Difference between revisions of "Team:UCSC/Experiments"

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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

We will first amplify our experimental plasmid as well as the riboswitch insert by transformation into E. coli<\i> and PCR, respectively. The protocol used for our transformations will be the high efficiency transformation protocol for DH5alpha competent E. coli<\i> cells from New England Biolabs. After running a selection on ampicillin plates and incubating our successfully transformed colonies in ampicillin-enriched LB broth, we will perform a plasmid DNA isolation using our Zymo miniprep kits. Our plasmid isolations will be confirmed for identity and quality using a combination of nanodrop analysis and Sanger sequencing. We will then create our reporter system plasmid by using PCR to linearize the pHR_D17_hrGFP<\i> plasmid as if we had placed a blunt-end double strand break in the 3’ UTR of the hrgfp gene. We will also order 5 different DNA oligos from Integrated DNA Technologies that will include our entire riboswitch construct with 1 of the 5 progesterone-specific aptamers described in the Jimenez paper as well as two 20 bp overhangs that are homologous with the blunt-end sequences of the linearized plasmid. We will then use the Gibson Assembly protocol described previously to incorporate our riboswitch insert into the reformed plasmid. We will then transform these plasmids into DH5alpha competent E. coli<\i> cells as previously described. The E. coli cells will be used both for cloning of the plasmid as well as testing the function of the riboswitch construct. Any colonies showing working riboswitch constructs will then have their plasmid DNA isolated using one of our minipreps. These plasmids will then be transformed into Y. lipolytica<\i> and assayed again to ensure continued function when transferred into eukaryotic cells. In the case that one of our five unaltered plasmids shows functionality with our riboswitch structure, we will then begin trials using CE-SELEX method (Mosing, 2009) and random mutagenesis via error-prone PCR (Mcullum, 2010) to identify variants that show a decreased sensitivity for progesterone in order to trigger fluorescence at higher concentrations.

Figure 5: Progesterone-dependent inactivation of hammerhead ribozyme in the 3’ UTR of GFP mRNA.

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        <p class="p-title">Quantification</p>
-        <p> Description in progress</p>
+        <p>We will first amplify our experimental plasmid as well as the riboswitch insert by transformation into <i>E. coli<\i> and PCR, respectively. The protocol used for our transformations will be the high efficiency transformation protocol for DH5alpha competent <i>E. coli<\i> cells from New England Biolabs. After running a selection on ampicillin plates and incubating our successfully transformed colonies in ampicillin-enriched LB broth, we will perform a plasmid DNA isolation using our Zymo miniprep kits. Our plasmid isolations will be confirmed for identity and quality using a combination of nanodrop analysis and Sanger sequencing. We will then create our reporter system plasmid by using PCR to linearize the <i>pHR_D17_hrGFP<\i> plasmid as if we had placed a blunt-end double strand break in the 3’ UTR of the hrgfp gene. We will also order 5 different DNA oligos from Integrated DNA Technologies that will include our entire riboswitch construct  with 1 of the 5 progesterone-specific aptamers described in the Jimenez paper as well as two 20 bp overhangs that are homologous with the blunt-end sequences of the linearized plasmid. We will then use the Gibson Assembly protocol described previously to incorporate our riboswitch insert into the reformed plasmid. We will then transform these plasmids into DH5alpha competent <i>E. coli<\i> cells as previously described. The E. coli cells will be used both for cloning of the plasmid as well as testing the function of the riboswitch construct. Any colonies showing working riboswitch constructs will then have their plasmid DNA isolated using one of our minipreps. These plasmids will then be transformed into <i>Y. lipolytica<\i> and assayed again to ensure continued function when transferred into eukaryotic cells. In the case that one of our five unaltered plasmids shows functionality with our riboswitch structure, we will then begin trials using CE-SELEX method (Mosing, 2009) and random mutagenesis via error-prone PCR (Mcullum, 2010)  to identify variants that show a decreased sensitivity for progesterone in order to trigger fluorescence at higher concentrations. </p>
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