Difference between revisions of "Team:UCSC/Experiments"
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| − | <p>Our team will use synthetic biology to address insufficient access to contraception by engineering a progesterone-producing yeast. We will construct our gene cassettes and insert them into the <i>Y. lipolytica</i> genome in three parallel experiments. In all experiments, we will use the strain FKP393 with the auxotrophic markers <i>LEU2</i> and <i>URA3</i> for selection. We will insert <i>URA3< | + | <p>Our team will use synthetic biology to address insufficient access to contraception by engineering a progesterone-producing yeast. We will construct our gene cassettes and insert them into the <i>Y. lipolytica</i> genome in three parallel experiments. In all experiments, we will use the strain FKP393 with the auxotrophic markers <i>LEU2</i> and <i>URA3</i> for selection. We will insert <i>URA3</i> into the <i>Y. lipolytica</i> genome using HR, and select using<i>URA3</i> deficient media. We have designed LoxP and Lox71 sites to flank <i>URA3</i> for use in Experiments 2 and 3. We will amplify out 1 kb areas upstream and downstream of the <i>ADE2</i> gene in <i>Y. lipolytica</i> for use as our HAs. </p> |
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Revision as of 19:35, 20 August 2018
Experiments
Overview
Our team will use synthetic biology to address insufficient access to contraception by engineering a progesterone-producing yeast. We will construct our gene cassettes and insert them into the Y. lipolytica genome in three parallel experiments. In all experiments, we will use the strain FKP393 with the auxotrophic markers LEU2 and URA3 for selection. We will insert URA3 into the Y. lipolytica genome using HR, and select usingURA3 deficient media. We have designed LoxP and Lox71 sites to flank URA3 for use in Experiments 2 and 3. We will amplify out 1 kb areas upstream and downstream of the ADE2 gene in Y. lipolytica for use as our HAs.
Experiment 0
Description in progress
Figure 1: Creation of pOPPY-UC19-yXXU containing URA3, pUC19 backbone, Lox sites and homologous arms using Gibson Assembly. Transformation of Y. lipolytica to create Y. lipolytica str. LipLox homologous recombination.
Experiment 1
Experiment 1 will use well-studied methods previously tested in Y. lipolytica. We will use Gibson cloning to assemble our five genes with our HAs into the linearized pUC19 plasmid. We will amplify this engineered plasmid in E. coli and then isolate the plasmids. We will linearize them using the restriction enzyme Sma1, which cuts the plasmid in the multiple cloning sites between the HAs, and then transform Y. lipolytica using HR. We will select for our transformed yeast on 5-Fluoroorotic Acid (5-FOA) enriched with URA3 to select for cells who have successfully exchanged the URA3 gene between the HAs for our gene insert.
Figure 2: Creation of pOPPY-19-yP via overlap extension PCR of genes involved in progesterone biosynthesis and gibson assembly of this gene cassette with homologous arms and linearized pUC19 backbone. Transformation of Y. lipolytica str. LipLox<\i> using homologous recombination to exchange gene cassette. Selection of Y. lipolytica str. PoPPY<\i> using 5FOA URA+.
Experiment 2
Experiment 2 will combine two well-studied and experimental techniques. We will use yeast-mediated cloning to assemble the five genes with the pXRL2 plasmid in S. cerevisiae<\i>. YMC experiments have been well documented in S. cerevisiae<\i> and have high levels of reliability. Next, we will isolate the engineered plasmids from S. cerevisiae<\i> and transform them into Y. lipolytica<\i> using the Cre-Lox recombinase method. The LoxP and Lox71 sites were placed on the ends of our five-gene construct during our design. Cre-Lox will integrate the DNA between the LoxP and Lox71 sites that flank the URA3<\i> gene in our engineered Y. lipolytica<\i> genome. To test for successful integration, we will grow those Y. lipolytica<\i> cells on 5-FOA enriched with URA3<\i> to select for cells that successfully exchange the URA3<\i> gene for our gene insert.
Figure 3: Creation of pOPPY-XRL2-yP<\i> via Yeast Mediated Cloning in S. cerevisiae<\i> using linearized pXRL2 and gene fragments. Transformation of Y. lipolytica str. LipLox<\i> using pOPPY-XRL2-yP<\i> followed by Cre-Lox Recombination and 5FOA URA+ selection to create Y. lipolytica str. PoPPY<\i>.
Experiment 3
Experiment 3 will be a completely novel experimental trial. Yeast-mediated cloning has not been tested in Y. lipolytica<\i>, nor has the Cre-Lox mechanism of integration. We will do the same steps as in Experiment 2, except we will perform the yeast-mediated cloning in Y. lipolytica<\i> directly. We will allow the yeast enough time to assemble the construct, and then add the Cre recombinase to activate the Lox site integration. If this experiment works, it will assemble the gene fragments and plasmid, and integrate said construct into the genome all in one organism, in very few steps. A successful Cre-Lox experiment would be a great advancement in this field.
Figure 4: Yeast Mediated Cloning in Y. lipolytica str. LipLox<\i> using linearized pXRL2 and gene fragments. This is followed by cre-recombination of Lip-Lox and 5FOA URA+ selection to form Y. lipolytica str. PoPPY<\i>.
Quantification
Description in progress
Figure 5: Progesterone-dependent inactivation of hammerhead ribozyme in the 3’ UTR of GFP mRNA.