Biotinylated Endolysin CBD for Gram-Positive Bacteria Detection
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Solution Overview
Problem
Current methods for detecting and enriching gram-positive bacteria, such as Bacillus cereus and Listeria, are time-consuming and inefficient, often requiring multiple steps and resulting in delayed detection, which is problematic for food safety and healthcare due to the risk of contamination and the need for timely treatment.
Innovation Solution
The use of biotinylated polypeptides comprising enzymatically non-active cell wall binding domains of endolysins or cell wall lysing enzymes, specifically the JS-tag fused with CBDs, for efficient binding, enrichment, and detection of gram-positive bacteria through a 2-step method involving incubation with a sample and subsequent immobilization on streptavidin-coated magnetic particles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional multi-step enrichment methods are used, then bacteria can be detected, but detection time is excessively long (4-7 days for listeria, 1.5-2.5 days for Bacillus cereus)
Solution Approach 1:
The invention extracts only the essential cell wall binding domain (CBD) from endolysins, separating it from the enzymatic domain. This CBD fragment retains the ability to specifically bind to gram-positive bacteria cell walls while being simpler and faster to produce and use in detection assays, thereby reducing detection time while maintaining sensitivity
Solution Approach 2:
The CBDs are pre-produced and pre-characterized for their specific binding affinities to different gram-positive bacteria. This preliminary preparation allows direct use in binding assays without requiring time-consuming culture enrichment steps, as the CBDs can directly bind to bacteria at very low concentrations present in infected samples
2Measurement precision
If conventional enrichment methods are used, then bacteria can be detected, but the process requires multiple complex steps including primary enrichment, selective secondary enrichment, and primary isolation
Solution Approach 1:
The detection method is segmented into distinct functional components: specific CBDs for different gram-positive bacteria species, each with defined binding specificities. This segmentation allows targeted detection of specific pathogens without requiring broad-spectrum enrichment steps, simplifying the overall process while maintaining reliability
Solution Approach 2:
The CBD acts as an intermediary binding molecule that directly connects the target bacteria to detection systems. This intermediary approach eliminates the need for multiple enrichment and isolation steps, as the CBD specifically binds to bacteria even at low concentrations and can be directly detected or used for further analysis
3Reliability
If antibody-based detection methods are used, then bacteria binding can be achieved, but recovery rates are poor (5-25%) and cross-reactions occur
Solution Approach 1:
The invention changes the binding parameter from antibody-antigen recognition to CBD-cell wall peptidoglycan recognition. This parameter change results in higher binding affinity and specificity, as CBDs directly bind to conserved cell wall components of gram-positive bacteria, achieving near-complete recovery rates without cross-reactions to gram-negative bacteria or other cell types
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 approach significantly reduces detection time, enhances the recovery rate of bacteria, and improves sensitivity, allowing for rapid identification of pathogens in complex samples like food and clinical specimens, thereby facilitating timely intervention.
Implementation Method 1
polypeptides comprising a enzymatically non-active cell wall binding domain of an endolysin or another cell wall lysing enzyme... for binding, enriching, removing from a sample, capturing and/or detecting bacteria
Implementation Method 2
immobilization on streptavidin-coated magnetic particles
Implementation Method 3
streptavidin-coated magnetic particles
Data Source
AI summary
The present invention relates to polypeptides comprising a enzymatically non-active cell wall binding domain of an endolysin or another cell wall lysing enzyme, and a sequence according to SEQ ID NO: 1 or derivatives thereof, wherein the polypeptide besides the cell wall binding domain comprises no further domains of an endolysin, as well as means for their preparation. The present invention further relates to methods for binding, enriching, removing from a sample, capturing and/or detecting bacteria, particularly gram positive bacteria.