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Related Experiment Video

Updated: May 29, 2025

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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Engineering a genomically recoded organism with one stop codon.

Michael W Grome^1,2, Michael T A Nguyen^1,2, Daniel W Moonan^2,3

¹Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA.

|February 5, 2025

Summary

This summary is machine-generated.

Researchers created Ochre, a genomically recoded organism (GRO), by compressing translation functions into single codons. This engineered bacterium uses UAA as its only stop codon, paving the way for a 64-codon non-degenerate genetic code.

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Published on: May 25, 2018

Area of Science:

Synthetic biology
Genomics
Molecular biology

Background:

The genetic code, while largely conserved, exhibits natural variations in codon assignments and translation factors.
Genomically recoded organisms (GROs) have been engineered with alternative genetic codes by replacing synonymous codons.
Previous efforts have not fully explored translation factor plasticity and codon degeneracy to achieve a single-codon translational function.

Purpose of the Study:

To construct and characterize a GRO, named Ochre, that compresses translational function into a single codon.
To assess the feasibility of a non-degenerate genetic code by leveraging codon degeneracy and translation factor engineering.
To enable precise production of synthetic proteins with unnatural amino acids for biotechnology and biotherapeutics.

Main Methods:

Replaced 1,195 TGA stop codons with synonymous TAA codons in Escherichia coli C321.∆A.
Engineered release factor 2 (RF2) and tRNA^Trp to prevent native UGA recognition.
Utilized engineered translation factors to isolate four codons for specific, non-degenerate functions.

Main Results:

Successfully constructed Ochre, a GRO with a compressed translational system.
Ochre utilizes UAA as the sole stop codon, UGG for tryptophan, and reassigned UAG/UGA codons.
Achieved >99% accuracy for multi-site incorporation of two distinct non-standard amino acids into single proteins.

Conclusions:

Ochre represents a significant advancement toward a 64-codon non-degenerate genetic code.
This engineered organism demonstrates the compression of degenerate stop codons into a single codon.
The development of Ochre facilitates the precise synthesis of multi-functional proteins with novel chemistries for broad biotechnological applications.