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Researchers Describe Approach for Developing Bacteria with Artificial DNA Base

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – In a study appearing online this week in the international edition of the journal Angewandte Chemie, an international group reported that it has developed Escherichia coli strains in which thymine nucleotides have been replaced with a synthetic base called 5-chlorouracil.

The researchers nudged the bacteria to evolve this synthetic version of a genome using a form of E. coli with a few genetic tweaks — including a gene deletion that left it dependent on external thymine sources. The bug was grown under controlled conditions in the lab, prompting some E. coli to adapt so it could withstand exposure to 5-chlorouracil and, eventually, incorporate the chemical into their genetic code in place of thymine.

"[W]e set out to evolve genomic DNA composed of the three canonical bases adenine, cytosine, and guanine and the artificial base 5-chlorouracil in an Escherichia coli strain lacking thymidylate synthase and requiring thymine," corresponding author Rupert Mutzel, a biology, chemistry, and pharmacy researcher at Freie Universität Berlin, and co-authors wrote.

"Chemically modified organisms, as embodied by our chlorouracil-requiring bacteria, could be systematically diversified in the future to block metabolic cross-feed and genetic cross-talk between synthetic and wild species," they noted.

Because it is found only in DNA and not in RNA, the researchers explained, it is easier to fiddle with thymine biosynthesis and replace the nucleotide than it is to sub out the other three DNA bases.

To do this, the researchers cycled E. coli cultures in and out of growth medium containing 5-chlorouracil in a so-called "pulse-feed regime," gradually increasing the concentration of 5-chlorouracil and decreasing the thymine concentration in their automated growth chamber system to maintain selection pressure on the bacteria.

The E. coli strain selected for the study was missing a few key genes, including the thymidylate synthase gene thyA, which codes for an enzyme involved in thymine biosynthesis.

"Our experimental plan consisted of the combination of tight metabolic selection with the long-term automated cultivation of fast-growing asexual bacterial populations to change a canonical DNA base for a chemical ersatz," they wrote.

Indeed, they reported, after around 25 weeks of cultivation or roughly 1,000 generations, the researchers had generated strains that could grow without external thymine sources and in which 5-chlorouracil had replaced thymine — findings that they verified using mass spectrometry.

When the researchers used Roche 454 GS FLX Titanium or Illumina platforms to sequence the genomes of their original E. coli strain and of two strains adapted to growth in chlorouracil, they found that numerous substitutions and rearrangements had sprung up in the bacterial genome over the course of this directed evolution. The researchers plan to explore the nature and consequences of these mutations in more detail in the future.

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