Team:Hendrix Arkansas/Project

This summer, Hendrix College entered the first Arkansas team to compete in the International Genetically Engineered Machine competition (iGEM). The overall goal of our project is to engineer a biological machine that can detect cancer. Skin cancers, such as melanoma, are known to give off volatile compounds that can be detected by trained dogs. The volatile compounds given off by cancer cells include several alkanes. The purpose of our experiment is to engineer a strain of Yarrowia lipolytica that is capable of detecting and growing on alkanes to turn color in the presence of these volatile compounds. It is our hope that we can use this yeast to cheaply and non-invasively (perhaps in a cancer detecting band-aid) to detect melanoma. In order to achieve our goal, we will build a reporter construct that uses an alkane sensitive promoter to drive the expression of a blue chromoprotein from the coral Acropora millepora. This construct should cause the yeast to turn blue in the presence of alkanes. We first want to test the ability of the blue chromoprotein to be expressed in yeast cells, so we have generated an expression vector to express the gene in Saccharomyces cerevisiae. We have also obtained a sample of Yarrowia lipolytica and are in the process of generating the parts required to build the reporter construct. These parts include an alkane response element (3xARE1), a leu minimal promoter, our reporter gene (blue chromoprotein), and a terminator (XPR2). Once the reporter construct is constructed, we will clone it into a Yarrowia lipolytica expression vector and integrate it into the yeast. If our experiment is successful, our cancer detecting band-aid could allow for easier and earlier detection of melanoma.

For the iGEM competition, the Hendrix team has decided to engineer an alkane responsive strain of yeast, Yarrowia lipolytica, to activate the expression of a visible reporter gene in the presence of volatile alkanes. We hope that this biological machine would be useful for the detection of skin cancer in a non-invasive approach. This approach was motivated by stories of dogs that could successfully identify cancer in humans. According to McCulloch et al. (2006), the first documented instance of dog-patient interactions leading to a diagnosis of cancer occurred in 1989. Canines are able to detect the presence of volatile compounds in concentrations as low as parts per trillion and have been trained to detect lung, breast, and ovarian cancer in exhaled breath samples (McCulloch et al., 2006). To determine which volatile compounds are given off by cancer cells, Abaffy et al. (2011) used gas chromatography/mass spectroscopy (GC/MS) on the volatile compounds given off by melanoma samples as compared to non-cancerous skin. They identified 23 volatile compounds that were detected only in melanoma samples and these compounds included the alkanes decane and undecane (Abaffy et al., 2011). Based on this research, we decided to try to develop a biological machine to detect the presence of alkanes in the hope that it will be able to specifically detect melanoma.

In order to detect the volatile alkanes given off by melanoma cells, we took advantage of a strain of yeast that can grow using alkanes as a sole carbon source. Yarrowia lipolytica is a non-conventional, non-pathogenic yeast that can utilize n-alkanes as a carbon source (Barth and Gaillardin, 1997). Once inside the cell, the n-alkanes induce the expression of cytochrome P-450 which hydroxylates the n-alkanes as the initial step in their utilization (Barth and Gaillardin, 1997). By comparing the promoter sequences of the cytochrome P-450 and other alkane inducible genes, Yamagami et al. (2004) identified a cis-acting alkane responsive element (ARE) and showed that it could drive the expression of a lacZ reporter gene in the presence of alkanes. We would like to take advantage of this finding and engineer a strain of Yarrowia lipolytica to express a visible reporter gene in the presence of alkanes. To accomplish this, we plan to generate a reporter construct that is similar to the one used by Yamagami et al. (2004)(Figure 2). Our construct will contain three copies of the alkane responsive element upstream of a minimal promoter sequence from the leu gene. We will use this promoter to drive the expression of a blue chromoprotein from the coral Acropora millepora (Alieva et al., 2008). After the coding region of the chromoprotein, we will include a terminator sequence from the alkaline extracellular protease gene (XPR2). This construct is similar to the one that was shown to work by Yamagami et al. (2004) except for the use of a different reporter gene (blue chromoprotein vs. lacZ). We will integrate the final construct into Yarrowia lipolytica and test for the ability of volatile alkanes to induce the expression of the reporter gene.

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