Team:RHIT/Project.html

The above simulation illustrates what will be occurring in a sample from our culture. The large red blobs symbolize the yeast, the green shapes act as the E. coli, and the purple “L”s symbolize lactate being secreted from the yeast. At the start, the yeast and E. coli are separated and randomly scattered throughout the solution, but as the yeast secretes the lactate, the E. coli will travel in the direction that it received the lactate via chemotaxis. They will continue to follow the trail of lactate until they make it to the source, the yeast, and then bind to its surface. However, if no lactate is detected after some time, the E. coli will die. Similarly, if no E. coli bind, the yeast dies after some time.

CUPID’s underlying concept is derived from the evolutionary theories of endosymbiosis and obligate mutualism. When first designing the project, our team aimed for something that we felt would be a great, paper-worthy goal, inducing an endosymbiotic event between E. coli and S. cerevisiae. Through the design process, we hypothesized that if we induced a scenario where the two organisms would depend on the other to live, we could put them in conditions averse to their cooperation to apply a selective pressure for uptake of the E. coli, or cause the two organisms to increase their binding affinities for one another.

To achieve this goal, we designed two constructs, one to be employed in each of the organisms. These constructs had to have two qualities: One, the ability to produce a product that would activate gene expression in the other organism, and Two, the ability to make the complementing organism dependent on this gene being activated. As a step towards this goal, we sought out inducible gene systems that we thought would be functional in each organism.

In yeast, we saw great potential to expand on the use of the FUS1 promoter the Rose-Hulman iGEM team worked with last year. If we could make the yeast dependent on the activation of this promoter sequence, then we should be able to make yeast dependent on E. coli. We used the gene encoding for His3p (Imidazoleglycerol-phosphate dehydratase), and hooked it up to this promoter, thus making the yeast dependent on the binding of its complementary mating factor.

In order to make the yeast dependent on E. coli, we needed a way to have it express the mating factor on its surface. Using the Ice Nucleation Protein-based surface display presented by the Penn iGEM team last year, we had a way to have the E. coli display the protein on its surface. We decided it would be best to constitutively express the mating factor on the surface of the E. coli.

To make E. coli dependent on the yeast, we could approach the auxotrophy from the same direction. Since there exist strains of E. coli that are mutants for the hisB gene, an analog to the HIS3 gene in yeast, we could complement the gene in the same way; we only needed to find an inducible system for its expression. In the registry, our team found a part that would allow growth on glucose, and be activated by lactate. Using this promoter, our team designed this portion of the E. coli construct to be dependent on lactate to live, unless supplemented with histadine.

We found papers describing the expression of Lactate Dehydrogenase (LDH) in yeast, and its subsequent production of lactate. With constitutive expression of LDH, we could cause the yeast to produce lactate, making the E. coli dependent on it. With this dependence, we hypothesized that the E. coli would undergo chemotaxis in order to reach the yeast, following the increasing lactate concentration, allowing for us to promote the two organisms coming together. This leads to the following genetic system.

E. coli Construct (2479 bp) E. coli construct composed of: constitutive promoter, ice nucleation protein linked to mating factor alpha with glycine-serine linker – used to induce FUS1 promoter, termination sequence, lactate sensitive promoter – induced by lactate produced by LDH, HIS3 gene complementing hisB mutation, and terminator.

S. cerevisiae Construct (2765 bp)

S. cerevisiae construct composed of: FUS1 promoter – sensitive to cascade produced in mating response, HIS3 gene complementing HIS3 mutation, termination sequence, constitutive terminal elongation factor promoter, lactate dehydrogenase to produce lactate – induces E. coli lactate promoter, and terminator.