Recombinant E. coli Fluorescent Screening for Erythritol
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Solution Overview
Problem
Current methods for screening erythritol-producing strains are inefficient, requiring complex sample processing and lengthy detection periods, making it difficult to achieve high-throughput screening.
Innovation Solution
A recombinant plasmid using the pET-22b(+) expression vector to express a transcriptional regulation factor and a marker gene, allowing for the detection of erythritol production by monitoring the expression of a fluorescent protein in the fermentation liquor using a microplate reader.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If high performance liquid chromatography (HPLC) is used to determine erythritol content, then measurement precision is improved, but device complexity and detection time increase
Solution Approach 1:
The patent replaces the mechanical HPLC system with a biological sensing system using recombinant E. coli that expresses erythritol-responsive transcriptional regulators and fluorescent reporter genes. The fluorescent signal directly indicates erythritol presence, eliminating complex chromatographic separation and detection equipment while maintaining measurement capability.
Solution Approach 2:
The patent introduces recombinant E. coli as an intermediary between the erythritol sample and the detection system. The bacteria express transcriptional regulators that bind to erythritol, triggering fluorescent protein expression that serves as a readable signal, thus mediating the detection process in a simplified manner.
2Measurement precision
If HPLC is used for erythritol detection, then measurement precision is improved, but detection time increases
Solution Approach 1:
The patent performs preliminary action by pre-engineering the recombinant E. coli with constitutive erythritol-responsive regulatory systems and fluorescent reporter genes before detection. This preparation allows direct incubation and reading without time-consuming sample processing steps, enabling rapid detection while maintaining accuracy.
Solution Approach 2:
The patent replaces the time-consuming HPLC mechanical detection process with a biological fluorescent reporting system that provides real-time or near-real-time signals upon erythritol binding, dramatically reducing detection time while preserving measurement precision.
3Measurement precision
If traditional screening methods are used, then measurement precision is improved, but productivity decreases
Solution Approach 1:
The patent enables self-service detection where the recombinant E. coli automatically sense erythritol in the environment and respond by expressing fluorescent proteins. This autonomous sensing and reporting capability allows multiple samples to be screened simultaneously without requiring complex automated HPLC systems, thereby increasing throughput while maintaining accuracy.
Solution Approach 2:
The patent creates a universal screening platform where the recombinant E. coli can detect erythritol across multiple samples simultaneously through simple incubation and fluorescent reading. This multi-functional approach replaces the sample-by-sample HPLC analysis, dramatically increasing screening productivity while maintaining detection accuracy.
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
This method enables rapid, efficient, and high-throughput screening of erythritol-producing strains with high sensitivity and accuracy, even in the presence of high-concentration glucose, by correlating fluorescence intensity with erythritol production.
Implementation Method 1
a gene encoding a transcriptional regulation factor and a marker gene; and a nucleotide sequence of the gene encoding the transcriptional regulation factor is shown as SEQ ID No.1
Implementation Method 2
the marker gene is a gene encoding a fluorescent protein. In one implementation of the present disclosure, a nucleotide sequence of the gene encoding the fluorescent protein is shown as SEQ ID No.2
Data Source
AI summary
The present disclosure discloses recombinant Escherichia coli and application thereof in screening erythritol-producing strains, and belongs to the technical field of microorganisms. The recombinant Escherichia coli used in a method for screening an erythritol-producing strain disclosed by the present disclosure can well perform positive correlation induction on erythritol with different concentrations, so that the method for screening the erythritol-producing strain has the advantage of high sensitivity. High-concentration glucose is usually adopted as a fermentation substrate when erythritol is produced in a fermentation mode in the industry, but the method for screening the erythritol-producing strain disclosed by the present disclosure can overcome the interference of the high-concentration glucose, and under the interference of the high-concentration glucose, the recombinant Escherichia coli used in the method for screening the erythritol-producing strain can still well perform positive correlation induction on erythritol with different concentrations, and the correlation is higher than that without the interference of the glucose. Therefore, the method for screening the erythritol-producing strain has the advantage of strong anti-interference capability.