Bt Toxin Variants via Phage-Assisted Continuous Evolution
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Solution Overview
Problem
The development of resistance in targeted pests to existing Bt toxins limits their effectiveness in pest control, necessitating the creation of novel Bt toxins with enhanced receptor binding capabilities.
Innovation Solution
The use of phage-assisted continuous evolution (PACE) to generate variant Bt toxins with altered amino acid sequences that bind with higher affinity to receptors in resistant pests, specifically through mutations in the receptor binding domain, such as those in the Cry1Ac toxin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type Bt toxins are used for pest control, then initial efficacy is achieved, but resistance develops in target pests over time
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of Bt toxin proteins through site-directed mutagenesis. Specific residues in the receptor binding domain are altered to change the binding characteristics, enabling the toxin to interact with modified or alternative receptors in resistant pest populations while maintaining toxicity to susceptible pests.
Solution Approach 2:
The patent creates Bt toxin variants that can bind to multiple receptor types or alternative receptors. By engineering toxins with broader receptor recognition capabilities, the invention enables a single toxin variant to effectively control both susceptible and resistant pest populations, providing multi-functional activity against diverse pest genotypes.
2Reliability
If Bt toxin amino acid sequences are modified to overcome resistance, then binding affinity to resistant pests improves, but protein stability may be compromised
Solution Approach 1:
The patent applies local quality by making targeted amino acid substitutions specifically in the receptor binding domain (particularly in domain II and domain III) while leaving the rest of the protein structure unchanged. This localized modification approach allows optimization of binding affinity to resistant pest receptors without disrupting the overall protein folding, stability, and crystalline inclusion body formation.
3Adaptability or versatility
If novel Bt toxin variants are developed through evolutionary methods, then diversity of effective toxins increases, but development time and complexity increase
Solution Approach 1:
The patent applies preliminary action by using in silico computational methods to predict which amino acid substitutions are likely to overcome resistance before conducting wet lab experiments. This preliminary computational screening guides the design of site-directed mutagenesis experiments, reducing the number of variants that need to be tested empirically and accelerating the overall development timeline.
Data Source
AI summary
The disclosure provides amino acid sequence variants of Bacillus thuringiensis (Bt) toxins and methods of producing the same. Some aspects of this disclosure provide methods for generating Bt toxin variants by continuous directed evolution. Some aspects of this disclosure provide compositions and methods for pest control using the disclosed variant Bt toxins.