Genomics and Cell Biology

Enhanced Assembly Of Bacteriophage T7 Produced In Cell-free Reactions Under Simulated Microgravity

By Keith Cowing
Press Release
biorxiv.org
December 27, 2022
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Enhanced Assembly Of Bacteriophage T7 Produced In Cell-free Reactions Under Simulated Microgravity
Experimental pipeline for testing the effect of simulated microgravity on the synthesis and assembly of bacteriophages in transcription-translation (TXTL) systems. A) Phage DNA is mixed with the TXTL components to start the reaction to produce bacteriophage T7. B) A 2D-clinostat is used to simulate microgravity (s-µg). C) TXTL reactions are performed in Polytetrafluoroethylene (PTFE) tubes fitted to the clinostat. Control reactions are performed in identical reaction tubes held static (1-g). D) Transmission electron microscopy (TEM) of T7 phages from the s-µg condition. Arrows indicate full size bacteriophage particles; boxes indicate phages enlarged below. E) Quantitative methods are used to assay protein and DNA content in TXTL samples (Dot-blot, mass spectrometry, fluorescence measurements, real-time quantitative PCR) and the assembly of infectious bacteriophages (plaque assays, TEM). — biorxiv.org

On-demand biomanufacturing has the potential to improve healthcare and self-sufficiency during space missions.

Cell-free transcription and translation reactions combined with DNA blueprints can produce promising therapeutics like bacteriophages and virus-like particles. However, how space conditions affect the synthesis and self-assembly of such complex multi-protein structures is unknown.

Here, we characterize the cell-free production of infectious bacteriophage T7 virions under simulated microgravity. Rotation in a 2D-clinostat increased the number of infectious particles compared to static controls. Quantitative analyses by mass spectrometry, immuno-dot-blot and real-time PCR showed no significant differences in protein and DNA contents, suggesting enhanced self-assembly of T7 phages in simulated microgravity.

While the effects of genuine space conditions on the cell-free synthesis and assembly of bacteriophages remain to be investigated, our findings support the vision of a cell-free synthesis-enabled “astropharmacy”.

Competing Interest Statement

The authors have declared no competing interest.

François-Xavier Lehr, Bruno Pavletić, Timo Glatter, Thomas Heimerl, Ralf Moeller, Henrike Niederholtmeyer
doi: https://doi.org/10.1101/2022.12.16.520761
https://www.biorxiv.org/content/10.1101/2022.12.16.520761v1

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