Brute Force Mutagenesis & Directed Evolution Flashcards
What are brute force mutagenesis & directed evolution
2 different approaches in the field of genetic engineering, and can be used for microbial strain improvement
Define mutagenesis
The process of intentionally inducing mutations in the genetic material of an organism
(Mutagenesis) mutations may be induced by various techniques such as
- chemical treatments
- radiation exposure
- genetic engineering methods
What are the 2 purposes of mutagenesis
- to study the effects of induced mutations on an organism’s traits
- to generate variants with desired characteristics
What does brute force mutagenesis involve
Introducing a large number of random, undirected mutations into a target molecule
What principle for brute force mutagenesis rely on
- That some of these mutations may lead to desired traits or properties
- explores a wide range of possibilities
What does the first stage of brute force mutagenesis involve
- introducing random, undirected mutations into target molecules
- generates mutant library
- using techniques like error-prone PCR or chemical mutasynthesis
What is the aim of the first stage of brute force mutagenesis
To create a diverse library of variants with different mutations
What are we doing in the second stage of brute force mutagenesis, and what do we use to accomplish this
- screening for variants with desired traits or properties
- use various assays to identify mutants exhibiting desired characteristics
What happens after potential mutants have been identified
- further characterised and analysed to understand the impact of the introduced mutations on target molecule
- helps us gain insight of relationship between genotype and phenotype
What is directed evolution compared to brute force mutagenesis
Directed evolution is a more focused and guided approach
As well as directed evolution, what are some other recent advances in strain improvement
- metabolic engineering
- molecular breeding (DNA shuffling & whole genome shuffling)
- combinatorial biosynthesis
What do these new advances in strain improvement mean
- achieve new and improved products
- direct research and development towards new targets
What is directed evolution essentially
- mimics the process of natural selection
- manipulates proteins/nucleic acids by giving them desired traits
- makes them better suited for specific applications
What does directed evolution involve
- designing specific mutations based on the understanding of the target molecule’s structure and function
- iterative mutagenesis, followed by selection and screening
What are the 4 main stages to directed evolution
- generation of diversity
- selection or screening
- amplification
- iteration
first stage of directed evolution (generation of diversity)
introducing mutations into target molecules such as proteins or nucleic acids
In directed evolution, what techniques can be implemented to mutate the genome
- error-prone PCR
- DNA shuffling
- site-directed mutagenesis
What is error-prone PCR
Accuracy of PCR is intentionally reduced, leading to the introduction of random mutations during amplification
What is DNA shuffling
The fragmentation and reassembly of DNA sequences from different variants, generating new combinations and mutations
What is site-directed mutagenesis
- specific mutations are introduced at positions in the target molecule
- using techniques like oligonucleotide-directed mutagenesis or PCR-based mutagenesis
- generate diverse library of variants for subsequent screening and selection processes
second stage of directed evolution
- screening for variants with desired traits or functions
- ones exhibiting desired traits are selected for further evolution
third stage of directed evolution
- selected variants are amplified (replicated) to increase their population size
- one common method is using PCR micro-reactors
- crucial for subsequent rounds of selection and evolution
What do PCR micro-reactors provide
- provide small-scale environment for reaction to take place
- allows for efficient & rapid amplification of DNA/gene of interest
advantages of PCR micro-reactors (4)
- designed to minimise sample volumes
- increase reaction speed
- reduce reagent consumption
- able to perform multiple reactions simultaneously
fourth stage of directed evolution
- iteration -> 3 processes are repeated for multiple rounds
- allows for further refinement and improved target molecules
