Weekly Summary

Amplified the empty pUC19 vector by transforming it into E.coli DH5α cells. We succeeded in our 2nd attempt and made primary inoculums of the colonies that we got followed by a plasmid miniprep and a confirmatory agarose gel. We received the fragments that we had ordered from Twist Bioscience (JcFATA, JcFATB and CvFAP) and resuspended them.

Weekly Summary

Resuspended our primers, did a gradient PCR followed by an agarose gel to find out the best annealing temperature for the primers to bind to our gene fragments. We faced a problem with PCR amplification of the JcFATB fragment as we were getting a non-specific binding of the primers. We tried it a couple of times and this time added DMSO to reduce the non-specific binding.

Weekly Summary

Linearization and purification of amplified pUC19, performed an NEBuilder® HiFi DNA Assembly of JcFATB, CvFAP part1, CvFAP part2 and JcFATA alongwith the linearized pUC19 vector followed by the transformation of E.coli DH5α cells with our NEBuilder® HiFi DNA Assembly assembled product. Got blue-white colonies and performed a plasmid miniprep followed by an agarose gel to confirm which colonies had our desired insert. We call these pUC19-JC. Sent it for sequencing.

Weekly Summary

Performed a gradient PCR to find the right annealing temperature of the primers to bind to the pUC19-JC and help us take out our insert from the plasmid. We also double digested our pUC19-JC and got very clear bands on the gel, again confirming that we have our desired insert. The sequencing results also came out positive. We could still see non-specific binding during PCR amplification of the JcFATB fragment, so we designed new primers and PCR amplified and purified them.

Weekly Summary

Performed another NEBuilder® HiFi DNA Assembly, this time only with newly purified JcFATB, CvFAP part 1 & 2 and JcFATA and stored the product. PCR amplified our construct from pUC19-JC. We were still getting a non-specific binding of the primers, so we proceeded with gel extraction of the desired fragment.

Weekly Summary

Performed another NEBuilder® HiFi DNA Assembly, this time only with newly purified JcFATB, CvFAP part 1 & 2 and JcFATA and stored the product. PCR amplified our construct from pUC19-JC. We were still getting a non-specific binding of the primers, so we proceeded with gel extraction of the desired fragment.

Weekly Summary

PCR amplified and purified our gel extracted 6kb construct. We double digested pYLEX with BamHI-HF and PmlI and our 6kb construct with BamHI-HF followed by gel extraction and purification and concentration estimation using a quantitative ladder. Got a very less concentration at around 12 ng/µl. We again PCR amplified our construct and after purification got a good conc of 287 ng/ul.

Weekly Summary

Gel extracted the 6kb product and proceeded with restriction digestion and ligation into pYLEX. Transformed E.coli DH5α cells with the product but got very few colonies. We made the primary inoculums and performed plasmid miniprep followed by a gel, but did not get the desired bands. Double digested the plasmid with NotI-HF and SalI-HF to verify whether ligation was successful. We did not get the desired bands.

Weekly Summary

Double digested pYLEX, confirmed it with a gel followed by PCR purification but got very less concentration, hence transformed DH5α cells with pYLEX. Did a plasmid miniprep and got good concentration of the plasmid, so proceeded with the double digestion and also digested the construct to create sticky ends and gel extracted it. Got a very low concentration so used centrivap to further concentrate it. We then proceeded with ligation with a control, 1:2 conc (vector:insert) and 1:3 conc. Transformed DH5α cells and made the master plate.

Deletions

We received all the constructs, ScRAD52 part 1 and part 2 in plasmids, Alk2, and FAA1 as Gblocks from IDT. Gradient PCR was done on each to figure out their annealing temperature. ScRAD52 parts 1 and 2 were taken out from the plasmids through PCR and purified; Alk2 and FAA1 cassettes were subjected to PCR to increase their concentration. FAA1 PCR was done using DMSO.

Weekly Summary

Performed colony PCR of colonies from all plates. We did not get satisfactory results. Made primary inoculums of 5 colonies that showed a darker band, followed by miniprep and double digestion and checked individually whether the Restriction enzymes were working or not by digesting empty pYLEX. The plasmid was getting self ligated and the REs were working perfectly. Made primary inoculums of the rest of the colonies on the master plate and did alkaline lysis to screen them. No colonies had our insert.

Deletions

ScRAD52 parts were joined through NEBuilder® HiFi DNA Assembly. The powdered antibiotics (mycophenolic acid, hygromycin, nourseothricin) were resuspended to make a solution. Primary and secondary inoculums were prepared for the transformation of ScRAD52. The OD⁶⁰⁰ reading was once again performed.

Weekly Summary

Performed colony PCR of colonies from all plates. We did not get satisfactory results. Made primary inoculums of 5 colonies that showed a darker band, followed by miniprep and double digestion and checked individually whether the Restriction enzymes were working or not by digesting empty pYLEX. The plasmid was getting self ligated and the REs were working perfectly. Made primary inoculums of the rest of the colonies on the master plate and did alkaline lysis to screen them. No colonies had our insert.

Deletions

The ScRAD52 NEBuilder® HiFi DNA Assembly product needed to be PCR amplified due to exonuclease activity in the NEBuilder® HiFi DNA Assembly. The NEBuilder® HiFi DNA Assembly product was confirmed using agarose gel and was PCR-purified. ScRAD52 transformation was performed using the YLOS transformation protocol and plated in mycophenolic acid-containing plates.

Weekly Summary

Gel extracted the digested pYLEX and proceeded with ligation with ratios of 1:1 and 2:1 (vector:insert) along with the control. Transformed DH5α cells with the ligation products followed by making a master plate and a primary inoculum. Performed plasmid miniprep followed by double digestion for confirming successful ligation of our 6kb construct into pYLEX. Did not achieve the desired results. We screened for more colonies but to no avail. We decided to try NEBuilder® HiFi DNA Assembly of our construct into pYLEX, and did a gPCR for introducing overhangs in our construct.

Deletions

Colony PCR was done for ScRAD52 colonies. The PCR failed, and the PCR was troubleshot. FAA1 transformation was performed. The transformation and integration check PCR (ICP) failed. FAA1 transformation was once again performed, which failed.

Weekly Summary

We then set up an NEBuilder® HiFi DNA Assembly reaction of our construct with the pYLEX overhangs and transformed DH5α cells with the NEBuilder® HiFi DNA Assembly mixture. We got 4 colonies after transformation and made inoculums and miniprepped those. We also screened more colonies from our previous ligation attempt and did an alkaline lysis only to get to know that the ligation had failed. The NEBuilder® HiFi DNA Assembly colonies showed us a good intensity band at 13kb. We also did double digestion and got the desired bands for 2 colonies (G1 and G3).

Deletions

Another trial for colony PCR was done with a different set of primers, which also failed. Gradient PCR was done for colony PCR to rule out the chances of wrong annealing temperature for primers, but we couldn't obtain any conclusive results from this. We did another trial at FAA1 deletion, but there was growth also on the control plates, so we decided to do the transformation again. Another trial at colony PCR with the next set of primers, and we started making Taq polymerase master mix for PCR, which also was a failure.

Weekly Summary

Mini-prepped G1 and G3 and sent it for sequencing. Made YNB selection plates for the transformed Po1g. Linearized G1, G3, and empty pYLEX and PCR purified them. We did the transformation of the Po1g strain following the YLEX manual and plated it on YNB media. After getting colonies on the YNB plate, we streaked some colonies on the YPD agar plates as well.

Deletions

We were not sure if the ScRAD had indeed gotten integrated or not since the FAA1 trails were failing, so we decided to make 4 plates with different concentrations of mycophenolic acid and streak untransformed strain on one side of the plate and transformed strain on the other side of the plate. We found out that 1000-2000ug/ml was the needed concentration of mycophenolic acid for the selection of Yarrowia lipolytica. Transformation of FAA1 was carried out with the colonies from 2000ug/ml mycophenolic acid plates; the transformation gave us inconclusive results, which we think might be due to the low concentration of DNA added.

Weekly Summary

Made inoculums from the colonies obtained from the YPD agar plates and pelleted them down with OD⁶⁰⁰ normalization. We then performed Thin Layer Chromatography(TLC) with 3 pellets and the C16:0 and C18:0 fatty acid standards - Po1g transformed with empty pYLEX, Po1g transformed with G1, Po1g transformed with G3, but we did not see a clear difference in the intensities of the spots in the TLC.

Deletions

Multiple trials of colony PCR were again done by tweaking different parameters, and none gave any results. Another trial of FAA1 deletion was done, and the transformation was successful with no colonies in the control strain. Another trial at colony PCR was done with Triton X-100 detergent to lyse the cells, which yielded no results.

Weekly Summary

Made new inoculums, normalized the OD⁶⁰⁰ for all samples (empty pYLEX, G1, and G3), made cell pellets, lysed them, and extracted the free fatty acids using the chloroform-methanol extraction method. We then dried the samples in a centrivap and proceeded with Liquid Chromatography - High Resolution Mass Spectrometry(LC-HRMS). We were able to see the difference in the fatty acid profiles of G1 and G3 (pYLEX-JC transformed Po1g) with respect to the control(empty pYLEX transformed Po1g). We also saw an increase in the fatty acid profile for Po1g in which we achieved FAA1 deletion. We also made cell pellets of the CvFAP-activated inoculum, extracted the hydrocarbons using hexane and isopropanol, and proceeded with Gas Chromatography(GC)-MS.

Weekly Summary

We did a Western blot to confirm if the proteins of interest were obtained by using 6X HIS and FLAG tags but we did not get a specific binding of either of these tags, we got the desired band sizes for the JcFatB (48.6 kDa), and CvFAP (62.9 kDa) protein but they also appeared in the control lane which shouldn’t be the case. However, there was no band obtained for JcFatA (42.9 kDa) protein in the western blot, but we couldn’t troubleshoot the protocol for it due to time constraints.