The Genetic Limiter

Our limiter device represents an example of a complex device built from smaller parts and circuits available to synthetic biology. At the same time, however, it also constitutes a departure from the iGEM tradition of building devices that function solely in a bacterial chassis.

Leading to the limiter

Consider a gene of interest (G) for which one inducing transcription factor (A) is known. In the presence of unnaturally-heightened levels of inducer A, our gene will experience excessive activation. A logical method for addressing this problem would be to introduce an inhibitor for the gene of interest to reduce its activity. Traditional interventions typically include the application of a chemical that can block the activity of either the inducer A or the gene G, or, with the use of genetic engineering, the introduction of a genetic inhibitor to hinder protein synthesis of gene G's encoded gene product. One concern that arises with the use of a chemical input is that, especially in the case of multi-cellular organisms, this often has diffuse, unspecified, and often undesirable effects. Chemicals that are introduced into biological systems under therapeutic conditions further require an understanding of the intricate balance between efficacy, . Although the application of a genetically-based inhibitor is often more difficult, such genetic parts afford a great deal of specificity to the system and are embedded in the cellular program, making their activity more pronounced. An issue with applying a

Instead of a constitutive promoter, one could ensure that the repressor for the gene of interest is under the control of a promoter that also takes the same inducer A as an input. This means that the amount of repressor in the system scales with the amount of inducer. However, the introduction of a single repressor construct in this fashion will also result in passively lowering the level of gene expression for any given level of inducer. This means that G will be present in lower than normal quantities for normal amounts of inducer.

To prevent such unwanted interference of endogenous transcription, one could incorporate a second repressor to inhibit the repressor.

At this point, it is important to turn to a concept from electrical engineering for inspiration.

The Bistable Switch

A bistable switch is a simple circuit in which, depending upon the nature or quantity of the input, two stable outcomes are possible. In one case, would allow the system to alternate between two modes of activity in response to varying levels of input. Dr. James Collins from Boston University developed a bistable switch capable of switching between two genetic outputs based upon the inputs to the system.

Driven by the desire to create a synthetic gene network that could act similarly to endogenous regulatory pathways, we developed a design for a system that had the ability to regulate

We began by developing a layout for

Diagram our network and explain why we laid it out this way.

• Bistable regulation of repression (mutual inhibition)
  • cite toggle switch
• Extensibility
  • upregulator
  • middle management