Engineering a two-gene system to operate as a highly sensitive biosensor or a sharp switch upon induction with...

Area of Science:

• Synthetic biology
• Molecular biology
• Biotechnology

Background:

• The human estrogen receptor (HER) in yeast (S. cerevisiae) has been used for decades in chimeric transcription factors.
• β-estradiol controls HER translocation to the nucleus, regulating target gene expression.
• Orthogonal activators are created by fusing HER with bacterial, viral, or eukaryotic domains.

Purpose of the Study:

• To optimize a β-estradiol-sensing device for enhanced detection range and maximal output signal.
• To engineer a chimeric activator and reporter protein expression cassette for improved biosensor performance.
• To fine-tune a synthetic promoter for precise gene expression control.

Main Methods:

• Engineering chimeric activators by fusing the human estrogen receptor with bacterial protein LexA and activation domains (VP64 or B42).
• Constructing a synthetic promoter based on the yeast CYC1 core sequence.
• Tuning promoter parameters: 5' UTR length, LexA operator spacing, and operator-TATA box distance.

Main Results:

• Optimized the β-estradiol-sensing device for improved detection range and signal output.
• Demonstrated enhanced biosensor performance through engineering of the chimeric activator and reporter cassette.
• Identified a synthetic promoter configuration enabling sensitive detection and sharp switching based on activator concentration and strength.

Conclusions:

• The engineered β-estradiol biosensor functions as a highly sensitive detection system.
• The system exhibits switch-like gene expression control dependent on activator concentration and activation domain strength.
• This optimized biosensor offers precise control over gene expression in yeast.