Barcoded Bacterial Promoter Profiling in Host RNA Backgrounds
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Solution Overview
Problem
Traditional methods for measuring transcriptional activity of plant-associated bacteria are inefficient and require lengthy sample preparation processes due to the overwhelming presence of host plant RNA, making genome-wide transcriptomic profiling challenging.
Innovation Solution
A method using a DNA-barcoded promoter library integrated into bacterial chromosomes via CRAGE, enabling the quantification of transcriptional activity by enriching bacterial transcripts and distinguishing them from host RNA, allowing rapid identification of biosensors for environmental conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods (fluorescent reporters and qPCR) are used to measure transcriptional activity, then measurement precision is improved, but productivity deteriorates because only a handful of promoter activities can be measured
Solution Approach 1:
The patent segments the promoter library into multiple members, each with a unique barcode sequence. This allows parallel measurement of many promoters simultaneously while maintaining individual identification through the barcode segments, resolving the contradiction between measuring precision and productivity
Solution Approach 2:
The patent uses barcode sequences as copies or identifiers for each promoter. Instead of directly measuring each promoter individually, the barcode serves as a unique copy that can be rapidly sequenced and identified, enabling high-throughput measurement of many promoters at once
2Productivity
If genome-wide transcriptomics is performed on plant-associated bacteria, then productivity is improved, but measurement precision deteriorates due to overwhelming host plant RNA
Solution Approach 1:
The patent extracts and enriches bacterial mRNA from the complex sample by using customized probes that specifically bind to bacterial RNA. This extraction step removes the overwhelming host plant RNA and isolates the bacterial transcripts of interest, enabling precise measurement despite the complex mixture
Solution Approach 2:
The patent uses barcode sequences as intermediaries between the promoters and the detection system. The barcodes are integrated into the bacterial mRNA and serve as detectable markers that can be identified through sequencing, allowing indirect but precise measurement of promoter activity without direct interference from host RNA
3Measurement precision
If physical separation of bacterial cells from host tissues is performed, then measurement precision is improved, but productivity deteriorates due to long sample preparation procedures
Solution Approach 1:
The patent replaces mechanical physical separation methods with a molecular-based enrichment approach using customized probes. Instead of physically isolating bacterial cells through complex mechanical procedures, the method uses molecular probes to selectively bind and enrich bacterial RNA, dramatically simplifying the sample preparation while maintaining precision
Data Source
AI summary
The present invention provides for methods and compositions for recovering high concentrations of transcripts is technically challenging for bacteria colonizing within the host environments. The invention uses Pi-seq technology in a DNA-barcoded promoter library to improve the ability to quantify transcriptional activity of bacterial genes. The invention offers a rapid way to screen and identify biosensors for chemicals and physical conditions associated with host physiology.