Eukaryotic Bioluminescence System Using Segmented Lux Operon
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing luciferase systems for bioluminescence in eukaryotic cells, such as those using IRES or 2A sequences, face inefficiencies due to decreased gene expression and protein misfolding, limiting their applicability in biosensors.
Innovation Solution
A nucleic acid with a continuous sequence containing genes encoding LuxA, LuxB, LuxC, LuxD, and LuxE, each under its own heterologous promoter, eliminating the need for IRES or 2A sequences, allowing for enhanced bioluminescence expression and substrate production within eukaryotic cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If IRES or 2A sequences are used to express multiple genes in eukaryotic cells, then gene expression can be achieved, but expression efficiency decreases and protein misfolding occurs
Solution Approach 1:
The patent divides the gene expression system into separate monocistronic units, with each gene (luxA, luxB, luxC, luxD, luxE) having its own promoter and terminator. This segmentation eliminates the need for IRES or 2A sequences, allowing each gene to be independently and efficiently expressed without causing protein misfolding or reduced expression efficiency.
2Adaptability or versatility
If bacterial lux operon structure is used in eukaryotes, then all genes can be regulated by one promoter, but direct gene transfer is difficult due to genetic organization differences
Solution Approach 1:
The patent transforms the bacterial polycistronic operon structure into multiple eukaryotic-compatible monocistronic units. Each gene is placed under its own eukaryotic promoter and terminator, making the system adaptable to eukaryotic genetic organization while maintaining the ability to regulate all genes through individual control elements that are easier to transfer and express in eukaryotic cells.
Solution Approach 2:
The patent changes the genetic organization parameters from prokaryotic operon structure to eukaryotic monocistronic structure, adapting the bacterial lux genes for expression in eukaryotic cells by introducing individual promoters and terminators for each gene, thereby facilitating easier gene transfer and expression.
3Illumination intensity
If external luciferin is added to Firefly luciferase systems, then bioluminescence can be achieved, but labor and costs increase
Solution Approach 1:
The patent implements a self-service system where the luxCDE genes encode enzymes that synthesize the luciferin substrate autonomously within the eukaryotic cell. This eliminates the need for external luciferin addition, reducing labor and costs while maintaining strong bioluminescence signals, as the system produces its own substrate internally.
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 results in a more stable and efficient bioluminescence system, with luminescence signals significantly stronger and more sustained than traditional Firefly luciferase systems, reducing labor and costs by eliminating the need for external luciferin and improving biosensor reliability.
Implementation Method 1
A luciferase catalyzes the reaction of luciferin with oxygen consumption to form a luciferase-bound peroxy-luciferin intermediate product, which releases its excess energy by emitting light
Data Source
AI summary
A nucleic acid comprising a continuous nucleotide sequence, containing: (i) a gene encoding LuxA, (ii) a gene encoding LuxB, (iii) a gene encoding LuxC, (iv) a gene encoding LuxD, (v) a gene encoding LuxE, wherein each of the genes is under the control of a promoter heterologous to the respective gene, and wherein all of the genes together with the promoter are contained in a single nucleotide sequence in a row. The vectors and host cells comprise the nucleic acid. Methods of producing a host cell and methods and uses for detecting an environmental effect and a kit therefor.