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.

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