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 Yarrowia 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 homologous recombination (HR) and select for recombinant strains using URA3-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 fragments upstream and downstream of the ADE2 gene in Y. lipolytica for use as our homology arms (HAs).

Experiment 0

In Experiment 0, we will use Gibson Assembly (GA) to assemble the gene block of URA3 flanked by two Lox sites, the amplified HAs from the Y. lipolytica genome, and the linearized pUC19 plasmid. To facilitate the GA, the HAs will have homologous regions attached to their ends using primer-flags. The 5’ end of the upstream arm will have homology with the pUC19 plasmid, and the 3’ end will have homology with the gene blocks which have homologous overlapping regions for the pXRL2 plasmid. On the downstream arm, the 5’ end will have homology with the gene blocks (pXRL2), and the 3’ end will have homology with the pUC19 plasmid. On the ends of the LoxP-URA3-Lox71 gene block, we will design homologous ends to the 3’ end of the upstream and to the 5’ end of the downstream HAs. Our LoxP-URA3-Lox71 gene block will ligate to the HAs, and the HAs will ligate to the pUC19 plasmid to assemble our full pOPPY-UC19-yXXU plasmid. We will then insert our Lox sites into the Y. lipolytica genome using homologous recombination to create the Y. lipolytica str. LipLox.

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

In Experiment 1, we will use GA to assemble our five progesterone pathway genes and our HAs into the linearized pUC19 plasmid to create the pOPPY-UC19-yP plasmid. We will transform this engineered plasmid into E. coli for replication and then isolate the plasmids. We will linearize pOPPY-UC19-yP using the restriction enzyme Sma1, which cuts the plasmid in the multiple cloning sites between the HAs, and then insert the progesterone genes into Y. lipolytica using HR. We will select for our recombinant yeast on 5-Fluoroorotic Acid (5-FOA) enriched with URA3 to select for cells that 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

In Experiment 2, we will use yeast-mediated cloning (YMC) in S. cerevisiae to assemble the five progesterone genes into the linearized pXRL2 plasmid to create the pOPPY-XRL2-yP plasmid. YMC experiments have been well documented in S. cerevisiae and have high levels of reliability. We will then isolate pOPPY-XRL2-yP from S. cerevisiae and transform it into Y. lipolytica str. LipLox using the Cre-Lox recombinase method. The LoxP and Lox71 sites were placed on the ends of our five-gene construct during our design process. Cre-Lox will integrate the DNA between the LoxP and Lox71 sites that flank the URA3 gene in the Y. lipolytica str. LipLox genome. To test for successful integration, we will grow the transformed Y. lipolytica cells on 5-FOA enriched with URA3 to select for cells that successfully exchange the URA3 gene for our five-gene insert.

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

Experiment 3 will be a completely novel experimental trial. Yeast-mediated cloning has not been tested in Y. lipolytica, nor has the Cre-Lox mechanism of integration. We will perform the same YMC steps in Experiment 2 using the Y. lipolytica str. LipLox to assemble the five-gene construct into pXRL2 to form the pOPPY-XRL2-yP plasmid. We will allow the yeast enough time to assemble the construct, and then we will add the Cre recombinase to activate the Lox site integration. If this experiment works, Y. lipolytica str. LipLox will have the ability to assemble and integrate pOPPY-XRL2-yP into its own genome to create the final Y. lipolytica str. PoPPY. A successful Cre-Lox experiment would be a great advancement in this field.

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

We will first amplify our experimental plasmid as well as the riboswitch insert by transformation into E. coli and PCR, respectively. The protocol used for our transformations will be the high efficiency transformation protocol for DH5alpha competent E. coli 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 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 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 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^[1] and random mutagenesis via error-prone PCR^[2] 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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1. Mosing, R. K. and Bowser, M. T. (2009). Isolating aptamers using capillary electrophoresis-SELEX (CE-SELEX). Nucleic Acid and Peptide Aptamers
2. McCullum, E. O., Williams, B. A., Zhang, J., and Chaput, J. C. (2010). Random mutagenesis by error-prone PCR. In Vitro Mutagenesis Protocols