Directed Evolution of Biomolecules Using Lentiviral Delivery
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Solution Overview
Problem
Current methods for directed evolution of biomolecules, such as error prone PCR, are time-consuming, require multiple molecular biology steps, and pose safety risks due to the use of viral vectors, necessitating a faster and safer approach.
Innovation Solution
A single PCR-based method that utilizes a DNA polymerase fused to a DNA binding protein, lacking editing activity, to introduce mutations into biomolecule-encoding plasmids, which are then circularized and expressed in mammalian cells, eliminating the need for viral vectors and reducing experimental time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional error prone PCR and viral vector methods are used for directed evolution, then biomolecule mutation and evolution can be achieved, but the process is time-consuming and requires multiple molecular biology steps
Solution Approach 1:
The patent combines multiple separate molecular biology steps (PCR amplification, viral vector packaging, transduction) into a single integrated system using lentiviral particles that directly deliver mutagenized DNA to mammalian cells, eliminating intermediate steps and reducing hands-on time by fourfold
Solution Approach 2:
The method performs mutagenesis and DNA preparation in advance using error-prone PCR on plasmid DNA before introducing it to mammalian cells, allowing the evolution process to be decoupled from cell culture steps and enabling parallel processing that accelerates overall productivity
2Ease of manufacture
If viral vectors are used for directed evolution, then efficient gene delivery to mammalian cells is achieved, but safety risks increase due to potential hazards
Solution Approach 1:
The patent extracts and utilizes only the beneficial gene delivery capability of viral vectors while removing the harmful components by using lentiviral particles as delivery vehicles for synthetic DNA constructs that lack viral genetic material, thereby maintaining transduction efficiency while eliminating biosafety risks
Solution Approach 2:
Lentiviral particles serve as an intermediary delivery system that transports mutagenized DNA sequences into mammalian cells without requiring the cells to be infected with live viruses, acting as a safe bridge between in vitro mutagenesis and in vivo expression
3Productivity
If multiple molecular biology steps are used for directed evolution, then comprehensive biomolecule optimization is achieved, but device complexity and procedural difficulty increase
Solution Approach 1:
The patent segments the directed evolution process into distinct modular phases: (1) in vitro plasmid mutagenesis by error-prone PCR, (2) lentiviral particle preparation with mutagenized DNA, and (3) mammalian cell transduction and selection, allowing each module to be optimized independently and simplifying the overall workflow
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 method significantly reduces hands-on time by fourfold, minimizes errors, and ensures safety by avoiding viral vectors, while efficiently evolving biomolecules like fluorescent proteins with enhanced properties.
Implementation Method 1
amplifying the entire circular plasmid DNA into an entire linear plasmid DNA by performing a single error prone PCR
Implementation Method 2
performing a single error prone PCR in the presence of a DNA polymerase fused to a DNA binding protein, wherein the DNA polymerase does not comprise any editing activity
Implementation Method 3
circularizing the linear plasmid DNA into a circular plasmid DNA in bacteria
Implementation Method 4
circularizing the linear plasmid DNA into a circular plasmid DNA in bacteria; incorporating the circular plasmid DNA into a host cell
Data Source
AI summary
Disclosed herein are compositions for and methods of directing evolution of biomolecules. The disclosed methods simplify traditional error prone PCR of a single or multiple genes expressed on plasmid DNA for directed evolution and remove several previously required molecular biology steps, thereby affording facile purification of plasmid DNA expressing biomolecules directly from mammalian cells.