CH391L/S12/MAGE lycopene production, CAGE "Amberless" E. coli
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MAGE Lycopene Production
Mulitplex Automated Genome Engineering
MAGE or multiplex automated genome engineering is a technique developed in George Church's lab at Harvard that can be used for large-scale programming and evolution of cells. Many directed evolution and in vitro technologies are limited to manipulations of single genes, making it slow if trying to alter much of the chromosome. MAGE has advantages over these techniques because it is able to simultaneously target many locations on chromosome for modification. Modification can be in the form of mismatch mutations, insertions, or deletions. Through the use of oligos with well-defined sequences, predictable modification can arise. However, through degenerate nucleotides, high-diversity chromosome modifications occur, producing a large variety of genetic variants. (Figure 1)
Modification in MAGE is done through oligo-mediated allelic replacement, which is controlled by the λ-Red single-stranded DNA binding protein β. This protein works by binding the single-stranded oligo and helping it to displace the okazaki fragment on the lagging strand. Normally, the cell's repair proteins would spot the mismatch, but one of the key genes for the repair mechanism has been knocked out. (More details) Upon the next round of DNA replication, the introduced oligo is copied and becomes part of genome. It is also possible that the fragments to be replaced by other near matching oligos, generating huge diversity. The Church group found using 90-mer oligos were the most efficient for replacement. This is likely due to the λ-Red protein needing at least 30 bps to complex DNA, and that 90bp presents a good chance of homology to the target.. As well, oligos larger than 90-mer have a higher chance of forming secondary structure, greatly reducing replacement effieciency.
Efficiency of MAGE
Through oligo-mediated allelic replacement with the λ-Red single-stranded DNA binding protein β, mismatch mutations up to 30bp, insertions up to 30bp, and deletions of up to 45 kbp were introduced to cell populations. (Image Figure 2) The efficiency of a mismatch or insertion is based on its homology to the genome target, and deletion efficiency is based on size. Since oligos with more homology to target sites are incorporated at a higher frequency, MAGE can be tuned to provide desired evolution.
Measuring Sequence Diversity Rate
In 2010 the Venter group of JCVI synthesized a 1.08–mega–base pair genome and transplanted it to M. capricolum to create the new M. mycoides. (Cite Venter) This synthetic biology achievement took 400 scientists year to create, along with a whopping price tag of $40 million. So unless you are Craig Venter, this approach to genome synthesis is likely unrealistic, but this is where MAGE may shine. The idea is that you add only DNA with the specific changes to the genome desired, rather than DNA with random mutations. Even if you are trying to change hundreds or thousands of genes at once, after a few cycles in the machine, a good proportion of the cells should have all the desired changes. This can be checked by sequencing. (Cite http://b-giar.blogspot.com/2011/07/evolution-machine-genetic-engineering.html)