MAGE procedure

The Multiplex Automated Genome Engineering (MAGE) workflow consists of 6 steps:

1. Setting the target phenotype or genotype

A certain phenotype might be the result of a certain enzyme or gene network. You should be able to describe your desired phenotype or genotype. For example: increasing beta-carotene production using E. coli. The desired phenotype would be E. coli colony with bright orange color.

2. Identify target loci

Identify all the genes related to the desired phenotype or genotype. There are several tools that use computational algorithms to help identify the relevant loci.

3. Design oligo

The synthetic single-stranded DNA (ssDNA) or oligonucleotides (oligo) are designed to carry the mutation. There are certain parameters in designing these oligos that should be met to achieve a high allelic replacement frequency (ARF).

4. Predict cycle time

The optimum amount of MAGE cycles should be investigated. Generally, more cycles increase the prevalence of allelic replacement and population diversity.

5. MAGE cycling

MAGE cycles involve a series of steps to allow the recombination of oligo(s) with the bacterial chromosome.

6. Screening

Bacterial cells are screened in order to check that they have the desired phenotype/genotype. For a genotype oriented project, the screening of mutant cells is performed using PCR-based methods or DNA sequencing. In the case of phenotype oriented projects, the screening is conducted by plating colonies for further morphology identification.