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High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Published on: February 7, 2019
Transcription factor shielding yields a highly specific and sensitive whole-cell biosensor for Pb2+ detection
Liang Shen1, Yubo Zhang2, Junhan Su2
1Wannan Medical College, Wuhu, 241002, China; Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, 230031, China.
Researchers developed a whole-cell biosensor (WCB) for detecting lead ions (Pb2+) with improved specificity. This biosensor engineering strategy enhances environmental contaminant detection, overcoming interference from mercury ions (Hg2+).
Area of Science:
- Environmental Science
- Biotechnology
- Analytical Chemistry
Background:
- Whole-cell biosensors (WCBs) offer cost-effective environmental contaminant detection.
- Limited specificity, especially interference from similar metal ions like mercury (Hg2+) for lead (Pb2+) detection, hinders WCB performance in complex samples.
Purpose of the Study:
- To engineer a highly specific whole-cell biosensor for lead ion (Pb2+) detection.
- To address and mitigate interference from mercury ions (Hg2+) in Pb2+ sensing.
Main Methods:
- Constructed a wild-type Pb2+ WCB using Klebsiella pneumoniae PbrR-kp.
- Employed directed evolution and a transcription factor (TF) shielding strategy to enhance sensitivity and specificity.
- Introduced a mercury-resistant TF mutant (MerR*) and optimized ribosomal binding site (RBS) library for improved specificity.
Main Results:
- Developed a mutant WCB (PbrR-3) with a low limit of detection (LOD) of 0.08 nM for Pb2+, though still responsive to Hg2+.
- Optimized WCB (RBSM480) achieved an LOD of 0.03 nM for Pb2+ and a 5-fold increase in specificity.
- Validated accurate detection of mixed Pb2+/Hg2+ samples against ICP-MS.
Conclusions:
- A high-performance Pb2+ WCB was successfully engineered.
- The TF shielding strategy is a versatile method for enhancing WCB specificity.
- This approach improves the reliability of WCBs for detecting environmental contaminants in complex matrices.
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