Difference between revisions of "Team:UCSC/Results"

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     <h2 id="Desi_pro">YEAST MEDIATED CLONING</h2>
 
     <h2 id="Desi_pro">YEAST MEDIATED CLONING</h2>
  
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     <p>Due to our extensive research on world issues, we chose to create a contraceptive in yeast: progesterone. Progesterone is a hormone produced by all mammals (men and women) naturally, and aids in many functions regulating fertility, menstruation/ovulation, pregnancy, and normal body function. "Progesterone prepares the endometrium for the potential of pregnancy after ovulation. It triggers the lining to thicken to accept a fertilized egg. It also prohibits the muscle contractions in the uterus that would cause the body to reject an egg. While the body is producing high levels of progesterone, the body will not ovulate" (hormone.org). Progestins are routinely used in contraceptive pills, vaginal rings, and IUDs. Progestins are a chemically formulated version of progesterone (CITATION!!!), that acts on progesterone receptors to increase cervical mucus and suppress ovulation to prevent pregnancy. They are not naturally made by the human body, and therefore have many ill effect on the women who take them (CITATION!!!!!). Progesterone is the molecule naturally made by the body, which when taken in excess, can perform the same function as progestins: preventing pregnancy. Progesterone can do so without the harmful effects that progestins cause (CITATION!!!!).  In mammals, the progesterone biosynthesis pathway begins with cholesterol. Enzymes convert cholesterol to pregnenolone to progesterone. Figure 1 shows a highly simplified version of the reference pathway for steroid hormone biosynthesis in mammals from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to highlight the progesterone biosynthesis pathway.</p>
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     <p>We concluded the successful removal of loxP from pXRL2 via site directed mutagenesis. The results of our colony PCR suggested that the product of the reaction was slightly smaller than the original plasmid. However, the positive control had a strange band higher than we expected. We sequenced to confirm our success and found that the product was missing the desired region in an alignment with the original plasmid. We also conducted attempts of yeast mediated cloning with successful growth in leucine deficient media. This result would suggest that we had a successful transformation and possible assembly of our gene cassette on to the plasmid backbone.</p>
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     <p>The organism we are engineering, Y. lipolytica, does not produce cholesterol naturally, but it does produce a cholesterol precursor: zymosterol. In Y. lipolytica however, only the zymosterol-ergosterol pathway exists. We considered engineering three non-native enzymes (circled in blue in Figure 2) into Y. lipolytica to complete the zymosterol-cholesterol pathway, due to the common pathway to progesterone in mammals starting from cholesterol. However, because ergosterol is the yeast’s preferred pathway product, navigating through the cholesterol pathway would require completely knocking out the pathway to ergosterol (starting from ERG6 on the right). The addition of those three genes, plus the additional genes needed to take cholesterol to progesterone, PLUS a pathway knockout required too much engineering for the timeframe of our project. The enzymes highlighted in green exist in Y. lipolytica, see the end product of ergosterol circled in pink. The enzymes highlighted with white do not exist, and would need to be engineered into the organism.</p>
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    <p>We needed to figure out how to create progesterone in a creative way. We found that we could create a synthetic pathway to progesterone, directly from ergosterol (circled in pink in Figure 2). This was beneficial because that pathway exists naturally in Y. lipolytica. In Y. lipolytica and other fungi, ergosterol serves similar functions in fungi as cholesterol does in animal cells (Money, 2016), meaning that while the pathway does not currently exist in the organism, it is possible to convert ergosterol to progesterone. We searched through research articles and found the work of Duport et al.(CITATION??!!) who outlined the possible pathway to convert ergosterol to progesterone using five genes. The first gene is for the enzyme ∆7-sterol reductase (∆7Red), isolated from Arabidopsis thaliana, which reduces ergosterol to ergosta-5-enol and ergosta-5,22-dienol (Waterham and Wan- ders, 2000). The next gene encodes for bovine side-chain cleavage cytochrome (P450scc), which converts ergosta-5-enol and ergosta-5,22-dienol to pregnenolone. P450scc requires the assistance of bovine ferredoxin-1 (FDX1) as an electron carrier (Grinberg et al., 2000), and adrenodoxin reductase (ADR) to reduce FDX1. The final gene is type II human 3β-hydroxysteroid dehydrogenase (3β-HSD) that converts pregnenolone to progesterone (Koritz, 1964). See the complete pathway to progesterone biosynthesis in Y. lipolytica below in Figure 3.</p>
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    <p>We found the sequences for each gene from either KEGG or UniProt and codon optimized them for Y. lipolytica using the DNA works system from the High-Performance Computing at the NIH website.</p>
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    <p>We conducted a colony PCR to confirm the successful site directed mutagenesis of loxP from pXRL2. We used a pair of primers: cloxP F and cloxP R to amplify the region that we removed and ran the samples on a gel. A successful trial would be a band 300 bp or lower since the fragment including the lox site would be 343 bp. Our results were puzzling due to the band being higher than 300 bp but we thought that this had occurred because the sample ran slower than the other lanes evident in the arc of the dye. We conducted another colony PCR with monoclonal colonies. Strangely, our positive control appeared to be much larger than the other samples but we concluded that it was due to issues with gel electrophoresis. We resolved to use the samples with the lowest bands or smallest size which were 5C, 5D, 21B and 23A. Upon further investigation by sequencing, we concluded that we had succeeded due to the region being missing in an alignment with the original plasmid. However, in sample A, more bases were deleted than expected so we did not use it because of risk of a frameshift.</p>
  
 
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Revision as of 17:22, 16 October 2018

Results

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    YEAST MEDIATED CLONING

    We concluded the successful removal of loxP from pXRL2 via site directed mutagenesis. The results of our colony PCR suggested that the product of the reaction was slightly smaller than the original plasmid. However, the positive control had a strange band higher than we expected. We sequenced to confirm our success and found that the product was missing the desired region in an alignment with the original plasmid. We also conducted attempts of yeast mediated cloning with successful growth in leucine deficient media. This result would suggest that we had a successful transformation and possible assembly of our gene cassette on to the plasmid backbone.

    Insert Image

    Insert Image

    Insert Image

    We conducted a colony PCR to confirm the successful site directed mutagenesis of loxP from pXRL2. We used a pair of primers: cloxP F and cloxP R to amplify the region that we removed and ran the samples on a gel. A successful trial would be a band 300 bp or lower since the fragment including the lox site would be 343 bp. Our results were puzzling due to the band being higher than 300 bp but we thought that this had occurred because the sample ran slower than the other lanes evident in the arc of the dye. We conducted another colony PCR with monoclonal colonies. Strangely, our positive control appeared to be much larger than the other samples but we concluded that it was due to issues with gel electrophoresis. We resolved to use the samples with the lowest bands or smallest size which were 5C, 5D, 21B and 23A. Upon further investigation by sequencing, we concluded that we had succeeded due to the region being missing in an alignment with the original plasmid. However, in sample A, more bases were deleted than expected so we did not use it because of risk of a frameshift.