Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biochemistry

Background:

• Cell-free systems offer flexibility for genetic code engineering.
• Reconstituted cell-free protein synthesis (PURE) systems enable precise control over biological processes.
• Synthetic biology aims to construct artificial cells for novel applications.

Purpose of the Study:

• To develop a cell-free system utilizing entirely in vitro transcribed transfer RNAs (iVTtRNAs) for active protein production.
• To demonstrate the capability of manipulating iVTtRNA composition for genetic code rewriting.
• To investigate the impact of nucleotide modifications on protein yield and decoding fidelity.

Main Methods:

• Development of a PURE system supplemented solely with 21 unmodified iVTtRNAs.
• Genetic code manipulation through altering iVTtRNA composition.
• Introduction of modified nucleotides into specific iVTtRNAs to assess effects on protein synthesis.

Main Results:

• The developed system synthesized active proteins using a redesigned genetic code.
• Genetic code rewriting was achieved by manipulating iVTtRNA composition.
• Modified nucleotides enhanced both protein yield (DHFR production ~40% of native tRNA) and decoding fidelity.

Conclusions:

• The iVTtRNA-based cell-free system provides a flexible platform for genetic code engineering.
• This system facilitates the study of decoding processes and enables protein engineering applications.
• The findings contribute to the advancement of cell-free synthetic biology and artificial cell construction.