Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell

Filippo Caschera, Vincent Noireaux

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Cell-free expression is a technology used to synthesize minimal biological cells from natural molecular components. We have developed a versatile and powerful all-E. coli cell-free transcriptiontranslation system energized by a robust metabolism, with the far objective of constructing a synthetic cell capable of self-reproduction. Inorganic phosphate (iP), a byproduct of protein synthesis, is recycled through polysugar catabolism to regenerate ATP (adenosine triphosphate) and thus supports long-lived and highly efficient protein synthesis in vitro. This cell-free TX-TL system is encapsulated into cell-sized unilamellar liposomes to express synthetic DNA programs. In this work, we study the compartmentalization of cell-free TX-TL reactions, one of the aspects of minimal cell module integration. We analyze the signals of various liposome populations by fluorescence microscopy for one and for two reporter genes, and for an inducible genetic circuit. We show that small nutrient molecules and proteins are encapsulated uniformly in the liposomes with small fluctuations. However, cell-free expression displays large fluctuations in signals among the same population, which are due to heterogeneous encapsulation of the DNA template. Consequently, the correlations of gene expression with the compartment dimension are difficult to predict accurately. Larger vesicles can have either low or high protein yields.

Original languageEnglish (US)
Pages (from-to)185-195
Number of pages11
JournalArtificial Life
Volume22
Issue number2
DOIs
StatePublished - May 1 2016

Fingerprint

Cell-Free System
Escherichia coli
Liposomes
Proteins
DNA
Unilamellar Liposomes
Fluorescence microscopy
Encapsulation
Metabolism
Gene expression
Nutrients
Byproducts
Phosphates
Artificial Cells
Genes
Adenosine Triphosphate
Molecules
Networks (circuits)
Reporter Genes
Fluorescence Microscopy

Keywords

  • Cell-free metabolism
  • Cell-free transcriptiontranslation
  • Encapsulation
  • Gene circuits
  • Minimal cell
  • Synthetic biology

Cite this

Compartmentalization of an all-E. coli Cell-Free Expression System for the Construction of a Minimal Cell. / Caschera, Filippo; Noireaux, Vincent.

In: Artificial Life, Vol. 22, No. 2, 01.05.2016, p. 185-195.

Research output: Contribution to journalArticle

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