Recombinant DNA Construct Switching Gene Expression
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Solution Overview
Problem
Current recombinant DNA constructs for plants often require separate constructs for overexpression and suppression of endogenous genes, leading to interactions at different chromosomal loci that complicate comparative trait analysis.
Innovation Solution
Development of recombinant DNA constructs with site-specific recombination technology that includes a promoter, sense-oriented DNA encoding an endogenous protein, an antisense fragment, and recombination sites recognized by site-specific recombinases, allowing for switching between expression and suppression of the endogenous gene by introducing a recombinase, thereby enabling dual function in a single chromosomal locus.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If separate constructs are used for overexpression and suppression of endogenous genes, then gene function assessment is possible, but device complexity and chromosomal locus interactions increase
Solution Approach 1:
The patent combines both overexpression and suppression capabilities into a single recombinant DNA construct. The construct contains sense-oriented DNA encoding an endogenous protein and anti-sense DNA fragment, along with recombination sites that allow switching between functions. This merging eliminates the need for multiple separate constructs and their associated chromosomal insertions, directly resolving the technical contradiction.
Solution Approach 2:
The single recombinant DNA construct is designed to perform multiple functions: it can both overexpress and suppress the endogenous gene depending on the presence or absence of specific recombination events. The construct contains all necessary elements (promoter, sense DNA, anti-sense fragment, recombination sites) to enable dual functionality, making one construct universal for both assessment modes.
2Device complexity
If a single construct is used for both expression and suppression, then device complexity is reduced, but reliability of function switching must be ensured
Solution Approach 1:
The construct is pre-configured with all necessary elements for function switching before transformation: recombination sites are placed at specific locations between the sense and anti-sense DNA, and the structure is designed to enable reliable switching. This preliminary arrangement ensures that when recombination occurs, the function switch is reliable and predictable, resolving the contradiction between simplicity and reliability.
Solution Approach 2:
Site-specific recombination systems act as intermediaries to control the switching between expression and suppression functions. The recombination sites and associated recombinases provide a reliable mechanism for switching, mediating the transition between functional states in a controlled and predictable manner, thus ensuring reliability while maintaining construct simplicity.
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 stable integration and switching between expression and suppression of endogenous genes in plants, allowing for efficient assessment and evaluation of gene function without the need for multiple constructs, using Cre recombinase or other site-specific recombinases to remove DNA sequences and alter gene expression.
Implementation Method 1
a pair of recombination sites which are recognized by a site-specific recombinase
Implementation Method 2
recombinase-mediated excision will remove the DNA between the recombination sites allowing for a change in original function
Data Source
AI summary
Recombinant DNA constructs, for use in plants and plant cells, have site-specific recombination sites that allow assessing phenotypes and modes of action by over expression or suppression of endogenous genes. In an aspect, a single DNA construct can be switched between over expression and suppression by the action of a recombinase such as the Cre recombinase on constructs having lox recombination sites. Other useful recombination systems include the Flp/frt system, the R/Rs system, the Dre/rox system, and the GIN/gix system.