Cogenerating Synthetic Parts toward a Self-Replicating System.

Area of Science:

• Synthetic biology
• Molecular biology
• Biochemistry

Background:

• Engineering biological systems requires strategic approaches.
• The Protein synthesis Using Recombinant Elements (PURE) system facilitates transcription, translation, and energy regeneration.
• Optimizing the PURE system is key for creating alterable, lifelike systems.

Purpose of the Study:

• To regenerate Escherichia coli ribosomal (r-) proteins individually using the PURE system.
• To enhance the coexpression and detection of ribosomal proteins within the PURE system.
• To assess the activity of reconstituted ribosomes for synthetic biology applications.

Main Methods:

• Individual regeneration of 54 E. coli r-proteins from DNA templates in the PURE system.
• Quantitative proteomics using stable isotope labeling to detect coexpressed r-proteins.
• Optimization of DNA template concentrations and use of a Fluid Array Device for improved protein yield.

Main Results:

• Detection of 26/33 50S and 20/21 30S subunit r-proteins in batch PURE system.
• Coexpression and detection of at least 29/33 50S and all 21 30S subunit r-proteins using optimized methods.
• Yield boost of 1.5 to 5-fold for single r-proteins, reaching up to 2.4 μM.
• Reconstituted ribosomes showed ~13% activity of native 70S ribosomes, increasing to 21% with GroEL/ES.

Conclusions:

• The study successfully regenerated and coexpressed a significant number of E. coli ribosomal proteins in the PURE system.
• Optimized PURE system conditions and a Fluid Array Device markedly improved ribosomal protein yield and detection.
• Reconstituted ribosomes exhibit partial activity, highlighting progress towards self-replicating synthetic ribosomes.