Latest revision as of 03:48, 18 October 2018

Results

Overview

LipLox

Yeast Mediated Cloning

Riboswitch

Quantification

Project Achievements

OVERVIEW

We constructed the biosynthetic pathway for progesterone, consisting of 5 genes ( Δ7 Red, P450scc, FDX1, ADR, 3Β-HSD). In addition, we created a riboswitch that will allow us to measure the concentration of progesterone.

LIPLOX

Homology Arms

To amplify out the HAs of Y. lipolytica str. FKP393, we lysed the cells and extracted the gDNA. We then performed touchdown PCR with the gDNA and confirmed the amplification of the HAs with gel electrophoresis. The UHA is 1014 bases long, and the DHA is 1000 bases long, so we expected bands at ~1 kb for both PCR samples. Figure 1, shows the expected bands just above 1 kb. It is likely that the bands are above 1 kb because the loaded samples contain Q5 reaction buffer and ran slower than the 2-log DNA ladder. We concluded that the bands produced were from the HAs. We sent the PCR products to be sequenced with primers that would identify regions of the homology arms and were able to confirm that our product were the HAs.

Figure 1: Gel of Upstream and Downstream HAs, 1% TBE gel electrophoresis of the upstream and downstream HA PCR amplifications. We dyed each 5 µL sample with 1 µL of 6X loading dye and loaded 5 µL of this mix into each well. Lane 1 contains the 2-log DNA ladder, lane 2 contains the amplified UHA, and lane 3 contains the amplified DHA.

Enter lox sites and ura3 into Y. lipolytica str. FKP393 genome

We attempted to assemble the loxP-URA3-lox71 geneblock with homology arms from the Y. lipolytica genome using Gibson Assembly (GA) to create pOPPY-UC19-yXXU. We performed GA using 1:10 molar ratio of plasmid backbone to insert, and transformed NEB 5-alpha E. coli with the product, and grew them on high copy LB/AMP plates for 14 hours at 37ºC. The E. coli plates grew colonies, shown below.

Figure 1: pOPPY-UC19-yX transformed in NEB 5-alpha E.coli grown on LB/AMP high copy plate.

We picked 12 colonies, with which we performed colony PCR, and created an index plate. Our colony PCR gel results indicated that the colonies we chose were not homogenous; while some colonies showed light bands at the appropriate size for pOPPY-UC19-yXXU (~4 kb), they also contained other bands around 0.5kb which is the size expected if there is no insert, and just pUC19 backbone.

Figure 1: 1% agarose gel containing samples from colony PCR of pOPPY-UC19-yXXU attempting to detect loxP-ura3-lox71 geneblock with homology arms to ade2.

The gel shows that we may have a mixed population in our colony picks. While there are light bands at 4kb, the bright bands at 0.5kb show that our insert was not included in the majority of the DNA we performed the PCR on. We created streak plates with the colonies on the index plate to attempt to screen out the mutants who had not successfully assembled our insert into the backbone. Unfortunately, after PCR of those, the results returned the same information that our colonies did not include the insert. This is most likely due to the plasmid somehow re-circularizing to itself, or DPN1 not completely digesting the original pUC19 plasmid after isolation from E.coli. Because we were not able to isolate pOPPY-UC19-yXXU, we chose a different route.

The Riboswitch team has been using a plasmid called D17, generously gifted from Cory Schwartz at UC Irvine, which contains homology arms for the pseudogene D17 in Y. lipolytica. We decided to try and insert loxP-ura3-lox71 into that plasmid. If successful, we would then amplify out to obtain a linear construct including the homology arms to D17, which we could perform homologous recombination into the Y. lipolytica genome with. We PCR amplified D17, cutting out plasmid backbone between the homology arms to create a construct with homology arms at each end of the linear product. We ordered primers with Gibson Overhangs (GO) to the D17 plasmid to amplify loxP-ura3-lox71 with. The PCR results were unsuccessful every try. Due to these issues, we decided to use KLD to insert the loxP-URA3-lox71 gene block into D17. We performed KLD treatment with linearized D17 backbone, and the loxP-URA3-lox71 gene block and transformed NEB 5-alpha E.coli. We saw many colonies, and we picked 15 for colony PCR to check if integration occurred.

Figure 1: 1% agarose gel containing colony PCR results from loxP-URA3-lox71 geneblock in between D17 homology arms.

The gel shows in lane 2 that we achieved a clean, single band at ~5kb. The primers bound to the D17 plasmid at the beginning and end of the D17 homologous arms, including the arms and our loxP-URA3-lox71 insert. The expected size of our product is 3.6 kb. The band is slightly higher than this size, which we believe is due to the salts in the cell causing it to run a bit slower than it should. Because of this we are confident that this band is our desired product. We have grown a liquid culture of Colony 2, miniprepped it, and performed PCR using Q5 polymerase and the same primers used for colony PCR to attempt to amplify out loxP-URA3-lox71 sandwiched between the D17 homology arms. The results have not yet shown any bands from this.

Once we are able to amplify loxP-URA3-lox71 with D17 homology arms, we will transform Y. lipolytica with this linear product and confirm success by growing the colonies on uracil-deficient media. Once the loxP and lox71 sites are integrated into the genome we can perform Recombinase mediated cassette exchange with the plasmid created by YMC with all pathway genes using the corresponding lox sites.

YEAST MEDIATED CLONING

We concluded the successful removal of loxP from pXRL2 via site directed mutagenesis. The results of our colony PCR (Figure 1) suggested that the product of the reaction was slightly smaller than the original plasmid. 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.

Figure 1: With the primers used in this colony PCR reaction, we should see a fragment of about 270bp if one loxP site was successfully removed from pXLR2 and a 340bp fragment if the loxP site was not removed. We initially believed that the bright bands between 300bp and 400bp in colony 2 and 5 (marked here in green rectangles) indicated failures to remove loxP because they appeared to be the 340bp fragments. We later realized that the gel did not run evenly, as can be seen in the arc of the dark line representing the dye. This resulted in the bands appearing higher than they should have, and these two bright bands likely indicated successful removal of loxP.

We conducted a colony PCR (Figure 1) to check 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 (Figure 2), 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. We, then relinearized the four sequenced samples for gene assembly via yeast mediated cloning. The results of this suggested a success since the bands resulting from gel electrophoresis were at the expected 8-10 kb.

Figure 2: We sequenced our relinearized samples of pOPPY-XRL2-yX and aligned the results with the original pXRL2 plasmid or negative control. The green lines indicate the successful removal of loxP by site directed mutagenesis while the red lines show the deletion of extra bases in plasmid A and 21.

Figure 3: After creating a set of liquid cultures of the two colonies we believed to contain plasmids with one loxP site removed (2 and 5), we miniprepped and then ran a PCR using the same primers used in the previous colony PCR (see figure 1). A band at 270bp indicates that loxP was removed, while a band at 340bp would indicate that loxP was not removed. The PCR products of “Miniprep C from Colony 5 liquid culture” (lane 1) showed a band just below the 300bp mark, suggesting that loxP was successfully removed from the plasmid in colony 5. The band in lane 4 also appeared promising, while we felt that the slightly higher bands appearing between 300bp and 400bp in lanes 5 and 9 seemed to indicate that loxP may not have been removed from the plasmid in colony 2. Our negative control, the PCR run on the unaltered pXLR2 plasmid backbone, surprisingly showed a band between 400bp and 500bp rather than the expected 340bp. We think this could potentially be due to errors made in addition of PCR reagents to this specific sample.

Figure 4: In this gel, we ran our samples that were relinearized with inverse PCR subsequent to confirming that loxP was removed. The PCR product resulting from the original plasmid shown in lane 5 appeared to be slightly higher than the pOPPY-XRL2-yX samples in lane 2, 3 and 4. This was expected since the linearization theoretically removed 230bp from the plasmid which was originally 6693bp.

We performed yeast mediated cloning to assemble delta 7-sterol reductase, ADR, FDX1, P450scc and 3ꞵ-HSD into linearized pOPPY-XRL2-yX. We plated the transformants and saw many colonies. We grew 30 colonies selected from leucine-deficient plates in leucine-deficient liquid media to mini-prep them and check for successful plasmid assembly. We rescued the plasmid and ran 250ng dilutions of our samples on a gel (Figure 5). The expected size of the desired plasmid with all pathway genes, pOPPY-XRL2-yP, was approximately 15kb in comparison to pXRL2 which is 6693bp.

Figure 5: All of the lanes contained circularized plasmids. Samples 1, 2, 9 and 14 had bands higher than the circular plasmid backbone control and above 10kb, suggesting they may have contained some number of genes. We selected the plasmid in Lane 2, 9, and 14 for sequencing to confirm their success.

RIBOSWITCH

For our part of the project, we intended to create a one-gene reporter system for progesterone concentration consisting of: a Renilla GFP gene (hrgfp), a cyc1 terminator, TEF(136) promoter, and a hammerhead ribozyme expression platform attached to a progesterone-specific aptamer.

Our first task in this project was to create a large stock of our pHR_D17_hrGFP plasmid (from here on referred to as D17), which we did by transforming a portion of our IDT D17 stock into chemicompetent E. coli, running a selection on an LB agar plate with a 100 ug/L concentration of ampicillin, and then growing out selected colonies in liquid LB medium. We then isolated our samples of D17 from the bacterial culture by using a Zymo plasmid miniprep kit.

In order to confirm the sequence of our hrgfp gene as well as the cyc1 terminator, we also designed a series of primers meant to be used in Sanger sequencing of our D17 plasmid. These primers were labeled Rib(f)-1 through Rib(f)-3 and Rib(r)-1 through Rib(r)-3 and encompassed the entire hrgfp gene as well as the cyc1 terminator with several hundred base pairs of overhang on either side. Sequencing data from the UC Berkeley DNA sequencing facility showed a successful primer design and an intact DNA sequence in our D17 plasmid.

Figure 1: Agarose gel confirming the presence of our linearized D17 plasmid at around the 9kb mark.

In order to carry out Gibson Assembly, we also had to amplify our stock of riboswitch inserts. To do this, we designed the Apt(f) and Apt(r) primers to anneal to the overhangs of all 5 different inserts. Running a series of PCR trials and imaging on an agarose gel in Figure 2 showed strong bands at the expected bp length, indicating successful amplification of our inserts.

Figure 2: Agarose gel confirming the amplification of riboswitch oligomers on lanes 2 through 5.

We then performed a Gibson assembly trial with our newly amplified D17 vector and riboswitch inserts. We attempted a confirmation PCR to check if our inserts were successfully integrated into the D17 backbone with the ampGB(f) and ampGB(r) primers. A successful integration event would result in amplicons approximately 4.2 kb in length, while a circular D17 template would create an amplicon 4.0 kp in length. Imaging of our gel in Figure 3 suggested the continued presence of D17 circular template along with our new pOPPY_GFP plasmids, even after digestion with >500-fold excess of DpnI.

Figure 3: Agarose gel comparing the presence of linear vs. circular D17 template amplification after Gibson assembly.

To remedy this, we performed a series of transformations and selective platings with our Gibson assembly product on 100 ug/mL LB-ampicillin medium and selected a total of 52 colonies--10 from each Gibson transformation and 2 from a D17 control plate--for colony PCR and spot plating on an index plate. The imaged gels in Figure 4 from the colony PCR trials allowed us to select single colonies we believed to be carrying our riboswitch insert.

Figure 4: Agarose gels of index plate colonies 1-52, 1-10 of pOPPY_GFP 1, 11-20 of pOPPY_GFP 2, and so on. Colonies 51 was our negative D17 control. We looked for a 200 base pair difference in each pOPPY_GFP variant to confirm presence of our riboswitch insert.

From these gels we selected colonies 2, 16, 27, 32, 43, as likely carriers of pOPPY_GFP(1)--pOPPY_GFP(5), respectively, and colony 51 as a control carrier of D17. We then used our previously grown index plate to create monoclonal liquid cultures of these colonies in LB medium. After overnight incubation, we then performed a plasmid isolation with all 6 cultures and transformed the isolated plasmids into our competent Y. lipolytica strain, which we created using the Zymo Frozen EZ-yeast transformation protocol.

QUANTIFICATION

From the pOPPY-XRL2-yP plasmids that YMC had assembled, supposedly including all of the progesterone pathway genes, we chose six colonies to inoculate and test for progesterone production. We chose the colonies YL 10, YL 11, YL 20, YL 21, YL 22, and YL 25, shown in Figure X. We chose these both arbitrarily and due to the presence of apparent plasmid bands at the appropriate size.

Figure 3: Colonies of Y. lipolytica transformed with progesterone pathway genes grown on leucine-deficient media. The pOPPY-XRL2-yP plasmid contains the gene completing the leucine pathway, acting as our auxotrophic marker.

After purification of progesterone according to the Progesterone Extraction protocol, we performed “dot blotting” on nitrocellulose membranes, and detected using anti-progesterone antibodies conjugated to HRP (sc-53423 HRP), gifted from Santa Cruz Biotech (Santa Cruz Biotechnology, Inc., TX, USA).

Figure 3: Dot blot of progesterone re-suspended in DMSO, isolated from colonies grown on leucine-deficient media after YMC with progesterone pathway genes. Negative control circled in black and marked with a dash (-). Positive controls circled in blue. Experimental samples circled in pink. Primary antibody used was anti-progesterone-HRP, blocked with BSA in TBS, washed with TBS/TWEEN. Positive controls are 1mg/100uL progesterone in DMSO with 1:100 dilutions. Negative control is Y. lipolytica transformed with pD17 with no pathway genes which underwent same extraction protocol as all other samples.

Our dot blot shows that we have high luminescence in multiple colonies, YL 10, YL 20, YL 21, and YL 25. We performed the extraction protocol on +1 positive control, and our negative control. The +1 positive is 1mg of pure progesterone extract from Sigma Aldrich (P8783 Sigma) that we added 1mL of MilliQ water and 1mL of hexane to, and then followed the rest of the extraction protocol for. The positive controls +3, +4 and +5 are 1:10 dilutions from +2 positive control of 1mg/1mL of pure progesterone in DMSO. These positives were not subjected to the extraction protocol. We believe that they may show no luminescence due to +2 being one order of magnitude less than detection that is possible for the antibodies. The negative control is pD17 transformed into Y. lipolytica with no pathway genes, and then subjected to the same extraction protocol.

We believe that these results suggest that we have at least four progesterone producing Y. lipolytica colonies. The positive control shows the level of luminescence for 1mg of progesterone, and the colonies that show luminescence as well are of similar magnitude. We inoculated 5 mL cultures with our colonies, so we find it a bit suggestive that we have similar levels of luminescence in our 1 mg positive control as we do in our colonies.

Due to the ability of our hexane extraction protocol to apparently purify our samples so greatly, we will perform mass spectrometry on these samples in later work.

PROJECT ACHIEVEMENTS

Successful Results

The following tasks were successfully completed by our team.

LoxP removal from pXRL2
Lox-URA3-Lox in D17
Amplification of HAs
Gibson Assembly of pOPPY_GFP plasmids

Once we received a set of improved primers, our D17 plasmid had overhangs that allowed for more efficient Gibson Assembly. We confirmed this by choosing 50 colonies from a series of transformations with our pOPPY_GFP plasmids for colony PCR. Running these individual PCRs on a gel allowed allowed us to select colonies that showed bands consistent with an insert of the correct size in the same location as our riboswitch insert.

Amplification of Δ7, P450scc, ADR, and 3ΒH genes
Successful Yeast-Mediated Cloning in Y. lipolitica
Made Progesterone

Unsuccessful Results

The following bullets include experiments and tasks that were not successful during the wetlab period.

Overlap Extension PCR

After many touchdown PCR trials, we were unable to assemble the full genes without seeing non-specific sequences in our PCR reactions. We tried using PCR without primers to anneal the overlapping regions of the gene fragments to create better DNA templates, but there were always non-specific bands alongside the full-length genes. Even after extracting the correct sized bands from agarose gels and using that DNA as a template resulted in the same non-specific banding patterns.

Attaching Homologous Arms to Lox-URA3-Lox
Gibson Assembly of Genes into pXRL2
P450 into PoPPY and D17 Plasmid
Lox-URA3-Lox into PoPPY Plasmid
Amplification of FDX1 gene

Ferrodoxin (FDX1) was one of the five genes in the progesterone pathway that we tried to amplify for Gibson Assembly and Yeast Mediated Cloning. After many touchdown PCR trials, we found a few thermocycler conditions that seemed to properly amplify FDX1. When we tried creating larger PCR reaction volumes, we saw bands that were much larger than the expected gene size for FDX1, and we couldn’t get the same amplification like in earlier PCR reactions.

Isolating pOPPY_GFP plasmids from D17 backbone

We had repeatedly tried to separate our pOPPY_GFP plasmids from their original D17 backbone upon completion of Gibson Assembly using both DpnI digests as well as gel electrophoresis. The small length difference between the plasmids (~200bp) did not allow for enough separation to perform a gel purification, and PCR repeatedly created amplicons indicative of both pOPPY_GFP plasmids as well as D17 backbone.

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      <h2 id="Overview">OVERVIEW</h2>
-     <p>We constructed the biosynthetic pathway for progesterone, consisting of 5 genes ( &#916;7 Red, P450scc, FDX1, ADR, 3&#914;-HSD). In addition, we created a riboswitch that will allow us to measure the concentration of progesterone.</p>
+     <p>We constructed the biosynthetic pathway for progesterone, consisting of 5 genes ( <i>&#916;7 Red, P450scc, FDX1, ADR, 3&#914;-HSD</i>). In addition, we created a riboswitch that will allow us to measure the concentration of progesterone.</p>
    </div>
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+      <p>We attempted to assemble the <i>loxP-URA3-lox71</i> geneblock with homology arms from the <i>Y. lipolytica</i> genome using Gibson Assembly (GA) to create pOPPY-UC19-yXXU. We performed GA using 1:10 molar ratio of plasmid backbone to insert, and transformed NEB 5-alpha <i>E. coli</i> with the product, and grew them on high copy LB/AMP plates for 14 hours at 37ºC. The <i>E. coli</i> plates grew colonies, shown below.</p>
+<div class="imageCont floatC">
+        <img src="https://static.igem.org/mediawiki/2018/c/c7/T--UCSC--Gibson-2.png" class="image-inpage vert large noBorder" width="60%">
+        <p><b>Figure 1</b>: pOPPY-UC19-yX transformed in NEB 5-alpha E.coli grown on LB/AMP high copy plate.</p>
+    </div>
+    <p>We picked 12 colonies, with which we performed colony PCR, and created an index plate. Our colony PCR gel results indicated that the colonies we chose were not homogenous; while some colonies showed light bands at the appropriate size for pOPPY-UC19-yXXU (~4 kb), they also contained other bands around 0.5kb which is the size expected if there is no insert, and just pUC19 backbone.</p>
+<div class="imageCont floatC">
+        <img src="https://static.igem.org/mediawiki/2018/8/86/T--UCSC--Gibson-3.png" class="image-inpage vert large noBorder" width="60%">
+        <p><b>Figure 1</b>: 1% agarose gel containing samples from colony PCR of pOPPY-UC19-yXXU attempting to detect <i>loxP-ura3-lox71</i> geneblock with homology arms to ade2.</p>
+    </div>
+    <p>The gel shows that we may have a mixed population in our colony picks. While there are light bands at 4kb, the bright bands at 0.5kb show that our insert was not included in the majority of the DNA we performed the PCR on. We created streak plates with the colonies on the index plate to attempt to screen out the mutants who had not successfully assembled our insert into the backbone. Unfortunately, after PCR of those, the results returned the same information that our colonies did not include the insert. This is most likely due to the plasmid somehow re-circularizing to itself, or DPN1 not completely digesting the original pUC19 plasmid after isolation from E.coli. Because we were not able to isolate pOPPY-UC19-yXXU, we chose a different route.</p>
+<p>The Riboswitch team has been using a plasmid called D17, generously gifted from Cory Schwartz at UC Irvine, which contains homology arms for the pseudogene D17 in <i>Y. lipolytica</i>. We decided to try and insert <i>loxP-ura3-lox71</i> into that plasmid. If successful, we would then amplify out to obtain a linear construct including the homology arms to D17, which we could perform homologous recombination into the <i>Y. lipolytica</i> genome with. We PCR amplified D17, cutting out plasmid backbone between the homology arms to create a construct with homology arms at each end of the linear product. We ordered primers with Gibson Overhangs (GO) to the D17 plasmid to amplify <i>loxP-ura3-lox71</i> with. The PCR results were unsuccessful every try. Due to these issues, we decided to use KLD to insert the loxP-URA3-lox71 gene block into D17. We performed KLD treatment with linearized D17 backbone, and the <i>loxP-URA3-lox71</i> gene block and transformed NEB 5-alpha <i>E.coli</i>. We saw many colonies, and we picked 15 for colony PCR to check if integration occurred.</p>
+    <div class="imageCont" style="float:center">
+        <img src="https://static.igem.org/mediawiki/2018/e/ea/T--UCSC--Gibson-1.png" class="image-inpage vert large noBorder" width="60%">
+        <p><b>Figure 1</b>: 1% agarose gel containing colony PCR results from <i>loxP-URA3-lox71</i> geneblock in between D17 homology arms.</p>
+    </div>
+    <p>The gel shows in lane 2 that we achieved a clean, single band at ~5kb. The primers bound to the D17 plasmid at the beginning and end of the D17 homologous arms, including the arms and our loxP-URA3-lox71 insert. The expected size of our product is 3.6 kb. The band is slightly higher than this size, which we believe is due to the salts in the cell causing it to run a bit slower than it should. Because of this we are confident that this band is our desired product. We have grown a liquid culture of Colony 2, miniprepped it, and performed PCR using Q5 polymerase and the same primers used for colony PCR to attempt to amplify out loxP-URA3-lox71 sandwiched between the D17 homology arms. The results have not yet shown any bands from this.</p>
+    <p>Once we are able to amplify loxP-URA3-lox71 with D17 homology arms, we will transform <i>Y. lipolytica</i> with this linear product and confirm success by growing the colonies on uracil-deficient media. Once the loxP and lox71 sites are integrated into the genome we can perform Recombinase mediated cassette exchange with the plasmid created by YMC with all pathway genes using the corresponding lox sites.</p>
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      <h2 id="YMC">YEAST MEDIATED CLONING</h2>
-     <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>
+     <p>We concluded the successful removal of loxP from pXRL2 via site directed mutagenesis. The results of our colony PCR (Figure 1) suggested that the product of the reaction was slightly smaller than the original plasmid. 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><b>Figure 1</b>: This is where a caption for the figure would be written.</p>
+         <p><b>Figure 1</b>: With the primers used in this colony PCR reaction, we should see a fragment of about 270bp if one loxP site was successfully removed from pXLR2 and a 340bp fragment if the loxP site was not removed. We initially believed that the bright bands between 300bp and 400bp in colony 2 and 5 (marked here in green rectangles) indicated failures to remove loxP because they appeared to be the 340bp fragments. We later realized that the gel did not run evenly, as can be seen in the arc of the dark line representing the dye. This resulted in the bands appearing higher than they should have, and these two bright bands likely indicated successful removal of loxP.</p>
      </div>
 </div></div>
+<div class="flex-row">
+  <div class="flex-col center">
+    <p>We conducted a colony PCR (Figure 1) to check 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 (Figure 2), 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. We, then relinearized the four sequenced samples for gene assembly via yeast mediated cloning. The results of this suggested a success since the bands resulting from gel electrophoresis were at the expected 8-10 kb.</p>
+  </div>
+</div>
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      <div class="imageCont floatC">
          <img src="https://static.igem.org/mediawiki/2018/2/20/T--UCSC--YMC-2.png" class="image-inpage vert large noBorder" width="60%">
-         <p><b>Figure 2</b>: This is where a caption for the figure would be written.</p>
+         <p><b>Figure 2</b>: We sequenced our relinearized samples of pOPPY-XRL2-yX and aligned the results with the original pXRL2 plasmid or negative control. The green lines indicate the successful removal of loxP by site directed mutagenesis while the red lines show the deletion of extra bases in plasmid A and 21.</p>
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-         <p><b>Figure 3</b>: This is where a caption for the figure would be written.</p>
+         <p><b>Figure 3</b>: After creating a set of liquid cultures of the two colonies we believed to contain plasmids with one loxP site removed (2 and 5), we miniprepped and then ran a PCR using the same primers used in the previous colony PCR (see figure 1). A band at 270bp indicates that loxP was removed, while a band at 340bp would indicate that loxP was not removed. The PCR products of “Miniprep C from Colony 5 liquid culture” (lane 1) showed a band just below the 300bp mark, suggesting that loxP was successfully removed from the plasmid in colony 5. The band in lane 4 also appeared promising, while we felt that the slightly higher bands appearing between 300bp and 400bp in lanes 5 and 9 seemed to indicate that loxP may not have been removed from the plasmid in colony 2. Our negative control, the PCR run on the unaltered pXLR2 plasmid backbone, surprisingly showed a band between 400bp and 500bp rather than the expected 340bp. We think this could potentially be due to errors made in addition of PCR reagents to this specific sample.</p>
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          <img src="https://static.igem.org/mediawiki/2018/e/ee/T--UCSC--YMC-4.png" class="image-inpage vert large noBorder" width="60%">
-         <p><b>Figure 4</b>: This is where a caption for the figure would be written.</p>
+         <p><b>Figure 4</b>: In this gel, we ran our samples that were relinearized with inverse PCR subsequent to confirming that loxP was removed. The PCR product resulting from the original plasmid shown in lane 5 appeared to be slightly higher than the pOPPY-XRL2-yX samples in lane 2, 3 and 4. This was expected since the linearization theoretically removed 230bp from the plasmid which was originally 6693bp.
+</p>
      </div>
    </div></div>
+<div class="flex-row"><div class="flex-col">
+<p>
+We performed yeast mediated cloning to assemble <i>delta 7-sterol reductase, ADR, FDX1, P450scc</i> and <i>3ꞵ-HSD</i> into linearized pOPPY-XRL2-yX. We plated the transformants and saw many colonies. We grew 30 colonies selected from leucine-deficient plates in leucine-deficient liquid media to mini-prep them and check for successful plasmid assembly. We rescued the plasmid and ran 250ng dilutions of our samples on a gel (Figure 5). The expected size of the desired plasmid with all pathway genes, pOPPY-XRL2-yP, was approximately 15kb in comparison to pXRL2 which is 6693bp.</p>
+</div></div>
 <div class="flex-row"><div class="flex-col">
      <div class="imageCont floatC">
          <img src="https://static.igem.org/mediawiki/2018/2/20/T--UCSC--YMC-5.png" class="image-inpage vert large noBorder" width="60%">
-         <p><b>Figure 5</b>: This is where a caption for the figure would be written.</p>
+         <p><b>Figure 5</b>: All of the lanes contained circularized plasmids. Samples 1, 2, 9 and 14 had bands higher than the circular plasmid backbone control and above 10kb, suggesting they may have contained some number of genes. We selected the plasmid in Lane 2, 9, and 14 for sequencing to confirm their success. </p>
      </div>
 </div></div>
@@ Line 141: / Line 189: @@
 -->
-<div class="flex-row">
-  <div class="flex-col center">
-    <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. We, then relinearized the four sequenced samples for gene assembly via yeast mediated cloning. The results of this suggested a success since the bands resulting from gel electrophoresis were at the expected 8-10 kb.</p>
-  </div>
-</div>
 <!--       ********************************** End Yeast Mediated Cloning **************************************      -->
-<!--       ********************************** Gibson **************************************      -->
-<div class="flex-row">
-  <div class="flex-col center">
-    <h2 id="Gibson">GIBSON</h2>
-<!--        <h3>pUC19 and PXRL2 Linearization</h3>
-    <p>We linearized plasmids pUC19 and pXRL2 to prepare for Gibson Assembly and to cut out the first loxP site in preparation for YMC. The first few times we tried, there were no bands of any length. This occurred for 5 rounds of PCR until we discovered there was an issue with our Q5 MasterMix, and resolved it. Once we fixed that, we got successful linearization results. \figref{PlGel} shows our gel with plasmid pUC19 linearization in Lane 1, with a band a little higher, but around the expected size of 2.7kb, and plasmid pXRL2 linearization in Lane 2, with a band around the expected size of 7kb.
-</p>
-<div class="flex-row"><div class="flex-col">
-    <div class="imageCont floatC">
-        <img src="https://static.igem.org/mediawiki/2018/e/ea/T--UCSC--Gibson-1.png" class="image-inpage vert large noBorder" width="60%">
-        <p><b>Figure 1</b>: This is where a caption for the figure would be written.</p>
-    </div>
-</div></div>
-</div>
-</div>
-<div class="flex-row">
-  <div class="flex-col hex-header center">
-    <p>We treated the samples with Dpn1, and we performed a Zymo Clean & Concentrate on the linearized plasmids. For the first 4 attempts, we were getting very low DNA ng/ul concentrations when we tested them on the NanoDrop. We asked McKenna and Dr. B for ways to fix this issue. We received advice to use hot MilliQ water (75ºC) as our elution buffer, and waiting 10 minutes for the DNA to elute before centrifuging. Unfortunately, these two pieces of advice contradict, and we still got bad results. After a few more tries and control tests, we found that using 75ºC Milli-Q and an elution time of 1 minute gave us much better results, seen in Table 1.</p>
-<table style="width:100%">
-  <tr>
-    <th colspan="4">NanoDrop Concentraitons of Cleaned-up Linearized pUC19 and pXRL2</th>
-  </tr>
-  <tr>
-    <th>Plasmid</th>
-    <th>Concentration (ng/uL)</th>
-    <th>260/280</th>
-    <th>260/230</th>
-  </tr>
-  <tr>
-    <th>pUC19</th>
-    <th>55.1</th>
-    <th>1.94</th>
-    <th>1.69</th>
-  </tr>
-  <tr>
-    <th>pXRL2</th>
-    <th>50.3</th>
-    <th>1.85</th>
-    <th>1.44</th>
-  </tr>
-</table>
-    <p>We will use the pUC19 plasmid to assemble our gene cassette, and loxP-URA3-lox71 into the plasmid for integration into the genome. The pXRL2 plasmid will be recircularized by site-directed mutagenesis, re-linearized in the proper location to be used for YMC in vivo assembly of gene cassette in <i>Y. lipolytica</i>.</p>
-    <h3>Lox-site genome integration</h3>
-    <p>We attempted to assemble the loxP-URA3-lox71 geneblock with homology arms from the <i>Y. lipolytica</i> genome using Gibson Assembly (GA) to create pOPPY-UC19-yXXU. We performed GA using 1:10 molar ratio of plasmid backbone to insert, and transformed NEB 5-alpha E. coli with the product, and grew them on high copy LB/AMP plates for 14 hours at 37ºC. The E. coli plates grew colonies, shown below.</p>
-<div class="flex-row">
-    <div class="flex-col">
-      <p> Image </p>
-      <img src=""  class="image-inpage large noBorder">
-    </div>
-    <p>Plate image</p>
-  </div>
-    <p>We picked 12 colonies, with which we performed colony PCR, and created an index plate. Our colony PCR gel results indicated that the colonies we chose were not homogenous; while some colonies showed light bands at the appropriate size for pOPPY-UC19-yXXU (~4 kb), they also contained other bands around 0.5kb which is the size expected if there is no insert, and just pUC19 backbone.</p>
-<div class="flex-row">
-    <div class="flex-col">
-      <p> Image </p>
-      <img src=""  class="image-inpage large noBorder">
-    </div>
-    <p>Gel image</p>
-  </div>
-    <p>The gel shows that we may have a mixed population in our colony picks. While there are light bands at 4kb, the bright bands at 0.5kb show that our insert was not included in the majority of the DNA we performed the PCR on. We created streak plates with the colonies on the index plate to attempt to screen out the mutants who had not successfully assembled our insert into the backbone. Unfortunately after PCR of those, the results returned the same information that our colonies did not include the insert. This is most likely due to the plasmid somehow re-circularizing to itself, or DPN1 not completely digesting the original pUC19 plasmid after isolation from E.coli. Because we were not able isolate pOPPY-UC19-yXXU, we chose a different route.</p>
--->
-    <p>The Riboswitch team has been using a plasmid called pD17, generously gifted from Cory Schwartz at UC Irvine, which contains homology arms for the pseudogene d17 in <i>Y. lipolytica</i>. We decided to try and insert loxP-URA3-lox71 into that plasmid. If successful, we would then amplify out to obtain a linear construct including the homology arms to d17, which we could perform homologous recombination into the <i>Y. lipolytica</i> genome with. We PCR amplified pD17, cutting out plasmid backbone between the homology arms to create a construct with homology arms at each end of the linear product. We ordered primers with Gibson Overhangs (GO) to the pD17 plasmid to amplify loxP-URA3-lox71 with. The PCR results were unsuccessful every try. Due to these issues, we decided to use KLD to insert the loxP-URA3-lox71 gene block into pD17. We performed KLD treatment with linearized pD17 backbone, and the loxP-URA3-lox71 gene block and transformed NEB 5-alpha E.coli. We saw many colonies, and we picked 15 for colony PCR to check if integration occurred.</p>
-<div class="flex-row">
-   <div class="flex-col">
-    <div class="imageCont" style="float:center">
-        <img src="https://static.igem.org/mediawiki/2018/e/ea/T--UCSC--Gibson-1.png" class="image-inpage vert large noBorder" width="60%">
-        <p><b>Figure 1</b>: This is where a caption for the figure would be written.</p>
-    </div>
-  </div>
-</div>
-    <p>The gel shows in lane 2 that we achieved a clean, single band at ~5kb. The primers bound to the pD17 plasmid at the beginning and end of the d17 homologous arms, including the arms and our loxP-URA3-lox71 insert. The expected size of our product is 3.6 kb. The band is slightly higher than this size, which we believe is due to the salts in the cell causing it to run a bit slower than it should. Because of this we are confident that this band is our desired product. We have grown a liquid culture of Colony 2, miniprepped it, and performed PCR using Q5 polymerase and the same primers used for colony PCR to attempt to amplify out loxP-URA3-lox71 sandwiched between the d17 homology arms. The results have not yet shown any bands from this.</p>
-    <p>Once we are able to amplify loxP-URA3-lox71 with d17 homology arms, we will transform <i>Y. lipolytica</i> with this linear product and confirm success by growing the colonies on uracil-deficient media. Once the loxP and lox71 sites are integrated into the genome we can perform Recombinase mediated cassette exchange with the plasmid created by YMC with all pathway genes using the corresponding lox sites.</p>
-<!--
-   <h3>P450scc Biobrick</h3>
-    <p>As a proof-of-concept for our project we decided to transform <i>Y. lipolytica</i> with one gene, P450scc. To be able detect P450scc we will add a HIS-tag to the end of the gene directly before the terminator, and use a His-HRP assay for detection. We chose this gene because the C-terminus of the final protein is exposed, and will not negatively affect the protein function. To be able to transform the yeast, and add the HIS-tag using site-directed mutagenesis, we needed the gene to be in a plasmid that could replicate in <i>Y. lipolytica</i> and <i>E.coli</i>. We have two plasmids that would work, and we first tried pXRL2. We tried to add Gibson Overhangs to P450scc using flagged primers, but the PCRs failed every time. To circumvent this, we decided to use KLD instead. We treated the linearized pXRL2 backbone and the P450scc gene with the KLD protocol, and transformed NEB 5-alpha E. coli, and grew the transformants on low copy LB/AMP plates. After five transformation tries we never got any colonies growing on the plates. Because of these issues, we decided to try using KLD to insert P450scc into pD17 instead, as it is also able to replicate in <i>Y. lipolytica</i> and E.coli. After one transformation, we saw many colonies, and picked 15 for colony PCR.</p>
-<div class="flex-row">
-    <div class="flex-col">
-      <p> Image </p>
-      <img src=""  class="image-inpage large noBorder">
-    </div>
-    <p>Gel image</p>
-  </div>
-    <p>The gel shows a clear band in lane 3. We used the same primers that bind on the edges of the d17 homology arms as we used for checking integration of loxP-URA3-lox71 into pD17. The expected size of P450scc is 4.3 kb, and the clear, defined band in lane 3 is ~6kb. We believe that the band ran slower than it should have due to the salts in the cell. We inoculated liquid cultures of Colony 3 and miniprepped the results. We attempted to perform site-directed mutagenesis on the miniprepped plasmids and the results showed no bands.</p>
-    <p>Once we achieve a successful mutagenesis plasmid that contains P450scc with a His-tag at the C-terminus, we will transform <i>Y. lipolytica</i> with the plasmid. The yeast will produce the protein, and we will perform an HisProbe-HRP assay using a nickel (Ni2+) activated derivative of horseradish peroxidase (HRP) from Thermo Fisher that is used for direct detection of recombinant poly-histidine-tagged fusion proteins.</p>
-    <h3>Progesterone Detection</h3>
--->
-  </div>
-</div>
-<!--       ********************************** End Gibson **************************************      -->
 <!--       ********************************** Riboswitch **************************************      -->
@@ Line 296: / Line 200: @@
      <p>For our part of the project, we intended to create a one-gene reporter system for progesterone concentration consisting of: a Renilla GFP gene (hrgfp), a cyc1 terminator, TEF(136) promoter, and a hammerhead ribozyme expression platform attached to a progesterone-specific aptamer.</p>
-     <p>Our first task in this project was to create a large stock of our pHR_D17_hrGFP plasmid (from here on referred to as D17), which we did by transforming a portion of our IDT D17 stock into chemicompetent E. coli, running a selection on an LB agar plate with a 100 ug/L concentration of ampicillin, and then growing out selected colonies in liquid LB medium. We then isolated our samples of D17 from the bacterial culture by using a Zymo plasmid miniprep kit.</p>
+     <p>Our first task in this project was to create a large stock of our pHR_D17_hrGFP plasmid (from here on referred to as D17), which we did by transforming a portion of our IDT D17 stock into chemicompetent <i>E. coli</i>, running a selection on an LB agar plate with a 100 ug/L concentration of ampicillin, and then growing out selected colonies in liquid LB medium. We then isolated our samples of D17 from the bacterial culture by using a Zymo plasmid miniprep kit.</p>
      <p>In order to confirm the sequence of our hrgfp gene as well as the cyc1 terminator, we also designed a series of primers meant to be used in Sanger sequencing of our D17 plasmid. These primers were labeled Rib(f)-1 through Rib(f)-3 and Rib(r)-1 through Rib(r)-3 and encompassed the entire hrgfp gene as well as the cyc1 terminator with several hundred base pairs of overhang on either side. Sequencing data from the UC Berkeley DNA sequencing facility showed a successful primer design and an intact DNA sequence in our D17 plasmid.</p>
@@ Line 337: / Line 241: @@
 <!--       ********************************** Quantification **************************************      -->
 <div class="flex-row">
@@ Line 342: / Line 249: @@
      <h2 id="Quantification">QUANTIFICATION</h2>
-    <p>We constructed the biosynthetic pathway for progesterone, consisting of 5 genes ( &#916;7 Red, P450scc, FDX1, ADR, 3&#914;-HSD). In addition we created a riboswitch that will allow us to measure the concentration of progesterone.</p>
+     <p>From the pOPPY-XRL2-yP plasmids that YMC had assembled, supposedly including all of the progesterone pathway genes, we chose six colonies to inoculate and test for progesterone production. We chose the colonies YL 10, YL 11, YL 20, YL 21, YL 22, and YL 25, shown in Figure X. We chose these both arbitrarily and due to the presence of apparent plasmid bands at the appropriate size.</p>
+<div class="imageCont floatC">
+        <img src="https://static.igem.org/mediawiki/2018/c/c5/T--UCSC--Quantification-plates.jpg" class="image-inpage vert large noBorder" width="60%">
+        <p><b>Figure 3</b>:  Colonies of Y. lipolytica transformed with progesterone pathway genes grown on leucine-deficient media. The pOPPY-XRL2-yP plasmid contains the gene completing the  leucine pathway, acting as our auxotrophic marker.
+</p>
+    </div>
+        <p>After purification of progesterone according to the Progesterone Extraction protocol, we performed “dot blotting” on nitrocellulose membranes, and detected using anti-progesterone antibodies conjugated to HRP (sc-53423 HRP), gifted from Santa Cruz Biotech (Santa Cruz Biotechnology, Inc., TX, USA).
+</p>
+<div class="imageCont floatC">
+        <img src="https://static.igem.org/mediawiki/2018/8/85/T--UCSC--Quantification-prog.jpg" class="image-inpage vert large noBorder" width="60%">
+        <p><b>Figure 3</b>:  Dot blot of progesterone re-suspended in DMSO, isolated from colonies grown on leucine-deficient media after YMC with progesterone pathway genes. Negative control circled in black and marked with a dash (-). Positive controls circled in blue. Experimental samples circled in pink. Primary antibody used was anti-progesterone-HRP, blocked with BSA in TBS, washed with TBS/TWEEN. Positive controls are 1mg/100uL progesterone in DMSO with 1:100 dilutions. Negative control is Y. lipolytica transformed with pD17 with no pathway genes which underwent same extraction protocol as all other samples.</p>
+    </div>
+<p>Our dot blot shows that we have high luminescence in multiple colonies, YL 10, YL 20, YL 21, and YL 25. We performed the extraction protocol on +1 positive control, and our negative control. The +1 positive is 1mg of pure progesterone extract from Sigma Aldrich (P8783 Sigma) that we added 1mL of MilliQ water and 1mL of hexane to, and then followed the rest of the extraction protocol for. The positive controls +3, +4 and +5 are 1:10 dilutions from +2 positive control of 1mg/1mL of pure progesterone in DMSO. These positives were not subjected to the extraction protocol. We believe that they may show no luminescence due to +2 being one order of magnitude less than detection that is possible for the antibodies. The negative control is pD17 transformed into Y. lipolytica with no pathway genes, and then subjected to the same extraction protocol.</p>
+<p>We believe that these results suggest that we have at least four progesterone producing Y. lipolytica colonies. The positive control shows the level of luminescence for 1mg of progesterone, and the colonies that show luminescence as well are of similar magnitude. We inoculated 5 mL cultures with our colonies, so we find it a bit suggestive that we have similar levels of luminescence in our 1 mg positive control as we do in our colonies.</p>
+<p>Due to the ability of our hexane extraction protocol to apparently purify our samples so greatly, we will perform mass spectrometry on these samples in later work.</p>
    </div>
@@ Line 355: / Line 285: @@
      <h2 id="ProjAchiev">PROJECT ACHIEVEMENTS</h2>
-           <button class="buttonnotebook2" onclick="openInfoBlock('DPP', this, '#e91b63')" id="defaultOpen">Designing PCR Primers</button>
+           <button class="buttonnotebook2" onclick="openInfoBlock('Success', this, '#e91b63')" id="defaultOpen">Successes</button>
-           <button class="buttonnotebook2" onclick="openInfoBlock('ECM', this, '#e91b63')"><i>E. Coli</i> Miniprep</button>
+           <button class="buttonnotebook2" onclick="openInfoBlock('Fail', this, '#e91b63')">Failures</button>
          <br> <br> <br>
-<!----------------------------------------Designing PCR Primers---------------------------------------------------->
+<!----------------------------------------Success---------------------------------------------------->
-         <div id="DPP" class="buttoncontent">
+         <div id="Success" class="buttoncontent">
              <br>
-             <h3> Designing PCR Primers </h3>
+             <h3> Successful Results </h3>
-             <p>Our teaching assistant, McKenna Hicks, taught us the following methods for primer design. </p>
+             <p>The following tasks were successfully completed by our team.</p>
-             <ol>
+             <ul>
-            <li>Identify the DNA sequence to amplify or stitch together with overlap extension PCR (OE-PCR). During PCR, primers anneal to their complementary sequence and recruit DNA polymerase to extend the sequence in the 5' to 3' Direction.</li>
+             <li>LoxP removal from pXRL2</li>
-            <li>Design a forward (5'- 3') and a reverse primer (3'- 5') to amplify both the top and bottom DNA strands. The ideal primer length is between 18 and 23 bp, but this can be changed to accommodate other constraints.</li>
+             <li>Lox-URA3-Lox in D17</li>
-            <li>For the forward primer, select a sequence of base pairs (bp) of optimal length.</li>
+             <li>Amplification of HAs</li>
-            <li>Repeat Step 3 for the reverse primer and generate the reverse complement of the reverse primer sequence.</li>
+             <li>Gibson Assembly of pOPPY_GFP plasmids</li>
-            <li>Analyze each sequence using a computational primer tool (such as IDT's Oligo Analyzer) and record the GC content and the melting temperature (Tm) for each primer.</li>
+             <p>Once we received a set of improved primers, our D17 plasmid had overhangs that allowed for more efficient Gibson Assembly. We confirmed this by choosing 50 colonies from a series of transformations with our pOPPY_GFP plasmids for colony PCR. Running these individual PCRs on a gel allowed allowed us to select colonies that showed bands consistent with an insert of the correct size in the same location as our riboswitch insert.</p>
-            <li>The optimal GC content is between 40-60%, and the optimal Tm range is between 52-62℃. Ideally, the difference in Tm for the forward and reverse primer pair should not exceed 5℃.</li>
+             <li>Amplification of &#916;7, P450scc, ADR, and 3&#914;H genes </li>
-            <li>If the GC content exceeds 60% for either primer, add High GC Enhancer to the PCR reaction mix.</li>
+             <li>Successful Yeast-Mediated Cloning in <i>Y. lipolitica</i></li>
-            <li>Check that the free energy (ΔG) value for self-dimers and hetero-dimers on each primer is above -10 kcal/mol to avoid primers spontaneously self-annealing or annealing to each other.</li>
+             <li>Made Progesterone</li>
-            <li>Check that hairpin formations do not exceed 30℃ in each primer. PCR uses different temperatures for each thermocycler step; the lowest temperature used during the replication step for primer annealing is 58-60℃. If hairpins form around this temperature, the primer binding efficiency may be reduced and yield less product. However, if the primer Tm is above the hairpin formation temperature, and if the potential hairpin leaves the 3' end of the primer free for annealing, the primer should still anneal properly without reducing PCR Efficiency.</li>
-           </ol>
+            </ul>
          </div>
-<!----------------------------------------E. Coli Miniprep---------------------------------------------------->
+<!---------------------------------------- Failures---------------------------------------------------->
-         <div id="ECM" class="buttoncontent">
+         <div id="Fail" class="buttoncontent">
              <br>
-             <h3> <i>E. Coli</i> Miniprep </h3>
+             <h3> Unsuccessful Results </h3>
-             <p>The following protocol was adapted from the ZyppyTM Plasmid Miniprep Kit procedure from Zymo Research.</p>
+             <p>The following bullets include experiments and tasks that were not successful during the wetlab period. </p>
-              <ol>
+              <ul>
-              <li>First prepare your cell samples: centrifuge the cell cultures (which should be 5 mL in conical tubes) for 15 minutes at 3,750 rpm and 30℃.</li>
+              <li>Overlap Extension PCR</li>
-              <li>During waiting time, grab a 250 mL beaker and add roughly 50 mL of 10% bleach. Grab some foil, label waste, and cover the top of the beaker. Use this beaker to discard biohazardous supernatants and flow-throughs throughout your miniprep.</li>
+              <p>After many touchdown PCR trials, we were unable to assemble the full genes without seeing non-specific sequences in our PCR reactions. We tried using PCR without primers to anneal the overlapping regions of the gene fragments to create better DNA templates, but there were always non-specific bands alongside the full-length genes. Even after extracting the correct sized bands from agarose gels and using that DNA as a template resulted in the same non-specific banding patterns.</p>
-            <li>Collect Zyppy columns, Zyppy collection tubes, and 2 sets of 1.5 mL microcentrifuge tubes to match the number of samples you centrifuged. Place the columns over the collection tubes. Be sure to label all vessels accordingly. Place a kimwipe over the open columns. </li>
+             <li>Attaching Homologous Arms to Lox-URA3-Lox</li>
-            <li>After centrifugation, discard the supernatants into your waste beaker. Be sure to replace the foil lid afterwards.</li>
+             <li>Gibson Assembly of Genes into pXRL2</li>
-            <li>Add 600 uL Milli-Q water into each of the conical tubes containing the cell pellets. Resuspend each by slowly pipetting up and down; use that same pipette to transfer the resuspended mixture to its respective clean microcentrifuge tube. Change tips between samples.</li>
+             <li>P450 into PoPPY and D17 Plasmid</li>
-            <li>Add 100 uL of 7X Lysis Buffer (blue) and mix by inverting the tube 4-6 times. Proceed to next step within 2 minutes. After addition of 7X Lysis Buffer, the solution should change from opaque to clear blue, indicating complete lysis. </li>
+             <li>Lox-URA3-Lox into PoPPY Plasmid</li>
-            <li>Add 350 uL of cold Neutralization Buffer and mix thoroughly. The sample will turn yellow when the neutralization is complete and a yellowish precipitate will form. Invert the sample an additional 2-3 times to ensure complete neutralization.</li>
+             <li>Amplification of FDX1 gene</li>
-            <li>Centrifuge at 13,000 rpm for 3 minutes.</li>
+             <p>Ferrodoxin (FDX1) was one of the five genes in the progesterone pathway that we tried to amplify for Gibson Assembly and Yeast Mediated Cloning. After many touchdown PCR trials, we found a few thermocycler conditions that seemed to properly amplify FDX1. When we tried creating larger PCR reaction volumes, we saw bands that were much larger than the expected gene size for FDX1, and we couldn’t get the same amplification like in earlier PCR reactions.</p>
-            <li>Transfer the supernatant into the provided Zymo-SpinTM IIN column, which should be on top of a collection tube. Avoid disturbing the cell debris pellet.</li>
+             <li>Isolating pOPPY_GFP plasmids from D17 backbone</li>
-            <li>Centrifuge the column + collection tube for 15 seconds at 13,000 rpm (stop the centrifuge when it reaches 15 sec). </li>
+             <p>We had repeatedly tried to separate our pOPPY_GFP plasmids from their original D17 backbone upon completion of Gibson Assembly using both DpnI digests as well as gel electrophoresis. The small length difference between the plasmids (~200bp) did not allow for enough separation to perform a gel purification, and PCR repeatedly created amplicons indicative of both pOPPY_GFP plasmids as well as D17 backbone.</p>
-            <li>Discard the flow-through and place the column back into the same Collection Tube.</li>
+             </ul>
-            <li>Add 200 uL of Endo-Wash Buffer to the column. Centrifuge for 30 seconds at 13,000 rpm. Discard the flow-through.</li>
-            <li>Add 400 uL of ZyppyTM Wash Buffer to the column. Centrifuge for 1 minute at 13,000 rpm. Discard flow-through. Be sure to close cap tightly on wash buffer so that ethanol does not evaporate. </li>
-           <li>Transfer the column into a clean 1.5 mL microcentrifuge tube then add 50 uL of Milli-Q water in place of Elution Buffer (EB) and add it as close to the filter as possible, being careful not to touch it (use Milli-Q instead of EB to prevent contamination from salts and EDTA). Let stand for at least 10 minutes at room temperature. Incubate longer if needed. </li>
-           <li>Centrifuge for 30 seconds at 13,000 rpm to elute the plasmid DNA. </li>
-           </ol>
        </div>
    </div>
 </div>
 <!--       ********************************** End Project Achievements **************************************      -->
 </div>
 </body>
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Difference between revisions of "Team:UCSC/Results"

Latest revision as of 03:48, 18 October 2018

Results

Results

OVERVIEW

LIPLOX

Homology Arms

Enter lox sites and ura3 into Y. lipolytica str. FKP393 genome

YEAST MEDIATED CLONING

RIBOSWITCH

QUANTIFICATION

PROJECT ACHIEVEMENTS

Successful Results

Unsuccessful Results