iGEM 2023 — Team iiser-pune-india

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## Notebook Calendar

Click on the dates highlighted on the calendar to access the pdfs enlisting a detailed account of the work done each day for the project.

February

March

April

May

S

M

T

W

T

F

S

[1](https://static.igem.wiki/teams/4879/wiki/meeting-notes-1-2-23.pdf)

2

[3](https://static.igem.wiki/teams/4879/wiki/meeting-notes-3-2-23.pdf)

4

5

6

7

8

9

[10](https://static.igem.wiki/teams/4879/wiki/meeting-notes-10-2-23.pdf)

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

[26](https://static.igem.wiki/teams/4879/wiki/meeting-notes-26-2-23.pdf)

27

[28](https://static.igem.wiki/teams/4879/wiki/meeting-notes-28-2-23.pdf)

[1](https://static.igem.wiki/teams/4879/wiki/meeting-notes-1-3-23.pdf)

2

3

4

[5](https://static.igem.wiki/teams/4879/wiki/meeting-notes-5-3-23.pdf)

6

[7](https://static.igem.wiki/teams/4879/wiki/meeting-notes-7-3-23.pdf)
[8](https://static.igem.wiki/teams/4879/wiki/meeting-notes-8-3-23.pdf)

9

10

11

[12](https://static.igem.wiki/teams/4879/wiki/meeting-notes-12-3-23.pdf)

13

14

[15](https://static.igem.wiki/teams/4879/wiki/meeting-notes-15-3-23.pdf)

16

17

18

[19](https://static.igem.wiki/teams/4879/wiki/meeting-notes-19-3-23.pdf)

20

[21](https://static.igem.wiki/teams/4879/wiki/meeting-notes-21-3-23.pdf)
[22](https://static.igem.wiki/teams/4879/wiki/meeting-notes-22-3-23.pdf)

23

[24](https://static.igem.wiki/teams/4879/wiki/meeting-notes-24-3-23.pdf)

25

26

[27](https://static.igem.wiki/teams/4879/wiki/meeting-notes-27-3-23.pdf)

28

29

30

31

1

2

[3](https://static.igem.wiki/teams/4879/wiki/meeting-3-4-23.pdf)

4

[5](https://static.igem.wiki/teams/4879/wiki/meeting-5-4-23.pdf)

6

[7](https://static.igem.wiki/teams/4879/wiki/meeting-7-4-23.pdf)

8

9

10

11

[12](https://static.igem.wiki/teams/4879/wiki/meeting-12-4-23.pdf)

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

[29](https://static.igem.wiki/teams/4879/wiki/meeting-29-4-23.pdf)

30

1

2

[3](https://static.igem.wiki/teams/4879/wiki/meeting-3-5-23.pdf)

4

5

6

7

[8](https://static.igem.wiki/teams/4879/wiki/meeting-8-5-23.pdf)

9

10

11

12

13

14

15

16

17

18

19

[20](https://static.igem.wiki/teams/4879/wiki/meeting-20-5-23.pdf)

21

22

23

24

[25](https://static.igem.wiki/teams/4879/wiki/meeting-25-5-23.pdf)
[26](https://static.igem.wiki/teams/4879/wiki/meeting-26-5-23.pdf)

27

28

29

30

31

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

DBTL Cycles

Overview

PCR Amplification

Construct Removal

Insertion

Deletions

## 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.

![](https://static.igem.wiki/teams/4879/wiki/dbtl-cycle.png)

The DBTL Cycle which was pivotal to our project

## 1. PCR Amplification of *JcFATB*

![](https://static.igem.wiki/teams/4879/wiki/note.png)

**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](https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492) 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.**

![](https://static.igem.wiki/teams/4879/wiki/en-1.jpg)

**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]

![](https://static.igem.wiki/teams/4879/wiki/en-2.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-3.jpg)

**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*.

![](https://static.igem.wiki/teams/4879/wiki/en-4.jpg)

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

![](https://static.igem.wiki/teams/4879/wiki/en-5.jpg)

**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]

![](https://static.igem.wiki/teams/4879/wiki/en-6.jpg)

**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 1

#### Design

#### D

We tried to get our assembled construct from pUC19-JC through its PCR amplification. We couldn’t take it out via restriction digestion as we didn’t want to include the SalI restriction site in our construct. When we want to ligate our construct into pYLEX plasmid, it should start with the **Kozak sequence**(CACA for *Y. lipolytica*) as we desire to initiate transcription.

#### Build

#### B

We did a gradient PCR to see at which annealing temperature the 6kb construct is getting amplified using our construct forward and construct reverse primers.

#### Test

#### T

On imaging the 0.8% agarose gel, we observed a **non-specific** band at around 3.5kb along with our desired construct band at 6kb at all temperatures which we used in the gradient. All the bands had quite faint intensity.

#### Learn

#### L

After discussing with our mentors we decided to repeat the PCR with 0%, 1%, and 2% DMSO, lower the annealing time, and set up each of them at 2 different temperatures (65.3 and 62.6 degrees Celsius).

### Cycle 2

#### Design

#### D

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

#### Build

#### B

We made 6 PCR reaction vials of 10 µl each. We made two reaction mixtures for each 0%, 1%, and 2% DMSO along with the other PCR reaction components from Q5 High-Fidelity 2X Master Mix protocol. We kept one set of 0%, 1%, and 2% DMSO PCR reaction mixtures at 65.3 degrees Celsius and the other set at 62.1 degrees Celsius. We also reduced the annealing time from 30 seconds to 10 seconds, hoping that it may decrease the non-specific binding.

#### Test

#### T

On imaging the 0.8% agarose gel, we didn’t get the desired construct bands; very faint bands appeared on the gel.

#### Learn

#### L

This probably occurred as the annealing time was too low.

### Cycle 3

#### Design

#### D

We decided to scale up the PCR reaction and as we were getting the non-specific 3.5kb band, we decided to **gel extract** our 6kb construct band.

#### Build

#### B

After the PCR amplification reaction, we loaded the PCR amplified product (6kb construct band along with the 3.5kb contamination band) in 0.8% agarose gel.

#### Test

#### T

After checking the concentration of gel extracted 6kb band using nanodrop technique, it gave us a **very low concentration**.

#### Learn

#### L

There was a huge loss of construct DNA (we got < 2ng/µl concentration) in the gel extraction protocol so we shifted our approach to doing the NEBuilder® HiFi DNA assembly of just the 4 gene fragments without the vector (pUC19) and then using the NEBuilder® HiFi DNA assembly of the 4 fragments to insert into the pYLEX vector.

### Cycle 4

#### Design

#### D

Since we wanted to do NEBuilder® HiFi DNA assembly of just the 4 fragments without the pUC19 vector, prior to that we had to amplify *JcFATB* without the SalI site so that when we insert our assembled construct into pYLEX, it starts with the Kozak sequence of *Yarrowia*.

#### Build

#### B

To remove the SalI site from *JcFATB*, we did PCR amplification of *JcFATB* using construct forward primer and *JcFATB* reverse primer.

#### Test

#### T

When we tested the PCR amplification of *JcFATB* on 0.8% gel using the construct forward primer and *JcFATB* reverse primer, we got a very smeary band as shown in Fig2(c).

#### Learn

#### L

The amplification of *JcFATB* without the SalI site didn’t happen as we wanted so we shifted our approach back to PCR optimization of procuring our construct from pUC19-JC through tweaking various PCR parameters.

### 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.

![](https://static.igem.wiki/teams/4879/wiki/en-7.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-8.jpg)

**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

![](https://static.igem.wiki/teams/4879/wiki/en-9.jpg)

**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

![](https://static.igem.wiki/teams/4879/wiki/en-10.jpg)

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

![](https://static.igem.wiki/teams/4879/wiki/en-11.jpg)

**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](https://www.neb.com/en/products/e5520-nebuilder-hifi-dna-assembly-cloning-kit#Product%20Information). 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

![](https://static.igem.wiki/teams/4879/wiki/en-12.jpg)

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

![](https://static.igem.wiki/teams/4879/wiki/en-13.jpg)

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

![](https://static.igem.wiki/teams/4879/wiki/en-14.jpg)

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

![](https://static.igem.wiki/teams/4879/wiki/en-15.jpg)

**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

![](https://static.igem.wiki/teams/4879/wiki/en-16.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-17.jpg)

**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

![](https://static.igem.wiki/teams/4879/wiki/en-18.jpg)

**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

![](https://static.igem.wiki/teams/4879/wiki/en-19.jpg)

**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.

### Cycle 1

#### Design

#### D

We decided to transform our linear gene cassette via the **YLOS transformation kit**.

#### Build

#### B

Since there was little information on how much concentration of mycophenolic acid was needed to make the plates, we went ahead with the standard **100µg/mL concentration**.

#### Test

#### T

The transformation was done, and after **incubating** the plates for 48 hours, we saw that both the control and the transformed plate had thick growth, which was not expected.

#### Learn

#### L

We concluded that either the antibiotic marker was **not potent** enough or we were not using enough marker.

### Cycle 2

#### Design

#### D

We decided to increase the concentration of mycophenolic acid to make the plates.

#### Build

#### D

We made another plate with **200µg/mL of mycophenolic acid** and proceeded with the transformation.

#### Test

#### D

We have discrete colonies on the transformation plate, but we also found thick growth on the control plate.

#### Learn

#### D

We decided we **couldn't conclude** with this result whether our transformation occurred or not.

### Cycle 3

#### Design

#### D

We wanted to know whether or not the suspected transformed colonies were indeed resistant to mycophenolic acid.

#### Build

#### D

We made **4 different plates** with different concentrations of mycophenolic acid - 500µg/mL,700µg/mL,1000µg/mL, and 2000µg/mL. We then split each of these plates into two halves. One side was streaked with the **transformed colonies**, and the other side with untransformed colonies.

#### Test

#### D

We found growth on the transformed side on all plates but no growth on the untransformed or control side on most plates except the **500µg/mL plate**.

#### Learn

#### D

We concluded that the transformation had indeed happened, and the concentration of mycophenolic acid to be used from now onwards is around **1000-2000µg/mL**.

![](https://static.igem.wiki/teams/4879/wiki/en-20.jpeg)

**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

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

July

Aug

Sep

Oct

S

M

T

W

T

F

S

25

26

27

28

29

30

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

1

2

3

4

5

30

31

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

1

2

27

28

29

30

31

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

1

2

3

4

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### June 25 - July 1

We setup our lab and cleaned it thoroughly. We learnt how to use the **laminar hood**, how to fill tip boxes, how to label plates, and how to use the micropipettes. We also took inventory of the supplies we had carried over from the previous year’s team.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### July 2 - July 8

**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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### July 9 - July 15

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### July 16 - July 22

**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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### July 23 - July 29

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### July 30 - August 5

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### July 30 - August 5

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### August 6 - August 12

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**.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### August 13 - August 19

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### August 20 - August 26

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**.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### August 27 - September 2

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### August 27 - September 2

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### September 3 - September 9

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### September 10 - September 16

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### September 17 - September 23

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### September 24 - September 30

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.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### October 1 - October 7

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**.

## Lab Protocol

[### PDF](https://static.igem.wiki/teams/4879/wiki/lab-protocols-merged-final.pdf)

## Weekly Summary

### October 8 - October 14

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.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## Overview

We achieved **successful NEBuilder® HiFi DNA assembly** of gene fragments, seamlessly combining Jatropha's thioesterases and the fatty acid photo decarboxylase from Chlorella variabilis. This construct found its home in the genome of Yarrowia lipolytica through precise homologous recombination. A significant feat was the targeted deletion of the faa1 gene, strategically preventing fatty acids from diverting into the beta-oxidation pathway, a key step validated by the observed increase in the fatty acid profile. Our **proof of concept became evident** through LC-HRMS results, closely mirroring Jatropha's fatty acid profile. Despite encountering challenges in detecting C15 and C17 hydrocarbons in GC-MS—attributed to time constraints and Yarrowia lipolytica’s adept utilization of hydrocarbons—we embrace these insights as integral steps in the iterative process of refining our biofuel synthesis strategy.

![](https://static.igem.wiki/teams/4879/wiki/prog.png)

### Note:

![](https://static.igem.wiki/teams/4879/wiki/note.png)

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

## 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.

![](https://static.igem.wiki/teams/4879/wiki/res-1.png)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-1.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-2.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-3.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-4.jpg)

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

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

![](https://static.igem.wiki/teams/4879/wiki/en-5.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-6.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-7.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-18.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/en-19.jpg)

**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*.

![](https://static.igem.wiki/teams/4879/wiki/res-2.png)

**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.

![](https://static.igem.wiki/teams/4879/wiki/tlc.jpeg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/poc-2.png)

**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.

![](https://static.igem.wiki/teams/4879/wiki/poc-3.png)

**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.

![](https://static.igem.wiki/teams/4879/wiki/poc-4.png)

**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.

![](https://static.igem.wiki/teams/4879/wiki/poc-122.png)

**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:

  ![](https://static.igem.wiki/teams/4879/wiki/res-9-1.png)

  **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:

  ![](https://static.igem.wiki/teams/4879/wiki/res-9-2.png)

  **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:

  ![](https://static.igem.wiki/teams/4879/wiki/res-9-3.png)

  **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:

  ![](https://static.igem.wiki/teams/4879/wiki/res-9-4.png)

  **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.

![](https://static.igem.wiki/teams/4879/wiki/del-1.jpg)

**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.

![](https://static.igem.wiki/teams/4879/wiki/del-2.jpg)

**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

![](https://static.igem.wiki/teams/4879/wiki/del-4.jpg)

**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

![](https://static.igem.wiki/teams/4879/wiki/del-3.jpg)

**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

![](https://static.igem.wiki/teams/4879/wiki/del-5.jpeg)

**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

![](https://static.igem.wiki/teams/4879/wiki/del-6.jpeg)

**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](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27poc%27%29%20%7D%7D).

{% endblock %}