CHE.496/2008/Responses/a11

Genetic circuit engineering (part 2)

• Discussion leader: Dan Tarjan (Discussion guide)

George McArthur's Response

• Environmentally controlled invasion of cancer cells by engineered bacteria
  • This paper describes the design of a very cool synthetic biological system that involves an engineered genetic circuit in bacteria. Essentially, the bacteria are equipped with sensing, processing and actuating abilities. This is the end goal of a genetic circuit engineering project - to have the cell behave like a programmed, yet biological, robot. In this case, the bacteria can sense cancer cells via heterologous environmental signals and, in response, invade and destroy these cells using invasin, a cytotoxic agent. The idea of programming a cell for a specific function is extremely exciting. Several project ideas should be generated with this in mind.
• Environmental signal integration by a modular AND gate
  • This paper is an extension of the previous paper and describes in detail the way in which their engineered bacteria integrate the heterologous environmental signals. The authors describe the moldular AND gate that is used to process the input signals and actuate the response, mammalian cell invasion/destruction. The AND gate uses "information from two promoters as input and activates a promoter output only when BOTH input promoters are transcriptionally active." This is a wonderfully detailed example of genetic circuit design and construction, and could serve as a guide to working through a new system design. In addition, the AND gate is modular so that the inputs and outputs can be changed by interchanging promoters and RBSs. Therefore, it could be used in a variety of engineering applications.
• GMcArthurIV 15:55, 26 February 2008 (EST)

Kevin Hershey's Response

• Environmentally controlled invasion of cancer cells by engineered bacteria
  • The article by Anderson et al describes a modification made to bacteria which allows it to swarm cancer cells and attack it. A modification was also made so that the blood cells don’t attack the E. coli. This can be applied to synthetic biology in several ways. First, standardizing the parts allows it to be used by other systems. For example, the Berkley team designed BactoBlood. This used the modified sensor in the E. coli to prevent human blood from attacking it. Also, with the principles of synthetic biology, modeling the E. coli is very important. In such a system as this, there must be extensive testing before it can be used in humans, whom the treatment is created for. The modeling can then be presented to the FDA to allow for clinical trials.
• Environmental signal integration by a modular AND gate
  • This article by Anderson describes an AND gate that reaches near-digital output. This can be very advantageous for the VGEM team, as this allows for another part in the ‘biological toolbox.’ Also, the modeling done by Anderson et al can be applied to a potential VGEM project. Their method of using fluorescence to model their output based on different inputs is a very common method. It is likely that a VGEM project will need to use their experimental design.
• KPHershey 16:17, 26 February 2008 (EST)

Patrick Gildea's Response

• Environmentally controlled invasion of cancer cells by engineered bacteria
  • The purpose of this paper is to describe a biological system composed of bacteria that can sense cancerous environments (via hypoxia detection) in a eukaryotic cell, invade the tumor, and kill it via invasion, a cytotoxic agent. The paper presents a bunch of good project ideas. For example, the sensing of a target environment; the papers we have reviewed mentioned quorum sensing, but this example is targeted toward a different environment. This is really cool since if we can identify a unique characteristic of whatever type of tissue environment or chemical related to the environment targeted; we can find a promoter that is sensitive for that. Another cool idea that comes from this paper is the fact that bacteria can move around in a biological organism carrying the genes to manufacture whatever chemical you wanted. For example I was thinking of heart disease – atherosclerosis, where bad cholesterol (LDL) is formed into oxidised-LDL when it gets past the wall of an artery. It would be cool if we could have some sort of bacteria that could sense the presence of oxidised-LDL and have a gene that could produce a protein that can break down the plaque.
• Environmental signal integration by a modular AND gate
  • The purpose of this article was to describe the construction of a AND gate circuit in E. Coli based on parts from the registry. One of the nice parts about the paper is the last section that describes the materials and methods. We have been complaining to George about the lack of protocols or any information of the “wet” work aspects of the research we’ve been reading. While it is not a step by step description like our manuals in chemistry, we can draw a picture through researching the endnotes mentioned specifically in that section. Furthermore, this AND gate is a useable biobrick, by looking at figure 1 in the paper, it is possible to add in genes that can be read off after the sensor is activated before the supD or T7ptag are reached, these genes could also activate other AND switches that have different sensors that test for the genes output. In other words, we could build a fairly complex circuit from this – maybe a Rube Goldberg device could inspire us to follow a Rube Goldberg kind of biological circuit where we can design as complex a circuit possible to accomplish a simple task. This would probably be research into the fundamentals side where bigger and more complex circuits will have to be designed. The benefit of a project like this will mainly be in terms of studying the stochastic effects and of course the modeling that would arise from understanding the behavior of said circuit.
• Patrick Gildea 18:33, 26 February 2008 (EST):

George Washington's Response

• Environmentally Controlled Invasion of Cancer Cells by Engineered Bacteria
  • This was an impressive article, describing three different mechanisms the team developed to make bacteria identify cancerous cells by various cues and invade them with Yersinia pseudotuberculosis's invasin. By making bacteria only invade cells when they're hypoxic, overly dense, or with a chemical cue, the system can selectively enter and destroy cancerous cells. However, I don't believe they demonstrated these three techniques in tandem, as would probably have to be implemented in any medical application, such that Type II error would be absolutely minimized. With the somewhat low invasion rates (0.2(+-0.1)% for some host strains), a composite of all three would likely give negligible invasion. I found it impressive that such complex behaviour as cell invasion can be induced by a single protein, much aiding efforts of synthetic biology in achieving modularity of design. We should watch for such superficially simple mechanisms as they will be invaluable in crthsalotoeating reliable systems.

• Environmental signal integration by a modular AND gate
  • Logic gates are going to be important in any kind of computation required in a synthetic biological system, so the development of robust AND gates is an essential step towards this. I, again, enjoyed the specificity of the paper's description, specifying exact techniques and methods used to effect their system. The technique used to integrate two signals, i.e. the T7 RNA polymerase with the amber stop codons, was impressive, and similar systems will be extremely useful in future applications. We may need to use logic circuits in our own system, so work like this should be observed as much as we can and understood to the best of our ability.

• George Washington 11:51, 27 February 2008 (EST)

Eyad Lababidi's Response

• Environmentally controlled invasion of cancer cells by engineered bacteria
  • This article describes E.coli cells that can sense an environment that has hypoxia and to use invasive to invade and destroy mammalian cells. This has the implication of being able to cure cancer. for our purposes we can use the same idea for a completely different project. Once our cell can seek out a certain environment it means it can use decision making on processing it should do and it will attempt to seek out the desired environment. I'm not really sure what project would be in our scope but seeing Patrick's idea about breaking up LDL plaque sounds really good. This seems easier then most cases because if the cell is travelling through the bloodstream it doesnt need a method of movement, just sensing and protein secretion which weve seen with the biofilm project.
• Environmental signal integration by a modular AND gate
  • This article describes a pretty succesful AND gate and it seems to me that we are beginning to have the tool to be able to create and actualy circuit with some complexity. I know we only have an inverter and an AND but with an inverted and (NAND) you can buiid any circuit although i get the feeling wed be limited on repetition of chemicals. To be honest building a circuit out of genetic parts seems like a cool idea to me but where we would improve the idea would have to be with speed of transition and with figuring out a way of being able to use more then one inverter or and gate. actually the combination of just an switch and an AND gate allows us to create what i mentioned in my other response with a cell that has 2 separate modes.

Eyad Lababidi 11:26, 27 February 2008 (EST)