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

Area of Science:

• Synthetic biology
• Microbiology

Background:

• Cell-free expression (CFE) systems are crucial for synthetic cell development.
• Orthogonality of CFE systems across different species remains a significant challenge.
• Genome-minimized bacteria like JCVI-syn3A offer potential platforms for novel CFE systems.

Purpose of the Study:

• To optimize a Mycoplasma bacterium-based CFE system using lysates from JCVI-syn3A and Mycoplasma capricolum.
• To overcome species-specific limitations in existing CFE platforms.
• To establish a compatible CFE system for minimal cell constructs.

Main Methods:

• Systematic testing of bacterial lysate production methods based on the Escherichia coli S30 protocol.
• Optimization of lysate production and feeding buffer composition.
• Investigation of ribonuclease (RNase) activity and alternative lysate preparation using digitonin.

Main Results:

• Mycoplasma capricolum and JCVI-syn3A lysates did not support cell-free gene expression.
• Only minimal in vitro transcription of RNA aptamers was observed.
• High ribonuclease activity was consistently detected in all tested lysates, degrading ribosomal RNAs and inhibiting CFE.

Conclusions:

• Developing Mycoplasma-based CFE systems is hindered by persistent, high ribonuclease activity.
• Surface-associated RNase activity in Mycoplasma cells, including minimal variants, poses a significant barrier.
• This research serves as a cautionary account for future efforts in creating novel CFE systems from Mycoplasma.