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.

To test the ability of yeast cells to express the Acropora millepora blue chromoprotein, we first constructed an expression vector that would express the reporter gene in the conventional yeast Saccharomyces cerevisiae. We obtained a plasmid vector, pSB1C3, containing the blue chromoprotein (part K592009, Figure 3) from the iGEM parts repository. We transformed the plasmid into bacteria, grew up overnight cultures, and performed a miniprep to purify the plasmid DNA. To isolate the coding sequence of the blue chromoprotein, we digested the construct with XbaI and SpeI. We also obtained an S. cerevisiae expression vector (p416Gal1, Figure 4) that uses the Gal1 inducible promoter to express heterologous genes (generous gift from Dr. Andrea Duina). We grew up an overnight culture and performed a miniprep to purify the plasmid DNA. To prepare the vector for subcloning, we digested the vector with XbaI and dephosphorylated the ends to prevent recircularization. We ligated the XbaI – SpeI fragment of the blue chromoprotein into the linearized p416Gal1 vector (SpeI and XbaI have compatible overhangs allowing hybridization to occur but both restriction enzyme recognition sites are lost after ligation) and transformed the construct into bacteria. Since the ligation was not directional, we screened the resulting colonies for the direction of the insert by digesting the miniprep DNA with XbaI and SpeI. Since the hybrid XbaI/SpeI site cannot be digested by either enzyme, we would expect to get an ~700bp fragment from an XbaI – SpeI digest only if the K592009 is in the correct orientation. Figure 6 shows that we obtained one clone with the insert in the correct orientation for expression.

To test whether the blue chromoprotein from Acropora millepora would be expressed in yeast cells, we transformed the blue chromoprotein expression construct and the p416Gal1 vector alone into a ura-minus strain of S. cerevisiae and selected for growth on SC -Ura media. We selected 3 colonies from each transformation and streaked them onto fresh SC -Ura plates to grow. We then made patches of each clone on an SC -Ura plate and after they had grown up, we replica plated the patches onto SC -Ura with glucose and SC -Ura with galactose and rafinose as a carbon source. In the presence of galactose, and the absence of glucose, the Gal1 promoter is activated and leads to the expression of the blue chromoprotein (Figure 7). While S. cerevisiae does express the blue chromoprotein, it took 72 hours for the color to be expressed. In the interest of finding a better reporter gene we are currently testing a GFP, an RFP, and a blue/green chromoprotein to see if they result in a more obvious color change more quickly.

To build our Yarrowia lipolytica expression construct, we have to generate several parts. These include an alkane response element (3xARE1), a leu minimal promoter, our reporter gene (blue chromoprotein), and a terminator (XPR2t) that we can assemble in the correct order. The 3xARE1 part is 157 bp in length so we will synthesize this part by synthesizing four oligonucleotides of 73+ base pairs each, and then annealing them in pairs to form two double stranded molecules approximately 80 base pairs long with compatible overhangs. We will then ligate these two fragments into the pSB1C3 vector to create the part. To generate the Leu minimal promoter and the XPR2t terminator, we have ordered oligonucleotides to PCR amplify the sequences from Y. lipolytica genomic DNA. We designed restriction sites into the primers so both parts can be cloned independently into pSB1C3. The blue chromoprotein was previously prepared by an iGEM team and is available from their parts registry. We will use either the blue chromoprotein, GFP, RFP, or the blue/green chromoprotein in our construct depending on which one shows the highest level and fastest expression in yeast cells. Once we have created each of the parts, we will assemble them using standard biobrick cloning techniques to generate a Y. lipolytica expression vector (Figure 2).

We successfully engineered a strain of S. cerevisiae that was able to express the blue chromoprotein (K592009) reporter gene. Although the yeast was able to express the blue chromoprotein, the blue color was not apparent until 72+ hours of incubation. Due to the extended incubation period necessary for expression of the blue chromoprotein, we have decided to explore other reporter genes such as the red fluorescent protein (RFP), green fluorescent protein (GFP), and a blue/green chromoprotein.

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