Team:Penn

What?

This summer, the UPenn 2014 iGEM team worked to engineer a complex genetic system that utilizes quorum sensing in bacteria. Our novel genetic pathway is designed to incorporate two orthogonal, or completely independent quorum sensing molecules to regulate the population levels of E. Coli and Streptomyces griesius in a co-culture. The population levels are determined by controlling the quorum sensing signals through external stimuli. Heat and IPTG act as tuning dials to dictate the exact composition of the composite population; adjustments can result in desired population ratios of ''E. Coli'' and ''Streptomyces griesiu''s.

How?

The goal of our project is to construct a noise-free, functional genetic circuit that uses two orthogonal, interspecies signaling molecules that can regulate the population levels of two different species of bacteria, E. coli and Streptomycces. Our inputs in the system are arabinose and salicylate.

Once arabinose has been added to the system, it will repress AraC, allowing for the production of tetracycline by Streptomycees. The tetracycline acts as an interspecies quorum sensing molecule and allows for the expression of Gene A or the methionine synthase gene in E.Coli. As an indicator that this has occurred, GFP will allow the bacteria to glow green. Upon production of methionine synthase, the E.Coli population will increase as methionine (an essential amino acid) availability increases.

Concurrently, salicylate will bind to nahR allowing for the production of LuxS. Expression of this gene allows for interspecies quorum sensing, signaling Streptomycces to express lysine synthase. Again, as an indicator, RFP allows for red fluorescence. Streptomycces population increases as lysine is also an essential amino acid.

In order to control population levels of both bacterial strains, we can incorporate external stimuli, namely heat and IPTG. Addition of heat to express TetX, which degrades tetracycline will decrease the expression of Methionine synthase (decrease the population of E. Coli). Similarly, addition of IPTG to express lsrK, which degrades AI-2 will decrease the expression of lysine synthase (decrease the population of Streptomyces griesius).

This circuit is displayed below:

Why?

The largest obstacle the medical field and the drug industry face today are the lack of testing options for new drugs and the latest treatments. Of the many animal models and other experimental methods that exist, none simulate the complexity of the microenvironments found in the human body like the gut or the mouth. This makes the testing and distribution of the most novel and potentially life saving treatments difficult and time consuming. At the same time, these testing methods may not provide the most accurate results of how treatments will affect the human body.

Microbiome?

Microbiomes are small scale environments found in nature that are filled with bacterial ecosystems. These miniscule systems are found in the soil, water sources, and even in the human body. The ecosystems found in microbiomes are organized by bacterial quorum sensing, chemical signaling between bacteria of the same and different species. By using quorum sensing, bacteria can tell species to grow and divide, secrete a particular compound, or to reduce populations by dieing. In this way, quorum sensing tunes the microenvironment by regulating the populations of certain bacteria by telling some to grow and others to die.

How will our project help?

The goal of our project is to use our genetic circuit that uses quorum sensing to control the populations of two different strains of bacteria in a coculture. By exploiting quorum sensing in a similar way bacteria found in the gut, mouth, and on skin do, we hope to create a microbiome that can someday be used to simulate those of the body. This microbiome will be the first step in using synthetic biology to create more accessible, safer, and more accurate drug testing platforms.