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Related Experiment Video

Updated: Jul 9, 2025

OnePot PURE Cell-Free System

OnePot PURE Cell-Free System

Published on: June 23, 2021

Changes in Coding and Efficiency through Modular Modifications to a One Pot PURE System for In Vitro Transcription

Phuoc H T Ngo¹, Satoshi Ishida¹, Bianca B Busogi¹

¹Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, United States.

ACS Synthetic Biology

|December 5, 2023

Summary

This study enhances the Protein Synthesis Using Recombinant Elements (PURE) system for efficient, site-specific incorporation of unnatural amino acids into proteins. The modified system demonstrates flexibility by using orthogonal aminoacyl-tRNA synthetase:tRNA pairs to suppress stop codons.

Keywords:

PURE system cell-free transcription and translation orthogonal aminoacyl-tRNA synthetase unnatural amino acids incorporation

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DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

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Published on: December 29, 2021

Area of Science:

Biochemistry
Molecular Biology
Synthetic Biology

Background:

Unnatural amino acid incorporation enhances protein functionality.
Cell-free protein synthesis, particularly the Protein Synthesis Using Recombinant Elements (PURE) system, is a powerful platform for this purpose.

Purpose of the Study:

To adapt and modify the One Pot PURE system for robust and modular single-site-specific unnatural amino acid incorporation.
To demonstrate the system's flexibility using orthogonal aminoacyl-tRNA synthetase:tRNA pairs.

Main Methods:

Modification of the One Pot PURE system.
Introduction of two distinct orthogonal aminoacyl-tRNA synthetase:tRNA pairs.
Utilizing stop codon suppression for site-specific incorporation.

Main Results:

A robust and modular system for enzymatic single-site-specific unnatural amino acid incorporation was developed.
The system successfully demonstrated flexibility through the suppression of two different stop codons in separate reactions.

Conclusions:

The enhanced One Pot PURE system provides a versatile platform for site-specific unnatural amino acid incorporation.
This advancement facilitates the creation of novel peptides and proteins with tailored functions.