MAGE to be the Gauge as we usher in new Page in Molecular Genetics Research?
The lure of the Human Genome Project had given the impetus for the advancement of high-throughput DNA sequencing technology. But what if your game is modifying genomes? Then you are in for a long day, with comparatively ancient technologies where you have to introduce single DNA constructs. This was the case until a group of researchers invented a method called the multiplex automated genome engineering (MAGE).
MAGE not only has a snappy acronym but is able to perform large-scale programming and evolution of cells. MAGE involves a process that targets many locations on the chromosome to modify a single or a population of cells simultaneously. Because this process is cyclical and scalable, it can be automated to produce mismatch, insertion or deletion genetic changes rapidly and continuously.
How is this done? SsDNA or oligonucleotides (oligos) is directed to the lagging strand of the replication fork during DNA replication in E. coli to cause allelic replacement. The oligos are mediated by Bacteriophage λ-Red ssDNA-binding protein β. Sequence diversity is ensured by repeatedly doing this process.
At optimized levels new genetic modifications are introduced in >30% of the cell population in 1 cycle (2-2.5 hours). The efficiency can be controlled by how homologous of the oligo to the chromosome target or in the case of deletion, the size of the deletion. Just to give an idea, at the end of 5 cycles an average change of 3.1 bp per cell across the population can be achieved, which equates to 4.3 X 109 bp variations/day.
What does all this mean? As an example of the power of this method, MAGE was used to modify gene expression to increase the yield of the red pigment, lycopene. Only 20 primary endogenous genes as well as an additional 4 genes through secondary pathways are known to affect lycopene yields. Previous molecular genetics methods have a limitation on the number of genetic components that can be manipulated at a time. With MAGE all 24 genes were manipulated and 15 billion genetic variants were produced in 35 cycles. Through this, genetic variants were identified that gave up to a 390% increase in yield compared to the wild-type yields
Not a bad result to achieve without having to be in the lab for 3 days.
Reference:
Wang, H. H., F. J. Isaacs, et al. (2009). "Programming cells by multiplex genome engineering and accelerated evolution." Nature 460(7257): 894-U133.
Reuben Durairatnam
42140867
MAGE not only has a snappy acronym but is able to perform large-scale programming and evolution of cells. MAGE involves a process that targets many locations on the chromosome to modify a single or a population of cells simultaneously. Because this process is cyclical and scalable, it can be automated to produce mismatch, insertion or deletion genetic changes rapidly and continuously.
How is this done? SsDNA or oligonucleotides (oligos) is directed to the lagging strand of the replication fork during DNA replication in E. coli to cause allelic replacement. The oligos are mediated by Bacteriophage λ-Red ssDNA-binding protein β. Sequence diversity is ensured by repeatedly doing this process.
At optimized levels new genetic modifications are introduced in >30% of the cell population in 1 cycle (2-2.5 hours). The efficiency can be controlled by how homologous of the oligo to the chromosome target or in the case of deletion, the size of the deletion. Just to give an idea, at the end of 5 cycles an average change of 3.1 bp per cell across the population can be achieved, which equates to 4.3 X 109 bp variations/day.
What does all this mean? As an example of the power of this method, MAGE was used to modify gene expression to increase the yield of the red pigment, lycopene. Only 20 primary endogenous genes as well as an additional 4 genes through secondary pathways are known to affect lycopene yields. Previous molecular genetics methods have a limitation on the number of genetic components that can be manipulated at a time. With MAGE all 24 genes were manipulated and 15 billion genetic variants were produced in 35 cycles. Through this, genetic variants were identified that gave up to a 390% increase in yield compared to the wild-type yields
Not a bad result to achieve without having to be in the lab for 3 days.
Reference:
Wang, H. H., F. J. Isaacs, et al. (2009). "Programming cells by multiplex genome engineering and accelerated evolution." Nature 460(7257): 894-U133.
Reuben Durairatnam
42140867
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