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Synthetic gene circuit allows precise dosing of gene expression
Date:3/10/2009

e network designed to repress yEGFP, a fluorescent reporter gene whose presence can be detected in each cell in a culture by flow cytometry. The gene circuit was then added to the growth medium, where it diffused into cells and blended into the yeast's DNA.

The gene circuit starts with a promoter that launches the tetracycline repressor gene, which then blocks yEGFP via a promoter for that gene, shutting down its expression. Adding anhydrotetracycline (ATc) to the cell culture in measured doses stifles the tetracycline repressor and permits expression of yEGFP.

When ATc was added to the culture, there was little or no response at low dosages, then a sudden, steep increase in yEGFP, which quickly hit a plateau at saturation. "This is called a sigmoidal response, which does not follow in a linear fashion the dose of ATc," Balzsi said.

Similar to distortion and an amplifier

The team then made the promoter for the repressor identical to that used for yEGFP. This, in effect, turns the tetracycline repressor back on itself, a negative feedback loop that results in automatic reduction of the repressor when it reaches high levels and an increase when levels drop.

They found that adding negative autoregulation makes yEGFP linearly responsive to ATc dose. So a 20 percent increase in ATc yields a 20 percent rise in gene expression, and so on.

Balzsi compares this to dealing with distortion in electronic circuits. Amplifiers strengthen a signal, but also distort it. By distorting the signal before it enters the amplifier in the opposite way that the amplifier will distort it, the two distortions cancel each other out, resulting in a clear signal. Making both gene promoters identical has the same effect in the gene network.

This linear dose-response relationship works while it achieves similar expression levels of yEGFP in all cells in the culture. Cell-to-cell differences are large in cell cultures tr
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Contact: Scott Merville
smerville@mdanderson.org
713-792-0661
University of Texas M. D. Anderson Cancer Center
Source:Eurekalert

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