Orthogonal DNA Binding Polypeptides for Genetic Circuit Design
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Solution Overview
Problem
Current methods for designing genetic circuits lack efficiency in creating orthogonal sequence-specific DNA binding polypeptides and target DNA sequences, leading to potential cross-talk and interference within genetic circuits, which complicates the development of reliable and predictable genetic programs in living cells.
Innovation Solution
The method involves providing a set of sequence-specific DNA binding polypeptides, optimizing their expression in a heterologous host cell, identifying target DNA sequences, and designing orthogonal sequence-specific DNA binding polypeptide-target DNA sequence pairs to generate a set of orthogonal pairs that do not interfere with each other, allowing for the creation of genetic circuits with specific logic gates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods for designing genetic circuits are used, then the design process can be completed, but cross-talk and interference occur within genetic circuits leading to unreliable operation
Solution Approach 1:
The patent applies segmentation by dividing the genetic circuit design into orthogonal modules, where each module uses unique, non-overlapping DNA binding specificities. This segmentation prevents cross-talk by ensuring that regulatory elements in one module do not inadvertently bind to or interfere with elements in other modules, thereby improving reliability while eliminating harmful interference.
Solution Approach 2:
The patent implements local quality by designing polypeptides with highly specific, localized DNA binding properties. Each polypeptide is engineered to recognize and bind only to its designated target sequence with high affinity and specificity, ensuring that binding events occur only at the intended location and not elsewhere in the genome, thus preventing cross-talk and enhancing circuit reliability.
2Reliability
If sequence-specific DNA binding polypeptides are designed without optimization, then the design process is simpler, but expression and functionality in heterologous host cells are compromised
Solution Approach 1:
The patent applies preliminary action by performing codon optimization and expression system compatibility adjustments before introducing the polypeptides into heterologous host cells. The coding sequences are pre-optimized to match the host cell's codon usage preferences, and regulatory elements are selected to ensure proper expression, thereby guaranteeing functionality while managing the complexity of the optimization process in advance.
3Object-generated harmful factors
If orthogonal polypeptide-target pairs are designed, then cross-talk is reduced, but the design and identification process becomes more complex
Solution Approach 1:
The patent implements feedback by using computational algorithms that predict potential cross-talk interactions and iteratively refine the selection of orthogonal polypeptide-target pairs. The design process incorporates feedback loops where predicted binding specificities are evaluated, and adjustments are made to ensure orthogonality, thereby reducing cross-talk while managing design complexity through systematic evaluation and refinement.
Data Source
AI summary
Methods for design of genetic circuits are provided.