Brute Force Mutagenesis & Directed Evolution

Question 1

What are brute force mutagenesis & directed evolution

Answer 1

2 different approaches in the field of genetic engineering, and can be used for microbial strain improvement

Question 2

Define mutagenesis

Answer 2

The process of intentionally inducing mutations in the genetic material of an organism

Question 3

(Mutagenesis) mutations may be induced by various techniques such as

Answer 3

• chemical treatments
• radiation exposure
• genetic engineering methods

Question 4

What are the 2 purposes of mutagenesis

Answer 4

• to study the effects of induced mutations on an organism’s traits
• to generate variants with desired characteristics

Question 5

What does brute force mutagenesis involve

Answer 5

Introducing a large number of random, undirected mutations into a target molecule

Question 6

What principle for brute force mutagenesis rely on

Answer 6

• That some of these mutations may lead to desired traits or properties
• explores a wide range of possibilities

Question 7

What does the first stage of brute force mutagenesis involve

Answer 7

• introducing random, undirected mutations into target molecules
• generates mutant library
• using techniques like error-prone PCR or chemical mutasynthesis

Question 8

What is the aim of the first stage of brute force mutagenesis

Answer 8

To create a diverse library of variants with different mutations

Question 9

What are we doing in the second stage of brute force mutagenesis, and what do we use to accomplish this

Answer 9

• screening for variants with desired traits or properties
• use various assays to identify mutants exhibiting desired characteristics

Question 10

What happens after potential mutants have been identified

Answer 10

• 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

Question 11

What is directed evolution compared to brute force mutagenesis

Answer 11

Directed evolution is a more focused and guided approach

Question 12

As well as directed evolution, what are some other recent advances in strain improvement

Answer 12

• metabolic engineering
• molecular breeding (DNA shuffling & whole genome shuffling)
• combinatorial biosynthesis

Question 13

What do these new advances in strain improvement mean

Answer 13

• achieve new and improved products
• direct research and development towards new targets

Question 14

What is directed evolution essentially

Answer 14

• mimics the process of natural selection
• manipulates proteins/nucleic acids by giving them desired traits
• makes them better suited for specific applications

Question 15

What does directed evolution involve

Answer 15

• designing specific mutations based on the understanding of the target molecule’s structure and function
• iterative mutagenesis, followed by selection and screening

Question 16

What are the 4 main stages to directed evolution

Answer 16

1. generation of diversity
2. selection or screening
3. amplification
4. iteration

Question 17

first stage of directed evolution (generation of diversity)

Answer 17

introducing mutations into target molecules such as proteins or nucleic acids

Question 18

In directed evolution, what techniques can be implemented to mutate the genome

Answer 18

• error-prone PCR
• DNA shuffling
• site-directed mutagenesis

Question 19

What is error-prone PCR

Answer 19

Accuracy of PCR is intentionally reduced, leading to the introduction of random mutations during amplification

Question 20

What is DNA shuffling

Answer 20

The fragmentation and reassembly of DNA sequences from different variants, generating new combinations and mutations

Question 21

What is site-directed mutagenesis

Answer 21

• 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

Question 22

second stage of directed evolution

Answer 22

• screening for variants with desired traits or functions
• ones exhibiting desired traits are selected for further evolution

Question 23

third stage of directed evolution

Answer 23

• 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

Question 24

What do PCR micro-reactors provide

Answer 24

• provide small-scale environment for reaction to take place
• allows for efficient & rapid amplification of DNA/gene of interest

Question 25

advantages of PCR micro-reactors (4)

Answer 25

• designed to minimise sample volumes
• increase reaction speed
• reduce reagent consumption
• able to perform multiple reactions simultaneously

Question 26

fourth stage of directed evolution

Answer 26

• iteration -> 3 processes are repeated for multiple rounds
• allows for further refinement and improved target molecules