Cloning of pUC19 Vector

Aim

• To procure higher reserves of the pUC19 vector that would be subsequently used for NEBuilder® HiFi DNA assembly.

Experiment

• We used the NEB® 5-alpha competent E.coli cells for cloning the pUC19 vector via chemical transformation, did plasmid miniprep to get the vector at a higher concentration, and then did gel electrophoresis with 0.8% agarose gel to verify the results of plasmid miniprep.

Result

• We were successful in cloning the empty pUC19 vector.


Fig 1: Gel electrophoresis of cloned empty pUC19 (2686 bp)

PCR Amplification of Gene Fragments

Aim

• To PCR amplify the 4 gene fragments (JcFATB, CvFAP fragment 1, CvFAP fragment 2, and JcFATA) so that we can proceed with NEBuilder® HiFi DNA assembly of them with the pUC19 vector.

Experiment

• We did gradient PCR with the respective forward and reverse primer for each fragment to figure out the optimal annealing temperature for amplification. Then we used the optimal annealing temperature of the respective fragments to PCR amplify them. For the JcFATB fragment, we encountered non-specific binding, which we managed to eliminate by using DMSO to reduce the annealing temperature for optimal binding of primers.

Result

• 62.1 degrees Celsius was an optimal annealing temperature for CvFAP fragments 1 and 2.


Fig 2(a): Lane 1: 1kb DNA ladder, Lane 2 to 6: Gradient PCR for CvFAP fragment 1 (62.1, 65.1, 66.4, 68.9, 71.9 degrees Celsius), Lane 7 to 11: Gradient PCR for CvFAP fragment 2 (62.1, 65.1, 66.4, 68.9, 71.9 degrees Celsius) [62.1 was an optimal annealing temperature for CvFAP fragment 1 and 2]

• Non-specific binding for JcFATB was observed, 65.8 degrees Celsius was the optimal annealing temperature for JcFATA, and PCR amplification of CvFAP fragments 1 and 2 at 62.1 degrees Celsius.


Fig 2(b): Lane 1,2: 1kb DNA ladder, Lane 3-6: gradient PCR product of JcFatB from 62 to 68 degrees Celsius, Lane 7-10: gradient PCR product of JcFatA from 62 to 68 degrees Celsius, Lane 12,13: PCR amplified CvFAP fragment 1 and 2 respectively at 62.1 degrees Celsius, Lane 14,15: gradient PCR product of JcFatB and JcFatA respectively at 71.3 degrees Celsius.

• Successful PCR amplification of JcFATB at 62.1 degrees Celsius with 1% DMSO eliminated the non-specific binding of the primer.


Fig 2(c): Lane 1: 1kb DNA ladder, Lane 2, 3, 4: 0%, 1%, and 2% DMSO JcFatB PCR amplification products at 62.1 degrees Celsius

NEBuilder® HiFi DNA assembly of pUC19-JC

Aim

• To assemble JcFATB, CvFAP fragments 1 and 2, and JcFATA in the pUC19 vector via NEBuilder® HiFi DNA assembly.

Experiment

• We double-digested the pUC19 vector with SalI-HF and BamHI and added it along with the 4 fragments in the NEBuilder® HiFi DNA assembly reaction as per NEBuilder®HiFi DNA Assembly Cloning Kit protocol. We used 2 µl of the Gibson assembly mixture for the transformation of DH5α.
• We did blue-white screening to select white colonies and did a plasmid miniprep to check if they had the recombinant pUC19-JC plasmid.
• Once we got the desired 6kb band for pUC19-JC, we also did a double-digestion of pUC19-JC to see the release of our 6kb NEBuilder® HiFi DNA assembled construct.

Result

• Successful transformation of NEBuilder® HiFi DNA assembly mixture into DH5α.


Fig 3(a): Blue-white screening of DH5α for pUC19-JC

• Plasmid miniprep of white colonies to procure recombinant pUC19-JC plasmid.


Fig 3(b): Lane 1: 1kb DNA ladder, Lane 2,3,4: Plasmid miniprep product from 3 of the white colonies from the blue-white screening plate. [Lane 2 shows an 8.6kb intense band implying the white colony which has our pUC19-JC recombinant plasmid]

• Successful double digestion of pUC19-JC to see the release of 6kb insert.


Fig 3(c): Lane 1: 1kb DNA ladder, Lane 2: Double digestion of pUC19-JC with SalI-HF and BamHI to verify the release of the insert from the recombinant plasmid (preliminary check whether pUC19 vector and accepted our NEBuilder® HiFi DNA assembly construct), Lane 3: PCR amplification of JcFATB with construct forward primer and JcFATB reverse primer

PCR amplification of Construct from pUC19-JC

Aim

• To get the 6kb NEBuilder® HiFi DNA assembled construct from the recombinant pUC19-JC plasmid through construct forward and reverse primers via PCR amplification.

Experiment

• We followed the Q5®High-Fidelity 2X Master Mix protocol to get the amplification of the 6kb construct without the SalI-HF site.
• We did an optimization of the PCR parameters to minimize the non-specific binding.

Result

• Successful PCR amplification of 6kb construct from pUC19-JC.


Fig 4(a): Lane 1: 1kb DNA ladder, Lane 2: optimized PCR reaction product (6kb construct successfully PCR amplified from pUC19-JC plasmid with minimal intensity of a 3.5kb contamination band)

Subcloning into pYLEX vector

Aim

• To insert the NEBuilder® HiFi DNA assembled construct of JcFATB, CvFAP fragments, and JcFATA into the pYLEX vector.

Experiment

• We did restriction-digestion-ligation to get the 6kb construct into pYLEX, by double-digesting the pYLEX vector with PmlI and BamHI-HF and single digesting the construct with BamHI-HF, however, all the 3 attempts for it failed, the reasons for which are elaborated in the engineering success page.
• We achieved successful sub-cloning of the construct into pYLEX by introducing overhangs in the construct and performing a second NEBuilder® HiFi DNA assembly reaction with double-digested pYLEX.
• After transforming DH5α with 2 µl of NEBuilder® HiFi DNA assembly mixture, we did a plasmid miniprep of the 4 colonies obtained on the LA-ampicillin plate.
• After getting the desired 13kb band for pYLEX-JC recombinant plasmid, we did double digestion of pYLEX-JC and empty pYLEX plasmid with SalI-HF and NotI-HF to see the shift between the recombinant and empty plasmid.

Result

• Successful NEBuilder® HiFi DNA assembly of 6kb construct and pYLEX vector to give the 13kb pYLEX-JC recombinant plasmid.


Fig 5(a): Lane 1: 1kb DNA ladder, Lane 2: Control for alkaline lysis, Lane 3 to 6: alkaline lysis products from 1:3 plate, Lane 8: Control for NEBuilder® HiFi DNA Assembly Miniprep products (Single digested pYLEX (7kb) vector with NotI-HF), Lane 9 to 12: Single digested (with NotI-HF) NEBuilder® Hi-Fi DNA Assembly miniprep products (13kb) of the 4 colonies

• Double-digestion of empty pYLEX and pYLEX-JC to see the shift between empty and recombinant plasmid.


Fig 5(b): Lane 1: Double-digested NEBuilder® HiFi DNA Assembly product 1, Lane 2: Control (double-digested empty pYLEX vector), Lane 3: Double-digested NEBuilder® HiFi DNA Assembly product 2, Lane 4: Double-digested NEBuilder® HiFi DNA Assembly product 3, Lane 5: 1kb DNA ladder

Transformation of Y.lipolytica with pYLEX-JC plasmid

Aim

• To achieve successful genomic integration of pYLEX-JC in Y.lipolytica via homologous recombination.

Experiment

• We linearized pYLEX-JC with NotI-HF, procured from two of the transformed DH5α colonies (labeled as G1 and G3) obtained after NEBuilder® HiFi DNA assembly reaction of construct and pYLEX.
• We then followed the YLEX kit protocol from Yeastern Biotech, Taipei, Taiwan, for transformation of Y.lipolytica with our recombinant pYLEX-JC linearized plasmid.
• We used the YNB medium, which uses leucine auxotrophy as a selection pressure for the growth of the Y.lipolytica transformants.

Result

• Successful transformation of control (empty pYLEX) and recombinant plasmid pYLEX-JC was achieved in Y.lipolytica.


Fig 6(a): Colonies of Y.lipolytica transformed with empty pYLEX (control) and the other two plates (G1 and G3) with pYLEX-JC (recombinant plasmid)

Thin-layer Chromatography

Aim

• To qualitatively check the increase in the free fatty acid composition of Po1g transformed with pYLEX-JC in comparison to Po1g transformed with empty pYLEX due to the expression of Jatropha’s thioesterases (JcFATA and JcFATB).

Experiment

• The colonies of Po1g transformed with pYLEX-JC (G1 and G3) and Po1g transformed with empty pYLEX were incubated in YPD broth for around 3 days to activate the expression of the hp4d promoter(expresses in stationary phase of culture).
• We normalized the OD⁶⁰⁰ values, made cell pellets of the samples, and extracted the FFA content using the chloroform-methanol-based method of extraction (Folch extraction).
• The samples after drying were resuspended in 100 µl of chloroform and then spotted on the silica TLC plate. We also spotted the C16:0 (palmitic) and C18:0 (stearic) fatty acid standards along with these samples to get to know if we have the desired fatty acids or not.
• The mobile phase used was 60% ethyl acetate in hexane.
• The TLC plate was then stained with PMA (Phosphomolybdic acid) and slightly heated over a flame to visualize the spots.

Result

• We cannot distinguish between the C16 and C18 fatty acid composition of the samples. The intensities of all the spots are quite similar but the FFA content in G1 was slightly higher as compared to P.
• TLC is unable to show the difference between C18:0, C18:1, and C18:2 FFA composition in the sample, so we had to proceed with LC-HRMS.
• From the TLC we can infer that we have the C16 and C18 FFA in our samples.


Fig 7(a): C16 -palmitic acid (C16:0) standard, C18 - stearic acid (C18:0) standard, P - Po1g with empty pYLEX (control), M - mix spot of P and G3, G3 - Po1g with pYLEX-JC insert (colony 3), and G1 - Po1g with pYLEX-JC insert (colony 1)

Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS)

Aim

• We aim to visualise the fatty acid compositions of 16:0, 18:0, 18:1 and 18:2 fatty acids for our empty plasmid as well as construct-containing pYLEX-JC. A significant change in composition resembling closer to Jatropha oil composition will give evidence for successful transformation.

Experiments

• We sent 5 samples to NCL-CSIR for LC-HRMS analysis:
  • Po1g transformed with empty pYLEX (A), Po1g transformed with G1 (A), Po1g transformed with G3 (A) - all normalized to OD=12.
  • Po1g transformed with empty pYLEX(B), FAA1(B) - normalized to OD=5.
• The FFAs were extracted from the cells using the same chloroform-methanol extraction and the sample was completely dried using a centrivap. The vials were then parafilmed and sent to the CSIR-National Chemical Laboratory’s(NCL) LC-HRMS facility for analysis. There they were resuspended in a 2:1 ratio in 200µl acetonitrile: methanol and 10µl of the sample was injected into the LC-HRMS equipment. A standard of known quantity of 15:0 fatty acid was used for quantification.
• Upon receiving the result, we isolated the peaks corresponding to our fatty acids using their molecular weights and formulae. We got the expected peaks for 16:0, 18:1 and 18:2 fatty acids, but did not get any peak for 18:0.


Fig 8(a): Representative peak of our LC-MS result for pYLEX-A at retention time approximately 16 minutes

• Upon receiving the raw data, we analysed it by extracting the areas under our relevant peaks, subtracting the area(if any) under blank, and then finding the total area(the given area values were only for 10 uL injection whereas the total resuspension in solvent mix was 200 uL) and then normalising the area with respect to the area under the standard. This gave us the amount of each of our components present in nanomoles. We normalised it further to find it for unit OD and per mL of culture(Note: Here OD value has been used as a proxy for cell number). This was then plotted for each fatty acid peak for each of our samples and we were in for a pleasant surprise.


Fig 8(b): This showcases the normalised amount of fatty acids against our required FAs

• Here we see that upon adding our thioesterases to the system, the composition of the fatty acids had an overall increase in fatty acid content in the organism. We hypothesise that this may be simply due to the addition of more thioesterases to the system. The native FAS complex of Y. lipolytica already makes fatty acids, but if there is excess of the Acyl-ACP/Acyl-CoA substrate leftover then these can be utilised by the transgenically expressed Jatropha curcas thioesterases to produce more fatty acids, thus increasing the overall content.
• One confounding result was that we did not get any peaks for 18:0(stearic) fatty acid in our LC-MS graph. We hypothesise that this happened because of the natural desaturases of the yeast which convert 18:0 fatty acid to 18:1 and 18:2 by dehydrogenation. So even if the Jatropha curcas thioesterases had produced 18:0 fatty acid, it may have been converted to the unsaturated 18:1 and 18:2. This could also help partly explain why there is a significantly higher yield of 18:1 and 18:2 fatty acids as compared to 16:0, which is not significant.
• To check how the composition(percentages) of our fatty acids changed, we plotted each fraction with respect to the total fatty acid composition we analysed. So for example, to compare 16:0, we divided 16:0 normalised area by (16:0 + 18:1 + 18:2) normalised area to get the percentage of our analyte mixture that was 16:0(palmitic) fatty acid. We did this for each sample and for each fatty acid. We also calculated the same values for the Jatropha oil fatty acid composition from literature(since the values varied among sources, we took acceptable ranges) and made the comparisons to obtain the following graph.


Fig 8(c): This showcases the change in total amount of fatty acids

• From this graph we can see that although we did not replicate the exact composition of the fatty acids in jatropha oil-based SAFs, we got intermediate values for the percentages of 18:1(oleic acid) and 18:2(linoleic acid) between our empty control and Jatropha oil-based SAFs. This is expected as we will not get the exact compositions of the Jatropha curcas plant’s fatty acids upon expressing it heterologously in a yeast. So we have got compositions which seem to be a combination between the two. We can’t see the same changes in 16:0 and we think this could be due to the already low abundance, so the differences may not be significant with respect to experimental error. Thus it is best not to make any comments about that data.
• We had also attempted performing deletions of genes of the native organism involved in fatty acid and degrading processes like FAA1 responsible for beta-oxidation and Alk1 for alkane metabolism to fatty alcohols. Upon talking to scientists we had learned that it usually takes years to perform such deletions, so we weren’t very hopeful about the success of this part of the project. However, we succeeded in the selection for the deletion strains one day before our LC-MS and thus were able to include this along with a separate control normalized at OD = 5 for comparing to see if our overall fatty acid production had increased in this strain. We were thrilled to see such strong results for this.


Fig 8(d): Increased fatty acid content upto three-fold for 18:1 and 18:2 fatty acids

• We can see a clear and significant increase in our total fatty acid content, especially 18:1 and 18:2, with 18:1 being greater than 3-fold! This clearly indicates that our deletion was a success and our new deletion strain produces much more fatty acid content and will be very helpful in taking our project to a commercial level.

Results

• We were successful in the transformation of our chassis Y. lipolytica with the thioesterases JcFATA and JcFATB from the plant Jatropha curcas as is clearly seen by the significantly altered fatty acid profiles of the transformed organism. We also succeeded in performing the deletion of FAA1 in our chassis to make it produce an overall larger amount of fatty acids and thus fuel.

Western Blot

Aim

• To check the expression of JcFatB (48.6 kDa), CvFAP (62.9 kDa), and JcFatA (42.9 kDa) using FLAG and 6X His tags.

Experiment

• We prepared the cell lysate for normalized cell pellets of Po1g transformed with empty pYLEX and pYLEX-JC (G1 and G3).
• We did protein estimation of the cell lysate using nanodrop so that we could load an equal amount of protein for each sample. Due to the use of SDS and other detergents in lysis buffer, it was difficult to proceed with Bradford or BCA assay for protein estimation.
• Western blot was performed as per the protocol stated in the Experiments and Journals Page, for JcFatB which was tagged with a FLAG tag, and CvFAP and JcFatA which were tagged with 6X-His tag.

Results

• [1] Checking the presence of JcFatB protein (48.6kDa) in G1 and G3 using FLAG tag:
  • Western blot attempt 1:
  

  Fig 9(a): Lane 1: Ladder, Lane 2: Control (Po1g transformed with empty pYLEX), Lane 3: G1 (Po1g transformed with pYLEX-JC), and Lane 4: G3 (Po1g transformed with pYLEX-JC)

  • Western blot attempt 2:
  

  Fig 9(b): Lane 1: Ladder, Lane 2: Control (Po1g transformed with empty pYLEX), Lane 3: G1 (Po1g transformed with pYLEX-JC), and Lane 4: G3 (Po1g transformed with pYLEX-JC)

• In both the western blot attempts, we detected the band at 48.6kDa in G1 and G3 with the FLAG tag, but the control (Po1g transformed with empty pYLEX) also showed the same band. We observed the presence of non-specific bands, apart from the desired 48.6kDa band.
• Probable reasons for the above observations:
  • There’s a possibility of contamination in the control sample (Po1g transformed with empty pYLEX) due to pippeting error or due to spillover from the wells of the gel from the experimental samples G1 or G3 (Po1g transformed with pYLEX-JC)
  • The pYLEX vector may have sequences that may be interfering with the JcFatB expression in the background, giving the non-specific bands and the 48.6kDa band in the control.
• Possible solutions and troubleshooting:
  • We need to minimize the chances of contamination in the control sample.
  • We can check the protein profile after FLAG tag incubation for untransformed Po1g to check if the Y.lipolytica expresses proteins of similar sizes natively and get a confirmation of whether the bands are appearing due to pYLEX vector background expression.
• [2] Checking the presence of CvFAP (62.9 kDa) and JcFatA (42.9 kDa) in G1 and G3 using 6X His tag:
  • Western blot attempt 1:
  

  Fig 9(c): Lane 1: Ladder, Lane 2: Control (Po1g transformed with empty pYLEX), Lane 3: G1 (Po1g transformed with pYLEX-JC), and Lane 4: G3 (Po1g transformed with pYLEX-JC)

  • Western blot attempt 2:
  

  Fig 9(d): Lane 1: Ladder, Lane 2: Control (Po1g transformed with empty pYLEX), Lane 3: G1 (Po1g transformed with pYLEX-JC), and Lane 4: G3 (Po1g transformed with pYLEX-JC)

• In both the western attempts, we detected the band at 62.9kDa for CvFAP in G1 and G3 with the 6X His tag, but the control (Po1g transformed with empty pYLEX) also showed the same band. We also observed the presence of non-specific bands, apart from the desired 62.9kDa band.
• However, we didn’t observe any band for JcFatA at 42.9kDa in G1 and G3. Probable reasons for the results:
  • There’s a possibility of contamination in the control sample (Po1g transformed with empty pYLEX) due to pippeting error or due to spillover from the wells of the gel from the experimental samples G1 or G3 (Po1g transformed with pYLEX-JC).
  • The pYLEX vector may have sequences that may be interfering with the CvFAP expression in the background, giving the non-specific bands and the 62.9kDa band in the control.
  • Generally, 6X His tag is known to have non-specific bands as most of the proteins have the basic amino acid residues.
  • For JcFatA expression, it may be a binding error with 6X His antibody due to its low specificity as we didn’t get the 42.9kDa band for it in both the western blots.

Possible Solutions and Troubleshooting

• We need to minimize the chances of contamination in the control sample.
• We can check the protein profile after 6X His tag incubation for untransformed Po1g to check if the Y.lipolytica expresses proteins of similar sizes natively and get a confirmation of whether the bands are appearing due to pYLEX vector background expression.

Deletions

Aim

• To extract linear ScRAD52 part1 and part2 from plasmids via PCR.

Experiment

• ScRAD52 homolgous recombination cassettes were ordered from IDT inside two separate plasmids . We used PCR to get both the parts as linear gene fragment.


Fig 10(a): 1-Ladder, 7-10- ScRAD part 1

• ScRAD part 2 (3Kb) showed amplification in gradient PCR, it was further then amplified with a higher reaction volume.


Fig 10(b): Lane 1-ladder, Lane 8-12 - ScRAD52 part 2

• We successfully PCR extracted both the parts and subjected the product to further PCR purification.

Aim

• To amplify the ALK2 and FAA1 gblocks via PCR

Experiment

• We received the ALK2 and FAA1 deletion cassettes as gblocks from IDT. We used PCR amplified all the fragments.

Result

Fig 11(a): Lane-1: Ladder,Lane 2-3: 1%DMSO ALK2(3Kb) and 2%DMSO ALK2, Lane 4-7: FAA1(3Kb)

• We successfully amplified both the fragments and subjected FAA1 to gel extraction and ALK2 to PCR purification.

Aim

• To join both the ScRAD52 part1 and part2 via gibson assembly

Experiment

• We decided to join both the fragmmets through gibson assembly, the gibson assembly protocol can be found on the protocol page. To check the success of the assembly we decided to do a PCR of the reaction mixture with the final construct primers and check via agarose gel.

Result

Fig 12(a): Lane-1: Ladder,Lane 2-3: 1%DMSO ALK2(3Kb) and 2%DMSO ALK2, Lane 4-7: FAA1(3Kb)

• We concluded that the whole ScRAD52 construct (6kb) has indeed been properly been assembled. We concluded this as we got a band at 6kb in the following gel below. It is also to be noted that gibson products cannot normally be visualized with agarose gel due to the low conconcentration of DNA added to the gibson reaction mixture.So since we get a band here the amplification indeed has happened.

Aim

• To transform the ScRAD52 into PO1g strain.

Experiment

• The assembled ScRAD52 strain was transformed in the Po1g strain by the YLOS transformation protocol and mycophenolic acid was as a selection marker.

Result

Fig 13(a): The ScRAD52 transformation plate.We got colonies on the transformed plate. Mycophenolic acid was used as selectable marker.

• We concluded that the whole ScRAD52 construct (6kb) has indeed been properly been assembled. We concluded this as we got a band at 6kb in the following gel below. It is also to be noted that gibson products cannot normally be visualized with agarose gel due to the low conconcentration of DNA added to the gibson reaction mixture.So since we get a band here the amplification indeed has happened.

Aim

• To delete the FAA1 gene in Po1g strain.

Experiment

• We transformed the PO1g strain with the FAA1 deletion cassette via YLOS protocol and we expect homolgous recombination to take place and replace the FAA1 gene with the hygromycin resistance gene.

Result

Fig 14(a): FAA1 transformation plates (with 200ug/ml hygromycin concentration) ,the upper plate is control plate which has no colonies as expected; the lower plate is the transformed plate with FAA1 colonies.

• The transformation was successful, we got distinct colonies on the mycophenolic acid plate which was expected instead of the normal dense growth when streaked.
• The LCMS result of FAA1 deleted strain showed a huge increase in the following fatty acids compared to the normal Po1g strain due to increased accumulation of fatty acid as convertion into Acyl CoA is inhibited, indicating that the deletion was indeed successful. The PoC page is linked here.