Transposon-Based Parallel DNA Functionality Testing
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Solution Overview
Problem
Current synthetic biology methods face challenges in efficiently diagnosing and debugging synthetic DNA modules, particularly at the genome scale, due to limited fast and cost-effective test methods for probing the functionality of synthetic genome constructs, making it difficult to determine which DNA sequence changes are tolerated or disruptive.
Innovation Solution
A transposon-based circuit testing strategy (Tncite) is employed to assess the functionality of synthetic DNA parts in parallel with base pair resolution, involving the introduction of synthetic DNA constructs into cells with ancestral parts, high-throughput transposon mutagenesis, and sequencing to map transposon insertion sites, allowing for precise measurement of part functionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional DNA sequencing methods are used to verify synthetic DNA constructs, then sequence fidelity is maintained, but functional testing capability is insufficient and the design-build-test cycle is slow
Solution Approach 1:
The patent replaces traditional mechanical/Sanger sequencing methods with next-generation sequencing (NGS) technology to enable parallel functional testing of synthetic DNA constructs. This substitution allows simultaneous sequencing of thousands of DNA variants, dramatically increasing testing throughput while maintaining accuracy through bioinformatic analysis of sequencing data
Solution Approach 2:
The patent segments the functional testing process into distinct components: (1) synthesis of DNA libraries with varied sequences, (2) transformation into host cells, (3) phenotypic selection under specific conditions, and (4) NGS-based sequencing and analysis. This segmentation enables each step to be optimized independently and facilitates high-throughput parallel processing
2Reliability
If synthetic DNA constructs are subjected to extensive functional testing, then functionality is verified, but time consumption and cost increase significantly
Solution Approach 1:
The patent applies partial action by testing only the essential functional aspects of synthetic DNA constructs relevant to the specific application. Rather than comprehensive testing of all possible functions, the method focuses on key phenotypic outcomes under defined selective conditions, achieving sufficient verification without exhaustive testing
Solution Approach 2:
The patent employs preliminary action through in silico design and prediction of synthetic DNA construct functionality before experimental testing. Bioinformatic tools predict expected phenotypes and essential functions, allowing the experimental testing to focus specifically on verifying these predictions rather than exploring all possible functions from scratch
3Productivity
If traditional DNA testing methods are used, then individual constructs are tested, but parallel testing capability and throughput are limited
Solution Approach 1:
The patent implements universality by designing a standardized testing platform that can evaluate multiple synthetic DNA constructs with different functions simultaneously. The same NGS-based workflow and analysis pipeline are applied universally across diverse DNA libraries, whether testing metabolic pathways, structural proteins, or regulatory elements, enabling high throughput without proportionally increasing system complexity
Solution Approach 2:
The patent uses copying by creating multiple identical replicates of synthetic DNA constructs in parallel cultures. Each construct is synthesized in duplicate or triplicate and transformed into identical host cell populations, allowing statistical analysis of functional data while maintaining experimental consistency. The NGS sequencing itself copies and amplifies DNA fragments for detection
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 effective and highly parallelized functionality testing of extensive genetic part libraries and synthetic genomes, accelerating the design-engineer-test cycle in synthetic genome construction and bio-synthetic pathway engineering, and allowing for radical sequence refactoring without affecting biological functions.
Implementation Method 1
Cells are subjected to high-throughput transposon mutagenesis, and cultivated under selective growth conditions
Data Source
AI summary
The invention relates to a process for determining the functionality of an artificial genetic element. An artificial genetic element designed to serve the same biological function as a native genetic element is introduced into cells, and the cells are subjected to conditions of high frequency transposon mutagenesis. Subsequently, a set of DNA sequences representative of a site of insertion of the transposable element is obtained from the cells and the frequency of insertion of the transposable element into the native genetic element and the artificial genetic element is determined. Comparing the frequency of transposon insertion allows for assigning a likelihood of functionality to said artificial functional element, which is high if the frequency of transposon insertion is essentially equal for both elements, and which is low if the frequency of transposon insertion is higher into the artificial genetic element.