Robust gene expression control in human cells with a novel universal TetR aptamer splicing module

Area of Science:

• Synthetic Biology
• Molecular Biology
• Gene Regulation

Background:

• Precise control of gene expression is crucial for synthetic biology applications.
• RNA regulatory devices offer a powerful mechanism for gene expression modulation.
• Existing methods often face challenges with dynamic range and basal activity in mammalian systems.

Purpose of the Study:

• To develop a versatile, robust, and reversible RNA regulatory device for controlling gene expression in human cells.
• To engineer a novel splicing device based on TetR-aptamer interaction for fine-tuning gene expression.
• To achieve high dynamic range and low basal activity in a synthetic gene regulation system.

Main Methods:

• Development of a TetR-aptamer recognition system integrated with a 5' splice site.
• Engineering of a portable splicing device incorporating exonic and intronic sequences for robust splicing.
• Utilizing a self-cleaving peptide for cleavage of exon-born amino acids.
• Testing the device's functionality and portability across different gene contexts in human cells.

Main Results:

• Demonstrated successful regulation of gene expression via intron retention controlled by TetR-aptamer binding.
• Identified and mitigated the impact of genomic context on initial regulatory device performance.
• Developed an advanced splicing device with enhanced portability and consistent functionality.
• Achieved a high dynamic range and low basal activity, overcoming common challenges in mammalian synthetic biology.

Conclusions:

• The novel splicing device provides a robust and reversible platform for precise gene expression control.
• The device's portability and performance characteristics make it highly valuable for synthetic biology applications in mammalian cells.
• This advancement addresses key limitations in current synthetic gene regulation strategies, enabling more sophisticated biological engineering.