attempted to optimize a Mycoplasma bacterium-based CFE system using lysates of the genome-minimized cell JCVI-syn3A (Syn3A) and its close phylogenetic relative Mycoplasma capricolum (Mcap). To produce mycoplasma-derived crude lysates, we systematically tested methods commonly used for bacteria, based on the S30 protocol of E. coli. Unexpectedly, after numerous attempts to optimize lysate production methods or composition of feeding buffer, none of the Mcap or Syn3A lysates supported cell-free gene expression. Only modest levels of in vitro transcription of RNA aptamers were observed. While our experimental systems were intended to perform transcription and translation, our assays focused on RNA. Further investigations identified persistently high ribonuclease activity in all lysates, despite removal of recognizable nucleases from the respective genomes and attempts to inhibit nuclease activities in assorted CFE preparations. An alternative method using digitonin to permeabilize the mycoplasma cell membrane produced a lysate with diminished RNAse activity, yet still was unable to support cell-free gene expression. We found that intact mycoplasma cells poisoned E. coli cell free extracts by degrading ribosomal RNAs, indicating that the mycoplasma cells, even the minimal cell, have a surface-associated RNAse activity. However, it is not clear which gene encodes the ribonuclease. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field.

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

“…Next, we tested common energy source molecules for CFE systems: 3-phosphoglyceric acid (3-PGA), phosphoenolpyruvate (PEP), sucrose, maltose, glucose, and glucose-6-phosphate (G6P). , As expected, 3-PGA and PEP performed better, and an equimolar combination of them showed the best expression levels (Figure b). The last component tested was CaCl 2 , which is a nonconventional element for CFE systems but was previously found as an inhibitor of Mcap nucleases. , Indeed, our experiments showed that 15–25 mM CaCl 2 improved mNG expression (Figure c). Therefore, CaCl 2 , 3-PGA, PEP, and Mcap tRNA were included as permanent components of the Syn3A CFE system (Table ).…”

“…The last component tested was CaCl 2 , which is a nonconventional element for CFE systems but was previously found as an inhibitor of Mcap nucleases. 11,21 Indeed, our experiments showed that 15−25 mM CaCl 2 improved mNG expression (Figure 3c). Therefore, CaCl 2 , 3-PGA, PEP, and Mcap tRNA were included as permanent components of the Syn3A CFE system (Table 1).…”

“…Previously, we reported considerable degradation levels of mRNA (mRNA) and rRNA (rRNA) in Mycoplasma lysate, which impeded in vitro expression . In a first breakthrough, we found functional ribosomes in a specific cell lysate derived from Syn3A cells trypsinized before cell disruption (French press) and centrifuged at high speeds (34,000–80,000 g ).…”

“…In previous attempts to derive a functional CFE system from Mycoplasma bacteria, we found high ribonuclease (RNase) activity in cell lysates, which impeded in vitro protein expression . We speculated that RNase activity arose from membrane-associated nucleases that were released into lysates during cell disruption.…”

“…9,10 In previous attempts to derive a functional CFE system from Mycoplasma bacteria, we found high ribonuclease (RNase) activity in cell lysates, which impeded in vitro protein expression. 11 We speculated that RNase activity arose from membrane-associated nucleases that were released into lysates during cell disruption. This hypothesis was supported by (i) the degradation of substrate RNA and ribosomes caused by intact Mycoplasma cells and (ii) mRNA degradation assays.…”