OVERVIEW

We attempted to construct a biosynthetic pathway consisting of 5 enzymes ( Δ7 Red, P450scc, FDX1, ADR, 3Β-HSD). In addition we are creating a riboswitch that will allow us to measure the concentration of Progesterone.

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.

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.

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.

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.

GIBSON

pUC19 and PXRL2 Linearization

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.

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pUC19 and pXRL2 Linearization Gel]{1\% agarose gel, NEB 2-Log DNA ladder in kb. Lane 1 contains linearized pUC19, Lane 2 contains linearized pXRL2.

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.

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 Y. lipolytica.

Lox-site genome integration

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.

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Plate image

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.

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Gel image

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.

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 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 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.

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3 Gel image

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.

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.

P450scc Biobrick

As a proof-of-concept for our project we decided to transform Y. lipolytica 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 Y. lipolytica and E.coli. 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 Y. lipolytica and E.coli. After one transformation, we saw many colonies, and picked 15 for colony PCR.

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Gel image

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.

Once we achieve a successful mutagenesis plasmid that contains P450scc with a His-tag at the C-terminus, we will transform Y. lipolytica 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.

Progesterone Detection

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.

We then had to linearize D17 in order to have a suitable linear vector for Gibson assembly (GA). We linearized our plasmid between the downstream end of the hrgfp open reading frame and the upstream end of our cyc1 terminator using the Ribo1(f) and Ribo1(r) primers ordered from IDT, which we also designed. We later substituted our Ribo1(r) primer with another primer, labeled as RiLin(r), in order to create better overhangs for GA. We also performed a DpnI digest on the PCR product in an attempt to remove any circular D17 before performing GA. Gel imaging of our PCR product showed a strong band near the 9kb band in the 2-log DNA ladder, suggesting a successful linearization of our plasmid at the correct length.

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Gel Image

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 showed strong bands at the expected bp length, suggesting successful amplification of our inserts.

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Gel Image

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 suggested the continued presence of D17 circular template along with our new pOPPY_GFP plasmids, even after digestion with >500-fold excess of DpnI.

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Gel Image

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 from the colony PCR trials allowed us to select single colonies we believed to be carrying our riboswitch insert.

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Gel Image

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Gel Image

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Gel Image

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.