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Plasmids

Plasmids

Plasmids are extrachromosomal DNA molecules found in bacteria, archaea, and some eukaryotic microbes like yeast. These small, circular DNA structures typically contain fewer than 30 genes, although some may exist linearly. Plasmids vary in their number within a cell, known as copy number. Single-copy plasmids are present in one copy per cell and multi-copy plasmids are present in multiple copies, reaching over 100 copies per cell.Plasmids usually replicate independently of the chromosomal DNA...

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Genetic code expansion in mammalian cells: A plasmid system comparison.

Wenyuan Zhou¹, Joshua S Wesalo¹, Jihe Liu¹

¹Department of Chemistry, University of Pittsburgh, Pittsburgh, PA 15260, USA.

Bioorganic & Medicinal Chemistry

|October 18, 2020

Summary

Genetic code expansion enables new protein capabilities in mammalian cells. This study compares plasmid-based systems for delivering unnatural amino acids (UAAs) and analyzes their efficiency across cell types.

Keywords:

Genetic code expansion Mammalian cell Protein Suppressor tRNA Unnatural amino acid tRNA synthetase

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Area of Science:

Biochemistry
Molecular Biology
Synthetic Biology

Background:

Genetic code expansion utilizes unnatural amino acids (UAAs) to enhance protein functionality.
Applications include probing molecular interactions, controlling biological processes, and developing novel therapeutics and vaccines.
Existing methods for transient UAA mutagenesis in mammalian cells require delivery of specialized protein biosynthetic machinery and the gene of interest.

Purpose of the Study:

To comparatively evaluate different plasmid-based genetic code expansion systems.
To analyze the suppression efficiency of various UAAs in mammalian cells.
To assess the performance of these systems across different cell lines.

Main Methods:

Utilized plasmid-based delivery systems for genetic code expansion.
Incorporated orthogonal tRNA/tRNA synthetase pairs and genes for proteins of interest.
Performed comparative analysis of suppression efficiency with diverse UAAs.
Tested system performance in multiple mammalian cell lines.

Main Results:

Demonstrated comparative performance of select plasmid-based genetic code expansion systems.
Provided a detailed analysis of UAA suppression efficiency.
Highlighted variations in efficiency across different cell lines.

Conclusions:

Plasmid-based systems offer viable strategies for genetic code expansion in mammalian cells.
UAA suppression efficiency is dependent on the specific UAA and cell line used.
Further optimization of these systems can advance protein engineering and therapeutic applications.