The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform.
David GarenneSeth ThompsonAmaury BrissonAset KhakimzhanVincent NoireauxPublished in: Synthetic biology (Oxford, England) (2021)
The new generation of cell-free gene expression systems enables the prototyping and engineering of biological systems in vitro over a remarkable scope of applications and physical scales. As the utilization of DNA-directed in vitro protein synthesis expands in scope, developing more powerful cell-free transcription-translation (TXTL) platforms remains a major goal to either execute larger DNA programs or improve cell-free biomanufacturing capabilities. In this work, we report the capabilities of the all-E. coli TXTL toolbox 3.0, a multipurpose cell-free expression system specifically developed for synthetic biology. In non-fed batch-mode reactions, the synthesis of the fluorescent reporter protein eGFP (enhanced green fluorescent protein) reaches 4 mg/ml. In synthetic cells, consisting of liposomes loaded with a TXTL reaction, eGFP is produced at concentrations of >8 mg/ml when the chemical building blocks feeding the reaction diffuse through membrane channels to facilitate exchanges with the outer solution. The bacteriophage T7, encoded by a genome of 40 kb and ∼60 genes, is produced at a concentration of 1013 PFU/ml (plaque forming unit/ml). This TXTL system extends the current cell-free expression capabilities by offering unique strength and properties, for testing regulatory elements and circuits, biomanufacturing biologics or building synthetic cells.
Keyphrases
- cell free
- circulating tumor
- gene expression
- induced apoptosis
- poor prognosis
- cell cycle arrest
- binding protein
- quantum dots
- drug delivery
- genome wide
- coronary artery disease
- transcription factor
- dna methylation
- protein protein
- public health
- physical activity
- signaling pathway
- endoplasmic reticulum stress
- small molecule
- low grade
- long non coding rna
- crispr cas
- cancer therapy
- wound healing
- label free