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One of the goals of synthetic biology is the construction of molecular circuits from genetic modules. A requirement for successful engineering of such systems is very tight control over the genetic elements. In simple organisms such as bacteria and yeast, scientists have successfully designed tight genetic switches. The challenge was to transport this principle to mammalian cells.

James Collins from Boston University previously developed a toggle switch in bacteria based on two co-repressive genes: activity of gene A turns off the activity of gene B and vice versa. But when his team tried to implement this switch in mammalian cells, the system failed. He explains the reason: “The options available in mammalian systems to repress or inhibit gene expression were not strong enough. We needed to have a very tight off switch; 80–90% [inhibition] was not enough.”

Inhibitors such as the tetracycline (tet) repressor or small interfering RNAs have a certain degree of leakiness, which make them unsuitable as sole regulators for tight switches. So Collins and his graduate student Tara Deans decided to follow the 'divide and conquer' paradigm; they combined repression at a lactose operon (lac) and a tet-controlled promoter with RNA interference as a means to get tight inhibition.

Their expression cassette consists of four modules, each driven by a promoter under the control of either a drug or the gene product of an upstream module (Fig. 1). In the 'off' state, the Lac inhibitor represses the lac operon, which prevents expression of the Tet repressor and the target gene. Additionally, a short hairpin RNA (shRNA) against the target gene is expressed from a tet-responsive promoter and removes any residual target-gene activity. To turn the switch on, a drug is added that inhibits expression of the Lac inhibitor, thus allowing expression of the target gene and the Tet repressor, which is needed to shut off shRNA expression from the tet-responsive promoter.

Figure 1: A synthetic gene network.
figure 1

In the off state the Lac repressor is expressed and inhibits the Tet repressor (tetR) as well as the gene of interest (GOI). shRNA prevents any residual gene expression. In the on state, a drug inhibits lacI expression, resulting in expression of the GOI and the Tet repressor, which inhibits the shRNA.

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Collins and Dean showed that this intricate system relying on cross-talk between the different modules does indeed tightly regulate gene expression in mammalian cells. They used diphtheria toxin to prove that in the off state there was no detectable gene expression; otherwise, the cells—sensitive to even small amounts of the toxin—would not have survived. The cells only died once the researchers flipped the switch on, resulting in the expression of the diphtheria toxin.

Other useful features of this genetic circuit are that it can be used with any gene of interest and expression can be fine-tuned as desired.

Collins anticipates applications of the system in functional genomics, as well as for the study of normal development or disease onset and progression. He says, “Starting from a very tight off state you can explore phenotypic responses to titrated levels of gene expression, to establish threshold responses.”

And of course it will be useful in synthetic biology. A well-characterized toggle switch can be incorporated into much more complicated circuit designs, which are needed to recapitulate pathways in mammalian cells.