Rewiring Two-Component Signal Transduction Systems for Biosensor Identification

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Solution Overview

Problem

Current methods are limited in identifying and engineering two-component signal transduction systems (TCSs) to function as biosensors, particularly due to challenges in transferring TCSs between bacterial species and identifying the inputs they sense, as well as difficulties in measuring output genes and ligands.

Innovation Solution

A method involving DNA synthesis and gene assembly to express computationally identified TCSs in standard laboratory bacteria, swapping DNA binding domains to control known output promoters, and using reporter genes to monitor activity, allowing for the identification of inputs and engineering of novel biosensors.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If TCSs are transferred between bacterial species, then the ability to sense and respond to environmental stimuli is improved, but incompatibilities silence their ability to respond to inputs

Engineering Contradiction:

Improvetransferability of TCSs between speciesVSAvoidresponse capability of TCSs

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The response regulator protein is divided into two functional domains: the receiver domain (which receives phosphoryl groups from the sensor kinase) and the DNA-binding domain (which binds to target promoters). By separating these domains and replacing only the DNA-binding domain with a heterologous one, the patent enables cross-species transfer of TCS sensing capability while maintaining reliability through domain compatibility.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A heterologous DNA-binding domain acts as an intermediary that bridges the sensor kinase from one species with the transcriptional machinery of another species. This intermediary domain allows the TCS to function across species boundaries by being compatible with both the phosphorylation signaling pathway and the target promoter recognition system.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If reporter genes are used to monitor TCS activity, then the measurement of output genes is improved, but the complexity of the system increases

Engineering Contradiction:

Improvedetection of TCS outputVSAvoidsystem construction complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

Fluorescent reporter genes (such as GFP) are used to convert the biochemical output of TCS activity into visible optical signals. When the response regulator binds to the reporter promoter, it drives expression of the fluorescent protein, allowing precise measurement of TCS activity through fluorescence intensity while maintaining relatively simple experimental procedures.

Inventive Principle:

Principle #32Color changes

3Measurement precision

If screens are performed with different possible input signals, then the identification of TCS inputs is improved, but the time and resources required increase

Engineering Contradiction:

Improveidentification of sensed inputsVSAvoidscreening duration

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The engineered TCS-reporter system performs self-detection of input signals through its natural sensing capability. When exposed to potential ligands, the system automatically responds by activating the reporter gene if the input is recognized, eliminating the need for complex analytical instruments or manual assessment methods.

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

Fluorescence activation serves as a rapid visual readout for positive interactions between TCS and input signals. This allows high-throughput screening of multiple compounds to be performed quickly by simply adding them to bacterial cultures and measuring fluorescence, dramatically reducing the time required compared to traditional characterization methods.

Inventive Principle:

Principle #32Color changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Enables the transfer and functional characterization of TCSs between bacterial species, facilitating the identification of sensed inputs and the creation of biosensors for various applications.

Implementation Method 1

signal transduction occurs through the transfer of phosphoryl groups from adenosine triphosphate (ATP) to a conserved histidine residue in the SK. This is an autophosphorylation reaction.

Methodology Applied

Scientific EffectAutophosphorylation:

Implementation Method 2

The RRs are phosphorylated on a conserved aspartate residue and are protein phosphatases for the SKs.

Methodology Applied

Scientific EffectPhosphorylation:

Implementation Method 3

Phosphorylation causes a change in the RR conformation, usually activating an attached output domain, which then leads to the activation (or repression) of transcription of target genes.

Methodology Applied

Scientific EffectConformational change:

Data Source

PatentUS10793840B2Identifying ligands for bacterial sensors

Publication Date: 2020.10.06 WILLIAM MARCH RICE UNIVERSITY

AI summary

Methods to create two component signal transduction systems by replace the DNA binding domains and output promoters in bacteria are described.