Antisense transcription as a tool to tune gene expression

Area of Science:

• Synthetic biology
• Molecular biology
• Genomics

Background:

• Genomic antisense transcription, occurring counter to gene orientation, is prevalent but its regulatory roles are poorly understood.
• Understanding antisense transcription is crucial for deciphering gene regulation and advancing genetic engineering.

Purpose of the Study:

• To investigate how antisense transcription impacts gene expression and regulatory circuit characteristics.
• To develop and validate a novel genetic part for controlling gene expression via antisense transcription.

Main Methods:

• Construction of a synthetic Escherichia coli system to study antisense transcription.
• Development of a genetic part combining a unidirectional terminator and a constitutive antisense promoter.
• Application of chip-based oligo synthesis to create a large library of 5,668 terminator-promoter combinations.
• Engineering a genetic NOT gate circuit with repressors (PhlF, SrpR, TarA) controlled by the antisense system.

Main Results:

• Demonstrated proportional repression of gene expression by the novel genetic part, dependent on antisense promoter strength.
• Successfully tuned the threshold of a genetic regulatory circuit using antisense promoters without altering other response functions.
• Quantified the contributions of antisense RNA and transcriptional interference to gene repression.
• Developed a biophysical model for RNA polymerase collisions in gene repression.

Conclusions:

• Antisense transcription offers a tunable mechanism for controlling gene expression in synthetic biology.
• The developed genetic part and library provide a new tool for precise gene expression modulation.
• This work elucidates the quantitative role of antisense transcription in regulatory networks and genetic engineering.