CHE.496/2008/Responses/a10

Genetic circuit engineering (part 1)

• Discussion leader: Kevin Hershey (Discussion guide)

Eyad Lababidi's Response

• A synthetic oscillatory network of transcriptional regulators
  • This article talks about creating an oscillatory network out of bricks that would act like inverters by alternating repressors. it seemed liek a pretty basic idea when you compare it to circuits but what the real goal seemed to be creating these inverters that would be in the network. The article talked about using well known bio bricks that would have the equivalent of being good at turning on and turning off. in chemical terms it talked about when being repressed the protein would not be synthesized and when it is being translated by mrna the protein would be in high production. This project goes back to the fundamentals so that later on a research could choose from a plethora of different proteins that can behave like inverters.
• Construction of a genetic toggle switch in Escherichia coli
  • This paper talks about a bistable switch that theyve created that is apparently very robust, although to switch the states of the switch it seems to be a rather involved process and takes several hours to switch. Beyond just using the switch as a demonstration it seems to be too slow to do anything really useful for the Vgem team. We could use it to change states of operation, in one state the cell could work in its primary function and maybe try to slow its metabolic rate in this state while in the other state the cell could either have a secondary function or just be in a sort of hibernate mode where its metabolic rate is back to normal, or some kind of combination of turning functions on and off.

Eyad Lababidi 10:36, 27 February 2008 (EST)

George McArthur's Response

• A synthetic oscillatory network of transcriptional regulators
  • This paper is a considered to be classic in the synthetic biology community and, in many ways, serves as a foundation from which to build. What we see presented here is Elowitz and Leibler's repressilator, a "cyclic negative-feedback loop composed of three repressor genes and their corresponding promoters," and an example of how we can build genetic circuits with novel functions/behavior from well understood, natural components.
• Construction of a genetic toggle switch in Escherichia coli
  • This paper by Collins et al. was published in the same issue of Nature as the repressilator paper and is also considered a foundational paper in the realm of synthetic biology. These papers paved the way for synthetic biology concepts (especially relating to genetic circuit engineering). A synthetic, bistable gene-regulatory network was implemented in E. coli. This genetic toggle switch is flipped using a chemical or thermal pulse and is extremely practical, promising countless biotechnological applications. The VGEM Team could use this or a similar approach to developing cellular memory. This would be useful in a variety of projects including a metabolic engineering project.
• GMcArthurIV 15:41, 26 February 2008 (EST)

Patrick Gildea's Response

• A synthetic oscillatory network of transcriptional regulators
  • The purpose of this article was to describe a genetic network in E. Coli that functions like an oscillatory circuit. This was achieved by coupling 3 repressor genes, where one gene would repress a second gene, and the third repress the first one in a cycle. All in all, a pretty neat circuit with GFP tied to the output of the second repressor with the end result of flickering on and off over a long time period. I think the most valuable part of this paper for us is the methodology they used to develop a circuit, in particular, box 1, that shows the mathematical model of the transcriptional system. While I am not qualified to judge whether the model is “good”, it is an example of how the behavior of a cellular circuit could be designed and could be enormous benefit when/if we decide to do a project like this. There was one thing I was disappointed in, that these folks didn’t show how they were able to link these three repressors to each other – I don’t mean the promoters or anything like that. What I mean is how do they know that LacI represses TetR and TetR represses gamma cL? If I had to do a project like this, I wouldn’t know which gene to pair with the other – perhaps this will be discussed in the meeting?

• Construction of a genetic toggle switch in Escherichia coli
  • Again, the purpose of the article was to describe a genetic network that functions like a toggle switch controlled via repressors, promoters, and inducers. Box 1 describes the model the researchers used to develop the behavior of the toggle switch in the cell. Stability of the switch was a major focus because a switch that randomly flickers between states defeats the purpose of a toggle switch. What is really cool (at least to me) is the fact that different promoters and repressors could be used as long as the cooperative repression of the promoters and degradation of the repressors. Another valuable point this paper makes is the Author’s use of the model to form a “toggle” theory that predicted the minimum requirements for a stable and functional switch. Furthermore, the authors of the paper made an important point about their research in that they focused on altering the network structure (the genes, etc.) as opposed to engineering proteins, which is what synthetic biology is all about!
• Patrick Gildea 17:11, 26 February 2008 (EST):

George Washington's Response

• A synthetic oscillatory network of transcriptional regulators
  • This was a useful article, especially since it directly provides the methods employed by the researchers. I believe the technique of artificially adjusting the half-life of a protein with an extra tag will be important, as persistence of chemistry will be a hindrance to any kind of fast-reacting system, unless we decided to use allosteric controls to suppress expression. Again, using a simple model that doesn't worry as deeply about the underlying kinetics gives reasonable predictions of the behaviour expected in the system, and I really think we should consider learning how to model stochastic effects as it may be important in our own system. The repressilator itself, being able to induce rhythms on an order greater than the cell-division cycle could possibly prove invaluable if we need that effect, but this mainly seemed a proof of concept on synthetic systems in that natural parts could effect rather specific, rationally designed behaviour.

• Construction of a genetic toggle switch in Escherichia coli
  • I enjoyed this article, as I was able to understand it fairly thoroughly since my Project had been on an extension of Gardner's work. He described his procedure in great detail, which will be useful if we do anything similar. Also, I believe it reasonably likely that we would be able to find a use for this switch or one like it in a wide range of project ideas, since it's such a general idea. Being able to have some cells in one state and others in another is such an enabling step if we do any multicellular system, and even if we use only single cell dynamics, we could still use it in a system that could, for instance, have the switch as an automatic check against overproduction of a product by tying the product itself to disabling the switch. This would enable the product to be removed before re-initiating production, thus establishing a fail-safe against product transport mechanisms failing. Although the paper doesn't overmuch emphasize the numerous practical applications, this is an essential device in demonstrating the power of synthetic biology.

• George Washington 09:46, 27 February 2008 (EST)