Difference between revisions of "Team:UCSC/Experiments"
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<p class="p-title">Experiment 1</p> | <p class="p-title">Experiment 1</p> | ||
| − | <p> | + | <p>Experiment 1 will use well-studied methods previously tested in <i>Y. lipolytica</i>. 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 <i>E. coli</i> and then isolate the plasmids. We will linearize them using the restriction enzyme <i>Sma1</i>, which cuts the plasmid in the multiple cloning sites between the HAs, and then transform <i>Y. lipolytica</i> using HR. We will select for our transformed yeast on 5-Fluoroorotic Acid (5-FOA) enriched with <i>URA3</i> to select for cells who have successfully exchanged the <i>URA3</i> gene between the HAs for our gene insert.</p> |
<img src="https://static.igem.org/mediawiki/2018/8/80/T--UCSC--Experiment_1_Overview.jpg" width="80%" class="image-inpage"> | <img src="https://static.igem.org/mediawiki/2018/8/80/T--UCSC--Experiment_1_Overview.jpg" width="80%" class="image-inpage"> | ||
<p class="image-inpage-caption" style="width:66%; font-size:85% !important"> 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 using homologous recombination to exchange gene cassette. Selection of Y. lipolytica str. PoPPY using 5FOA URA+.</p> | <p class="image-inpage-caption" style="width:66%; font-size:85% !important"> 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 using homologous recombination to exchange gene cassette. Selection of Y. lipolytica str. PoPPY using 5FOA URA+.</p> | ||
Revision as of 19:22, 20 August 2018
Experiments
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 using homologous recombination to exchange gene cassette. Selection of Y. lipolytica str. PoPPY using 5FOA URA+.
Experiment 2
Description in progress
Figure 3: Creation of pOPPY-XRL2-yP via Yeast Mediated Cloning in S. cerevisiae using linearized pXRL2 and gene fragments. Transformation of Y. lipolytica str. LipLox using pOPPY-XRL2-yP followed by Cre-Lox Recombination and 5FOA URA+ selection to create Y. lipolytica str. PoPPY.
Experiment 3
Description in progress
Figure 4: Yeast Mediated Cloning in Y. lipolytica str. LipLox 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.
Quantification
Description in progress
Figure 5: Progesterone-dependent inactivation of hammerhead ribozyme in the 3’ UTR of GFP mRNA.