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Researchers divide enzyme to conquer genetic puzzle
Date:3/14/2013

alves of the split RNAP were present. Bennett and Shis modified the bacterium E. coli to produce the RNAP segments in response to specific sugar molecules found in the environment. One half of the RNAP is produced only in the presence of arabinose and the other in the presence of lactose. When both sugars were found, their proof-of-concept polymerase would turn on a target gene, in this case a reporter gene that encodes a green fluorescent protein.

Better yet, the RNAP was orthogonal; that is, it didn't fraternize with the E. coli's native protein pathways. "This special T7 RNAP will not turn on any other gene but its specific target," Bennett said. "In that way, it's transparent to the host. This gives us an easy way to determine whether or not it's working."

The researchers found that while split T7 RNAP was not as active in expressing protein products as the full-length polymerase, the pieces were more stable and less prone to mutations that could affect a gene circuit's function.

Bennett said the next step would be to test the split RNAP in hosts other than E. coli. "We want to port this into more complicated organisms: eukaryotes like yeast or zebrafish or mammalian cells. It will take a little bit of engineering to get them to work in more complicated organisms."

He sees advanced diagnostics as a reasonable goal for gene circuits using AND gates. "The two inputs can be programmed to respond to separate conditions, whether they're environment factors outside the organism or tissue-specific markers within a multicellular organism," Bennett said.

"For instance, your inputs could be cancer markers that would trigger a fluorescent reporter gene for diagnosis or tumor suppressors for treatment," he said. In addition, multiple gates from the library could be combined and layered to create more complicated circuits that simultaneously monitor many variables. "This means you could build circuits that turn on only in ve
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Contact: Jeff Falk
jfalk@rice.edu
713-348-6775
Rice University
Source:Eurekalert

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