Overview

Embarking on the journey of JetroEco, our project is intricately woven into the fabric of Design, Build, Test, Learn (DBTL) cycles — the foundational framework of synthetic biology engineering.

We navigate the complexities of engineering principles by integrating our gene construct of JcFATA, JcFATB (genes coding for the thioesterases of Jatropha curcas), and CvFAP (gene coding for fatty acid photo-decarboxylase in Chlorella variabilis) into the Po1g expression plasmid, pYLEX. Our chassis, primarily the Po1g strain of Yarrowia lipolytica, serves as the fertile ground where Jatropha curcas composition fatty acids and fatty acid-derived hydrocarbons come to life.

In the unique context of JetroEco, these engineering principles, punctuated by specific actions such as PCR amplification, NEBuilder® HiFi DNA Assembly, and gene construct integration into pYLEX, become the compass guiding us through the complexities of synthetic biology. This propels our project towards groundbreaking success, utilizing pYLEX and pUC19 plasmids as indispensable tools in our pursuit of sustainable biofuel solutions.

The DBTL Cycle which was pivotal to our project

1. PCR Amplification of JcFATB

Fig: NEB 1kb DNA ladder (For all the agarose gels, we used this DNA ladder)

We started with PCR amplification of each of our 4 gene fragments (JcFATB, CvFAP fragment 1, CvFAP fragment 2, and JcFATA) procured from Twist Bioscience. We did gradient PCRs for each of these fragments to confirm the annealing temperature and later did a PCR amplification reaction at the annealing temperature, giving us the best intensity band in respective cases. There was a successful gradient PCR and PCR amplification for CvFAP fragment 1, CvFAP fragment 2, and JcFATA. However, the PCR amplification of JcFATB was especially tricky.

Cycle 1

Design

D

We wanted to get PCR amplified product for each of the 4 fragments which we can subsequently use in our NEBuilder® HiFi DNA assembly reaction.

Build

B

For the gradient PCR and PCR amplification reaction of each fragment, we made a 50 µl reaction mixture and then divided it into 5 aliquots of 10ul each and set the PCR parameters as per Q5 High-Fidelity 2X Master Mix protocol.

Test

T

We did a 0.8% agarose gel electrophoresis to verify the PCR results. The 3 fragments (CvFAP fragment 1, CvFAP fragment 2, and JcFATA) showed us perfect results but gradient PCR of JcFATB showed us a non-specific band apart from our desired band.

Learn

L

There was a non-specific binding of primers for the JcFATB fragment. Upon discussion with our mentors, we decided to lower the annealing temperature for JcFATB and try out the PCR again with 0%, 1%, and 2% DMSO.

Fig 1(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]

Fig 1(b): Lane 1-2: 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 11-12: PCR amplified cvFAP fragment 1 and 2 respectively at 62.1 degrees Celsius, Lane 13-14: gradient PCR product of JcFATB and JcFATA respectively at 71.3 degrees Celsius

Cycle 2

Design

D

We integrated the advice of our mentors from the first round into our experiment plan.

Build

B

We set up three 10µl reactions for JcFATB at 0%, 1%, and 2% DMSO with the other components as per Q5 High-Fidelity 2X Master Mix protocol at 62.1 degrees Celsius (reduced annealing temperature).

Test

T

When the 0.8% gel was imaged, we saw that the non-specific band had disappeared and the desired band of amplified JcFATB was obtained. The best intensity band was observed in the case of 1% DMSO as shown in Fig1(c).

Learn

L

Finally, we set up a higher-volume PCR amplification reaction for JcFATB with 1% DMSO at 62.1 degrees Celsius. Hence, we successfully PCR-amplified all 4 of our gene fragments.

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

2. Procuring the Construct from pUC19-JC

After PCR amplification of all 4 of the gene fragments, we proceeded with NEBuilder® HiFi DNA assembly after double digesting the pUC19 vector with SalI-HF and BamHI. We transformed NEB® 5-alpha Competent E. coli (High Efficiency) with 2 µl of NEBuilder® HiFi DNA assembly mixture and did blue-white screening (Fig2(a)) for the desired colonies. Since our assembled construct had been inserted in the lacZ gene, we screened only the white colonies. After successfully screening one of the white colonies with the construct into the pUC19 plasmid, we renamed the recombinant plasmid as pUC19-JC (Fig2(b)). We verified whether we had the correct assembled construct via sequencing and also did double digestion tests to see the release of our assembled construct DNA (Fig2(c)). Now, the next step was to get our assembled construct out of pUC19-JC so that we could insert it into the pYLEX plasmid through restriction-digestion-ligation for its expression in the Po1g strain of Yarrowia lipolytica.

Fig 2(a): Blue-white screening for colonies having pUC19-JC

Fig 2(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]

Fig 2(c): Lane 1: 1kb DNA ladder, Lane 2: Double digestion of pUC19-JC with Sal1-HF and BamH1 to verify release of the insert from the recombinant plasmid (preliminary check whether pUC19 vector and accepted our recombinant construct), Lane 3: PCR amplification of JcFATB with construct forward primer and JcFATB reverse primer

Cycle 5

Design

D

After discussion with our mentors, we tweaked a lot of PCR parameters to get the 6kb construct out of pUC19-JC like annealing temperature and annealing time, reaction volume in each PCR vial, and the amount of template DNA (pUC19-JC plasmid) in the PCR reaction.

Build

B

Since our construct band was slightly greater than 6kb, we increased the annealing time and did a lot of gradient PCR trials to optimize the annealing temperature which resulted to be 65.7 degrees Celsius.

Test

T

After PCR reaction optimization, we were able to minimize the intensity of the non-specific 3.5kb band and increase the intensity of our 6kb assembled construct which we verified on 0.8% agarose gel as shown in Fig2(d).

Learn

L

Even though there was a very faint intensity of the 3.5kb contamination band along with our 6kb intense construct band after PCR optimization, we decided to proceed with restriction-digestion-ligation with the pYLEX vector as there was a high chance that only our construct would participate in ligation reaction due to the BamHI sticky end created in the restriction digestion prior to the ligation reaction.

Fig 2(d): 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)

3. Insertion of the Assembled Construct into pYLEX

After successfully minimizing the 3.5kb non-specific band and getting a high-intensity band of our 6kb assembled construct via PCR amplification from pUC19-JC plasmid, we moved on to getting the construct into the pYLEX expression plasmid of our chassis Y.lipolytica (Po1g strain). We followed the restriction-digestion-ligation protocol for the same, where we double-digested our pYLEX vector (procured from Yeastern Biotech, Taipei, Taiwan) with PmlI and BamHI-HF to create a blunt end and a sticky end respectively. The PmlI digestion enzyme was irreplaceable as it was the only enzyme that we could use to get the desired Kozak sequence “CACA” for Y.lipolytica between our promoter and coding sequence of the gene. We also digested the 6kb assembled construct with BamHI-HF to create the complementary sticky end for ligation. At the end of this DBTL cycle, we successfully managed to get the 6kb insert into the pYLEX vector and renamed the recombinant plasmid as pYLEX-JC.

Before linearizing the pYLEX-JC plasmid so that it can integrate into the Po1g genome via homologous recombination, we transformed DH5-Alpha cells to clone the pYLEX-JC plasmid so that we had sufficient reserves for it and we selected the transformed DH5-Alpha via ampicillin resistance. However, after integrating the pYLEX-JC into the Po1g genome, we used leucine auxotrophy to select the transformants.

Cycle 1

Design

D

We wanted to get the 6kb assembled construct obtained after PCR amplification from pUC19-JC into the pYLEX vector via restriction-digestion-ligation protocol.

Build

B

Protocol followed: Double-digestion of 1µg pYLEX vector with PmlI and BamHI-HF to create blunt and sticky ends respectively. Single digestion of 1µg 6kb Construct with BamHI-HF to create a complementary sticky end. CIP(phosphatase enzyme) treatment of digested pYLEX to prevent self-ligation, and then gel extraction of the digested pYLEX vector and 6kb insert. We set up a control (pYLEX without the insert), 1:1, and 1:2 (vector: insert ratio) for the ligation reaction using the T4 DNA ligase enzyme. We transformed DH5-Alpha cells with these 3 ligation products.

Test

T

We obtained only 6 DH5-alpha colonies for 1:2 ligation reaction and no colonies on the plates that were transformed with control and 1:1 ligation reaction product. We did miniprep for those 6 DH5 alpha colonies and ran the miniprep products along with the empty pYLEX on 0.8% agarose gel to see the shift between the 1:2 ligation product and empty pYLEX. pYLEX-JC should be a 13kb band and the empty pYLEX vector should be a 7kb band. The shift of 6kb between pYLEX and pYLEX-JC is due to the insert. We obtained a very inconclusive and shabby gel for it (Fig 3(a)). Since we didn’t do linearization of the miniprep products before running the gel, that might be a possible reason for the shabby gel, so we double-digested two of the miniprep products (as they gave us a faint 13kb band in the previous gel as shown in Fig 3(a)) and empty pYLEX with SalI-HF and NotI-HF to see if the insert was present in those miniprep products as shown in Fig 3(b).

Learn

L

Since the first gel (Fig 3(a)) was inconclusive and the second gel (Fig 3(b))upon double digestion with SalI-HF and NotI-HF, did not show us the desired 6 kbp shift, we concluded that the miniprep products were the same as empty pYLEX and there was no shift from the control (empty pYLEX) band as we desired. Fig 3(c) and Fig 3(d) denote the bands that we should get ideally after double digesting pYLEX-JC and empty pYLEX with SalI-HF and NotI-HF respectively. Ligation might have failed since we got a very low concentration after gel extraction of the digested pYLEX vector and insert, we decided to scale up the quantity of digestion reaction for the insert, then do gel extraction of 6kb band for it, and for the empty pYLEX vector, we decided to do PCR purification after digestion and CIP treatment reaction to avoid DNA loss in gel extraction. We also tried to alter the vector:insert ratio in our next restriction-digestion-ligation attempt.

Fig 3(a): Lane 1: empty pYLEX, Lane 2 to 7: Plasmid miniprep products obtained from DH5-Alpha transformed with 1:2 ligation reaction products, Lane 9: 1kb DNA ladder

Fig 3(b): Lane 1: 1kb DNA ladder, Lane 2: double digested empty pYLEX with Sal1-HF and Not1-HF, Lane 3 and 4: double digested miniprep products with Sal1-HF and Not1-HF

Fig 3(c): Simulated gel for double-digested pYLEX-JC with Sal1-HF and Not1-HF

Fig 3(d): Lane 1: Simulated gel for double-digested empty pYLEX with Sal1-HF and Not1-HF

Cycle 2

Design

D

Troubleshooting measures were incorporated into the restriction-digestion-ligation protocol to get a high concentration of digested vector and insert which we can use in ligation reaction and also their vector:insert ratios were changed to favor ligation of our insert into the vector pYLEX.

Build

D

We scaled up the double-digestion reaction to 5µg for the pYLEX vector and almost 3µg for single digestion of the 6kb construct. We did PCR purification for the digested pYLEX vector, once the CIP treatment was done, to avoid DNA loss that we encountered in gel extraction in our last attempt but we continued gel extraction for our 6kb insert band as there was a very faint intensity 3.5kb non-specific band in it from the PCR amplification from pUC19-JC as described in DBTL 2. We also altered the vector:insert ratio for the ligation reaction this time to 1:2 and 1:3 since we didn’t get any colonies on the 1:1 plate in our previous attempt.

Test

D

After the transformation of the control (double-digested empty pYLEX), 1:2, and 1:3 ligation reaction products into DH5 alpha, we got many colonies on the 1:2 (Fig 3(f)) and 1:3 plate (Fig 3(g)), but we also got a lot of colonies on the control plate which wasn’t expected (as shown in Fig 3(e)). We did the screening of 10 colonies each from 1:2 and 1:3 plates via colony plasmid protocol to see the shift from the control plasmid in 0.8% agarose gel as shown in Fig 3(h) and Fig 3(i). We didn’t get any shift of the plasmid band relative to the control for our ligation product transformed colonies.

Learn

D

The probable problem in the protocol could be in the phosphatase enzyme treatment of the pYLEX vector backbone after double digestion. It might be removing the phosphate from the blunt end created by PmlI which is making it even more difficult to ligate. After discussion with our mentors, we thought of doing more screening of colonies from 1:2 and 1:3 plates via alkaline lysis. As an alternative, we thought of introducing overhangs in our 6kb assembled insert so that we can do NEBuilder® HiFi DNA assembly of our insert and the double digested pYLEX vector directly through the NEBuilder HiFi DNA Assembly Cloning Kit. The only reason why we were skeptical about doing NEBuilder® HiFi DNA assembly was whether it would allow us to respect the fusion promoter-gene without modification of the correct sequence of hp4d: CAC-A-ATG

Fig 3(e): Control Plate from restriction-digestion-ligation attempt 2

Fig 3(f): 1:2 plate from restriction-digestion-ligation attempt 2

Fig 3(g): 1:3 plate from restriction-digestion-ligation attempt 2

Fig 3(h): Lane 1: 1kb DNA ladder, Lane 2: Colony plasmid product of control colony, Lane 3 to 10: Colony plasmid products from 1:2 plate

Fig 3(i): Lane 1: 1kb DNA ladder, Lane 2: Colony plasmid product from control colony, Lane 3 and 4: Colony plasmid products from 1:2 plate, and Lanes 5 to 14: Colony plasmid products from 1:3 plate

Cycle 3

Design

D

Since the restriction-digestion-ligation wasn't working, we decided to do 2 fragment NEBuilder® HiFi DNA assembly by introducing overhangs in our 6kb insert (1st fragment) and double-digested pYLEX vector with PmlI and BamHI-HF (2nd fragment). We also did more screening through alkaline lysis for the colonies from the previous ligation attempt from 1:2 and 1:3 plates according to our mentors' suggestions.

Build

D

We did gradient PCR to get the appropriate annealing temperature for the primers which helped in introducing the pYLEX overhangs in our insert. We then PCR amplified the 6kb insert to introduce overhangs at 65.4 degrees Celsius annealing temperature. We did the NEBuilder® HiFi DNA assembly reaction for the double-digested pYLEX vector with PmlI and BamHI-HF, and 6kb insert with the introduced overhangs. Then we transformed the DH5-Alpha cells with this NEBuilder® HiFi DNA assembly reaction mixture. We obtained only 4 colonies after this transformation and we did plasmid miniprep for all of them.

Test

D

We did single digestion of the 4 miniprep products obtained from the transformation of NEBuilder® HiFi DNA assembly mixture in DH5-Alpha with NotI-HF and ran them along with the alkaline lysis screening products of the ligation attempt 2 (10 colonies each from 1:2 and 1:3 plate were screened) on 0.8% agarose gel (Fig 3(j) and Fig 3(k)). As shown in Fig 3(j) and 3(k), we are not able to see the shift between the control and ligation products in the screening of 1:2 and 1:3 colonies after alkaline lysis. However, we are clearly able to see the shift between the control (single-digested pYLEX vector(7kb)) and single-digested NEBuilder® HiFi DNA assembly miniprep products (13kb) in Fig 3(k). This confirms preliminarily that NEBuilder® HiFi DNA assembly has worked and we have a successful insertion in the pYLEX vector. We also did double-digestion tests with SalI-HF and NotI-HF to replicate the gels as shown in Fig 3(c) for pYLEX-JC and Fig 3(d) for empty pYLEX. The results for double-digestion with SalI-HF and NotI-HF of empty pYLEX and NEBuilder® HiFi DNA assembly miniprep products are shown in Fig 3(l) which clearly shows that we have the insert in pYLEX vector.

Learn

D

NEBuilder® HiFi DNA assembly of vector and insert worked successfully as shown in Fig 3(k) and Fig 3(l). We also sent the pYLEX-JC plasmid obtained from the NEBuilder® HiFi DNA assembly reaction for sequencing to get the final confirmation.

Fig 3(j): Lane 1: 1kb DNA ladder, Lane 2: Control (colony transformed with empty pYLEX), Lane 3 to 13: Alkaline lysis products of colonies from 1:2 plate, Lane 14 to 20: Alkaline lysis products of colonies from 1:3 plate

Fig 3(k): 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 vector with Not1-HF), Lane 9 to 12: Single digested (with Not1-HF) NEBuilder® Hi-Fi DNA Assembly miniprep products of the 4 colonies

Fig 3(l): 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

4. Growing the Modified Strain on a Selectable Marker

We integrated the ScRAD52 gene into the Po1g strain to increase the HR efficiency. Since recombination efficiency was very low for this transformation, we wanted to use a cheaper antibiotic, so we chose mycophenolic acid. The deletion cassette was designed so that the mycophenolic acid resistance gene was bound by the ScRAD52 gene downstream, along with 1000bp of AO8 gene homology flanking both sides.

Fig 4(a): The part labeled CT is the control side, where the untransformed colony was streaked, and the other side, TF, is the transformed side. The concentration of the plates is bottom right - 500µg/mL, top right - 700 µg/mL, top left - 1000µg/mL, bottom left - 2000µg/mL