Harnessing split fluorescent proteins in modular protein logic for advanced whole-cell detection

Area of Science:

• Synthetic Biology
• Molecular Biology
• Biotechnology

Background:

• Whole-cell biosensors offer potential for molecular detection but suffer from limited selectivity due to natural protein substrate tolerance.
• Enhancing biosensor precision is crucial for reliable molecular detection and analysis.

Purpose of the Study:

• To improve whole-cell biosensor performance by integrating logic AND gates.
• To engineer an orthogonal AND gate in Escherichia coli using the HrpR/S system and self-associating split fluorescent proteins (Spy Tag/Spy Catcher).
• To selectively activate a reporter gene in response to both IPTG and Hg(II) ions.

Main Methods:

• Construction and comparison of HrpR/S and self-associating split fluorescent protein systems for AND gate functionality.
• Systematic genetic engineering and evaluation of biological parts under various conditions.
• Utilizing Spy Tag/Spy Catcher system for split protein complementation.

Main Results:

• Self-associating split fluorescent proteins demonstrate utility in developing high-performance whole-cell biosensors.
• This approach offers advantages including engineering simplicity, reduced basal activity, and enhanced selectivity.
• The HrpR/S system served as a control, providing comparative insights into system performance.

Conclusions:

• Self-associating split fluorescent proteins provide an effective strategy for creating selective whole-cell biosensors.
• This method addresses the challenge of broad substrate tolerance in natural proteins.
• The findings offer a systematic and adaptable approach for advancing biosensor technology.