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Scientists Develop In Vivo Genome-Wide Codon Replacement Technique

July 20th, 2011 Medgadget Editors News

Researchers from MIT and Harvard are reporting the development of a new technology to selectively edit small identical sections of DNA within batches of living bacteria.

Among other things, the technique should prove useful in engineering microorganisms that produce useful byproducts for science, medicine, and industry.

MIT News Office with some details:

To [translate DNA into amino acids], nearly all living cells use the same genetic code, which has 64 codons — three-letter DNA “words.” While most of them specify an amino acid, there are also a few codons that tell the cell when to stop adding amino acids to a protein chain. The MIT and Harvard researchers targeted one of these “stop” codons, which consists of the letters TAG. With just 314 occurrences, the TAG stop codon is the rarest in the E. coli genome, making it a prime target for replacement.

To make their edits, the researchers combined a technique they previously unveiled in 2009, called multiplex automated genome engineering (MAGE), with a new technology dubbed conjugative assembly genome engineering (CAGE).

MAGE, which has been called an “evolution machine” for its ability to accelerate targeted genetic change in living cells, locates specific DNA sequences and replaces them with a new sequence as the cell copies its DNA. This allows scientists to precisely control the types of genetic changes that occur in cells: The targets are replaced, while the rest of the genome is left untouched.

In this case, the researchers replaced the TAG codon with another stop codon, TAA, in living E. coli cells. To make the process more manageable, the researchers first used MAGE to engineer 32 strains of E. coli, each of which has 10 TAG codons replaced.

To combine those strains and eventually end up with one that has all 314 edits, the researchers then developed CAGE, which allows them to precisely control a naturally occurring process that bacteria use to exchange genetic material. One bacterium builds an extension to a neighboring cell, then passes a piece of genetic material — in this case, TAA codons — to its neighbor.

The researchers set up a playoff-like system in which each strain shares its DNA with one other strain. After the first round of CAGE, the researchers had 16 strains, each of which had double the number of TAG edits that it started with. Those 16 strains then went into a second round of CAGE, producing eight strains.

At this point, the researchers have four strains, each of which has about one-quarter of the possible TAG substitutions; they believe they are on track to produce the single combined strain with all 314 of the substitutions, Carr [Peter Carr, senior researcher at MIT’s Lincoln Laboratory] says.

Press release: Scientists unveil tools for rewriting the code of life

Abstract in Science: Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement

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