Synthetic Bt Gene Codon Optimization for Plant Expression
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Solution Overview
Problem
The expression of Bacillus thuringiensis (B.t.) genes in plants is problematic, resulting in low levels of stable mRNA and protein production, leading to inadequate insecticidal efficacy, due to factors such as high A+T content, potential polyadenylation signals, and ATTTA sequences affecting mRNA stability and processing.
Innovation Solution
Modification of the B.t. gene sequences by removing ATTTA sequences and potential polyadenylation signals through site-directed mutagenesis, and optimizing codon usage to enhance mRNA stability and translation efficiency, while maintaining the amino acid sequence and avoiding restriction sites, to create synthetic genes with improved expression levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If B.t. genes with high A+T content are introduced into plants, then the genes can be successfully transformed and expressed, but the mRNA stability decreases and protein production levels remain low
Solution Approach 1:
The patent removes destabilizing sequences (ATTTA motifs and polyadenylation signals) from the B.t. gene coding region to eliminate their harmful effects on mRNA stability while preserving the essential insecticidal function of the toxin protein
Solution Approach 2:
The patent modifies the nucleotide sequence parameters of the B.t. gene by replacing A+T rich codons with G+C containing codons that encode the same amino acids, thereby changing the mRNA sequence composition to improve stability without altering the protein product
2Manufacturing precision
If the native B.t. gene sequence is used, then the amino acid sequence is preserved, but the translation efficiency and protein expression levels are low
Solution Approach 1:
The patent optimizes codon usage by replacing rare B.t. codons with plant-preferred codons, thereby improving translation efficiency and protein expression levels while maintaining the exact same amino acid sequence through synonymous codon substitutions
Solution Approach 2:
The patent creates a synthetic copy of the B.t. gene with optimized nucleotide sequence that is transcriptionally and translationally optimized for plant cells, while the amino acid sequence remains identical to the native toxin
3Stability of the object's composition
If ATTTA sequences are present in the B.t. gene, then the gene maintains its native structure, but mRNA degradation is accelerated and expression levels decrease
Solution Approach 1:
The patent specifically removes ATTTA destabilizing sequences from the B.t. gene coding region by site-directed mutagenesis, eliminating their capacity to accelerate mRNA degradation while preserving the functional integrity of the toxin protein
Solution Approach 2:
The patent identifies and exploits the harmful effect of ATTTA sequences on mRNA stability by deliberately removing them, thereby converting a potentially detrimental feature into a benefit through targeted sequence modification
4Manufacturing precision
If potential polyadenylation signals are present in the B.t. gene, then the gene sequence remains authentic, but premature transcription termination occurs and full-length mRNA is reduced
Solution Approach 1:
The patent removes potential polyadenylation signals (such as AATAAA and variants) from the B.t. gene coding sequence through site-directed mutagenesis, preventing premature transcription termination and ensuring production of full-length mRNA
Solution Approach 2:
The patent introduces point mutations at specific nucleotide positions within polyadenylation signal motifs, thereby interfering with their function as transcription termination signals while maintaining the reading frame and amino acid sequence
Data Source
AI summary
A method for modifying structural gene sequences to enhance the expression of the protein product is disclosed. Also disclosed are novel structural genes which encode insecticidal proteins of B.t.k. HD-1, B.t.k. HD-73, B.t. tenebrionis, B.t. entomocidus, 2 protein of B.t.k. HD-1, and the coat protein of potato leaf roll virus.