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

Andrei Sakai, Christopher R. Deich, Frank H.T. Nelissen, Aafke J. Jonker, Daniela M.De C. Bittencourt, Christopher P. Kempes, Kim S. Wise, Hans A. Heus, Wilhelm T.S. Huck, Katarzyna P. Adamala, John I. Glass

Research output: Contribution to journalArticlepeer-review

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 Escherichia 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 (RNase) 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 RNase. This work summarizes attempts to produce mycoplasma-based CFE and serves as a cautionary tale for researchers entering this field.

Original languageEnglish (US)
JournalSynthetic Biology
Volume7
Issue number1
DOIs
StatePublished - 2022

Bibliographical note

Publisher Copyright:
© 2022 The Author(s) 2022.

Keywords

  • Cell-free expression system
  • In vitro transcription
  • In vitro translation
  • Mycoplasma
  • Ribonuclease

PubMed: MeSH publication types

  • Journal Article

Fingerprint

Dive into the research topics of 'Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform'. Together they form a unique fingerprint.

Cite this