Codon-Optimized Yeast Red Fluorescent Protein for AT-Rich Genomes
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Solution Overview
Problem
Current genetic studies in Candida albicans are hindered by the challenges of expressing red fluorescent proteins, particularly due to difficulties in heterologous protein expression and the lack of effective fluorescent markers for analyzing gene expression, protein localization, and protein-protein interactions, as existing red fluorescent proteins are not well expressed in yeast with AT-rich genomes like Saccharomyces cerevisiae and Candida albicans.
Innovation Solution
Development of a yeast-enhanced red fluorescent protein (yeRFP) that is optimized for expression in Candida albicans and Saccharomyces cerevisiae, allowing for visual detection under natural light and offering various UV excitation and emission wavelengths, enabling applications such as FACS sorting and monitoring of plasmid stability and gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional red fluorescent proteins are used in yeast with AT-rich genomes, then the fluorescent marker function is available, but the expression level is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying the nucleotide sequence of red fluorescent protein genes to optimize codon usage for yeast genomes. Specifically, the invention recodes the DNA sequence to use codons that are more frequently used in Saccharomyces cerevisiae and Candida albicans, while maintaining the same amino acid sequence. This codon optimization dramatically improves translation efficiency and protein expression levels in these yeast systems, resolving the contradiction between having a functional fluorescent marker and achieving sufficient expression.
2Adaptability or versatility
If heterologous protein expression is attempted in Candida albicans, then fluorescent markers can be introduced, but expression difficulties prevent successful implementation
Solution Approach 1:
The patent modifies the nucleotide sequence parameters of red fluorescent protein genes to match the codon usage preferences of Candida albicans. By changing the DNA sequence composition while preserving the amino acid sequence, the invention makes heterologous expression feasible in C. albicans, transforming it from a difficult to an achievable process.
Solution Approach 2:
The patent uses codon-optimized nucleotide sequences as an intermediary between the original Discosoma sp. RFP gene and the yeast expression system. This intermediary sequence acts as a bridge that allows the protein to be correctly synthesized in yeast by using codons that the yeast translation machinery efficiently recognizes, thereby eliminating the expression barrier.
3Ease of operation
If Saccharomyces cerevisiae expression is used for genetic studies, then the model organism advantage is available, but lack of effective fluorescent markers limits analysis capabilities
Solution Approach 1:
The patent optimizes the nucleotide sequence of red fluorescent protein genes for Saccharomyces cerevisiae codon usage, creating versions that are efficiently expressed in this model organism. This enables S. cerevisiae to serve as an effective model system for genetic studies with fluorescent markers, combining the ease of genetic manipulation with reliable visualization capabilities.
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
The yeast-enhanced red fluorescent protein provides a robust and visible marker for genetic and pathological studies in yeast, enabling efficient expression, visualization, and analysis of gene expression and protein localization, overcoming previous limitations in using red fluorescent proteins in these organisms.
Implementation Method 1
Red fluorescent protein (RFP) was first isolated and sequenced from a Discosoma sp.
Implementation Method 2
offering various UV excitation and emission wavelengths
Data Source
AI summary
The invention provides a yeast-enhanced red fluorescent protein. In an embodiment of the invention, the yeast-enhanced red fluorescent protein is monomeric and is expressible in Candida albicans. The invention also provides a novel visible color marker for plasmid expression in yeast, particularly Saccharomyces cerevisiae and Candida albicans.