Materials

Reagent	Volume/mass
Tryptone	10 g/L
Yeast Extract	5 g/L
NaCl	10 g/L
ddH2O	Add to 1000 ml
Kana	100 µg/ml
Agar powder (for plate)	15 g/L

Apparatus

	Amount
Beaker	1
Balance	1
Weighing paper	1
Autoclave	1
Flask	1
plate	20

Procedure

-Liquid

1) Weigh 10g Tryptone, 5g Yeast Extract, and 10g NaCl in a 1L beaker.

2) Add about 800ml of deionized water, and adjust the pH to 7.0.

3) Adjust the solution to a final volume of 1L by adding deionized water.

4) Solution aliquot into 50ml bottles and sterilizing under high pressure and at 121℃ for 20 minutes.

5) Add kana before using.

-Solid

1) 1.5g of agar powder was added to 100ml of LB liquid medium

2) Shaken and autoclaved at 121℃ for 20 minutes.

3) When cooling to about 50℃, add the correct concentration of antibiotics (such as 100µg/ml, 1000X)

4) Mix and pour it into the sterile plate for bacteria.

5) Use it after the agar solidifies.

-Cultivation of GFP and pET-28a in Kana and LB broth

Materials

Reagent	Volume/mass
LB media	45 ml
Kana	100 ug/ml
pET-28a E. coli Glycerol Stocks	100 ul
GFP E. coli Glycerol Stocks	100 ul

Apparatus

	Amount
Pipette	1
Incubator-shaker	1

Procedure

1) Use 45 mL of LB and 45  100 ug/ml Kana

2) Make a mixture with 6mL of Kana+ LB and 100  of pET-28a and GFP E. coli glycerol stocks

3) Place the mixture into a shaker incubator shaker at 37℃ overnight

-Cultivate E. coli

Materials

Reagent	Volume/mass
Kanamycin	150mg
DdH2O	1500µl
Kana+ LB liquid media containing GFP-E. coli	10µl+ 4ml

Apparatus

	Amount
Pipette (0.5-10µl)	1
Pipette (100-1000µl)	1
Incubator-shaker	1

Procedure

a) Prepare diluted Kanamycin solution

- Mix 150mg of Kana and 1500µl of ddH₂O

- Vortex the tube

- Filter the Kanamycin solution for sterilization

b) Mix Kanamycin solution into the LB liquid media containing DH5-E. coli

- Mix 10 µl of kana solution and 10 mL of LB liquid media containing GFP- E. coli

- Pipette 10µl of Kana+ LB liquid media containing GFP-E. coli onto the petri dish

- Place the LB liquid media containing GFP-E. coli back into the shaker set at 37℃ and 220rpm

- Cultivation of Petri dishes

1) Take out five LB Petri dishes

2) Label the LB Petri dishes

3) Transfer the GFP-E. coli to the corresponding LB Petri dishes

4) Use a plastic spreader to spread the cells evenly on the Petri dishes

6) Stack them together

 

 

1. Prepare for PCR:

Materials

Reagent	Volume/mass
Primer-F	2.5µl
Primer-R	2.5µl
2×Master mix	25µl
Water	19.5µl
Templet	0.5µl

Apparatus

	Amount
Pipette(2-20µl)	10
Pipette(20-100µl)	10
PCR tube	3

Procedure

Two tubes of 50µl of mixture, one labeled E1, the other labeled E2

RCR procedure

For ELOVA5:

94℃ for 5 min;

94℃ for 30 s, 60℃ for 30 s,72℃ for 60s Cycles 35;

72℃ for 10 min 4℃∞

For FAT:

94℃ for 5 min;

94℃ for 30 s, 60℃ for 30 s, 72℃ for 80 s Cycles 35;

72℃ for 10 min 4℃∞

For GFP:

94℃ for 5 min;

94℃ for 30 s, 60℃ for 30 s,72℃ for 80 s Cycles 35;

72℃ for 10 min 4℃∞

-Gel electrophoresis of Fat1 and ELOVL5 yield from PCR

Materials

Reagent	Volume/mass
1*TAE	100 ml
Agarose(powder)	2g
ELOVL5	20 ul
FAT1	20 ul
DNA ladder	10 ul

Apparatus

	Amount
Flask	1
Weighing paper	1
Pipette(20µl)	10
Gel box	1
UV Transilluminator	1

1. Prepare for gel electrophoresis

Procedure

1) Add 980ml of water into flask

2) Add 20ml of 50*TAE

3) Add 100ml 1*TAE

4) Add 2g Agarose powder

5) Use a microwave oven to heat the mixture until it dissolved

6) Add the solution to the gel box

2. Run Gel Electrophoresis

Procedure

1) Remove the comb that is used to make the wells

2) Pipette 2 samples of FAT1 in wells 2 and 3

3) Pipette 2 samples of 20µl ELOVL5 in wells 4 and 5

4) Pipette 10µl DNA ladder in well 1

5) Run the gel at 180V for about 30 min

6)  analysis of the result from the UV transilluminator.

3. Gel DNA extraction

Procedure

1) Excise the DNA fragment from agarose gel (FAT1)

2) Place the 0.3g extracted gel into a tube

3) Add 1.2ml of Buffer B2 into the dry bath set at 50℃ for 10 min.

-Run gel electrophoresis to determine Fat1 and ELOVL5 gene fragment size

Materials

Reagent	Volume/mass
Loading dye	10µl
Digested Fat1	50µl
ELOVL5	50µl
Plasmids vec N/H	50µl
Plasmids vec B/N	50µl
DNA ladder	10µl

Apparatus

	Amount
Pipette(50µl)	1
Pipette(10µl)	1
UV transilluminator	1
Gel box	1

Procedure

1) Pipette 10µl of loading dye in the tubes containing Fat1, ELOVL5, and corresponding plasmids (vec N/H, vec B/N)

2) Pipette 50 µl of digested Fat1 and ELOVL5 in wells 2 and 3

3) Pipette 50 µl of plasmids vec N/H and vec B/N in wells 6 and 7

4) Pipette 10 µl DNA ladder in well 1

5) Run the gel at 180V for about 30 minutes

6) Separate DNA fragments from agarose gel

7) Place the gel under a UV transilluminator

8) Cut out the portions of gel that contain DNA, indicated by fluorescence under a UV transilluminator.

 

 

Materials

Reagent	Volume/mass
Overnight bacteria solution	5 ml
Plasmid DNA extraction kit	1
ddH2O	40µl

Apparatus

	Amount
Spin column	3
1.5 ml tube	3
Centrifugal machine	1
Pipette	2

Procedure

-Purification of digested Fat1, ELOVL5, and corresponding plasmids

1. Transfer

1) Place a spin column into a collection tube

2) Transfer the mixture of buffer B2 and sample genes to the spin column

3) Centrifuge the spin column with the collection tube at 8000G for 30s

4) Discard the flow-through in the collection tube

2. Wash

1) Pipette 500 µl of wash buffer into the zymo-spin column

2) Centrifuge at 9000 X g for 30 seconds

3) Discard the flow-through in the collection tube

4) Repeat the above washing procedures

5) Centrifuge the spin column with an emptied collection tube at 9000 X g for 1min

6) Pipette 20 µl of ddH₂O in the spin column

7) Make sure the ddH₂O drops to the center of the columns’ filter

8) Let the column and tube sit for 1min, after that centrifuge at 9000 X g for 1 min

9) Save the flow-through in the collection tube

 

Materials

Reagent	Volume/mass
sample	2µl
ddH2O	10 µl

Apparatus

	Amount
Pipette	1
nanodrop	1

Procedure

1) Use ddH₂O to clean the petal pedestal

2) Pipette 2 µl of ddH₂O onto the petal pedestal to serve as a blank control

3) Close the lever and initialize the program, clean the petal pedestal

4) Pipette 2 µl of sample onto the petal pedestal to test its yield and purity

Materials

Reagent	Volume/mass
ddH2O	31.7 µl
10X Cut smart solution	5 µl
BamHI restriction enzyme	1 µl
Not I restrictive enzyme	1 µl
Fat1 plasmid	12.3 µl (500 ng)

Apparatus

	Amount
PCR tube	1
PCR apparatus	1

Procedure

1) Add 31.7  of ddH2O and 12.3  of extracted Fat1 containing 500 ng of pure Fat1 gene

2) Add 5  of 10X Cutsmart solution

3) Add 1  of BamH I restriction enzyme and 1  of Not I restrictive enzyme

4) Vortex the mixture

5) Run PCR of the mixture for 1h at 37 ℃

Materials

Reagent	Volume/mass
GFP-E. coli	4ml
Buffer S	500µl
Buffer SP1	250µl
Buffer SP2	250µl
Buffer SP3	300µl
Wash solution	1000µl
ddH2O	20µl

Apparatus

	Amount
Centrifuge tube	2
spin column	1
Pipette(100-1000µl)	1
Filter	1

Procedure

1)  Transfer 2 mL of GFP into a centrifuge tube

2)  Centrifuge at 4,000 rpm for two minutes

3)  Transfer another 2 ml of GFT and centrifuge at 4,000 rpm for two minutes again

4)  Activate the spin column: add 500µl of Buffer S to the zymo-spin column on top of a collection tube. Centrifuge at 12,000 rpm for 1 min. Discard the supernatant and column in the collection tube

5)  Add 250µl of BufferSP1 to the centrifuge tube containing cell sediments

6)  Add 250 µl of buffer SP2 to the centrifuge tube and let it sit for 2 minutes

7)  Add 300µl of buffer SP3 to the centrifuge tube and invert it a couple of times

8)  Centrifuge the mix at 8,000 rpm for 30 seconds

9)  Pipette the 800 ul supernatant of the mix to a spin column on top of another centrifuge tube

10)  Add 500 µl of wash solution to the zymo-spin column and centrifuge at 9000 rpm for 30

11)  Repeat the step 10. Discard the flow-through in the centrifuge tube

12)  Centrifuge the spin column and the centrifuge tube at 9000 rpm for 1 minute

13)  Add 20 µl of ddH2O onto the middle of the spin column filter

 

Materials

Reagent	Volume/mass
pET-28a vector N/H	25µl
ddH2O	15.3µl
T4-Buffer	5µl
EVOLA5	1.2µl
T4 ligase	1µl
Fat1	2.5µl

Apparatus

	Amount
Pipette	2
PCR tube	1

Procedure

For pet28a-EVOLA5:

1) Add 12.5 µl of pET-28a vector N/H (E2)

2) Add 8.3 µl of ddH₂O

3) Add 2.5 µl of T4-Buffer

4) Add 1.2 µl of EVOLA5 (E1)

5) Add 0.5 µl of T4 ligase  

For pet28a-Fat1:

1) Add 12.5 µl of pET-28a vector B/N (F2)

2) Add 2.5 µl of Fat1 (F1)

3) Add 2.5 µl of T4-buffer

4) Add 0.5 µl of T4 ligase

5) Add 7 µl of ddH₂O

Then put the PCR tube at 16 ℃ for 30 min

                      

Materials

Reagent	Volume/mass
Clone Mix	13µl
ELOVL5	0.5µl
GFP	0.2µl
pET-28a-fat	12µl

Apparatus

	Amount
Pipette (0.5-10µl)	1
Pipette (10-100µl)	1

Procedure

1) Add 13µl of Mix into the test tube

2) Add 0.5µl of ELOVL5 into the mix

3) Add 0.2µl of GFP into the mix

4) Add 12µl of pET-28a-fat into the test tube

5) Place the test tube under 37 ℃ for 30 minutes

6) Place the test tube on ice

Materials

Reagent	Volume/mass
pET-28a-ElOVL5	10 µl
pET-28a-Fat	10 µl
pET-28a-ElOVL5-Fat-GFP	10 µl
Kana-LB agar plate	3
LB liquid media	1500 µl
Competent cells (BL21 and DH5α)	100 ul/tube

Apparatus

	Amount
Tube	9
Incubator-shaker	1
Dry bath	1

Procedure

1) Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).

2) Mix 10 μl of DNA into 100 μL of competent cells in a falcon tube.

3) The competent cell/DNA mixture on ice for 20-30 mins.

4) Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 45 seconds

5) Put the tubes back on ice for 2 min

6) Add 500 μl LB into this tube and shake at 37°C for 1 hour

7) Use 100 μl LB media on an LB agar plate then put it at 37°C overnight

Materials

Reagent	Volume/mass
2*15% bottom gel solution	18ml
Bottom gel buffer	18ml
Top gel solution	6ml
2*top gel buffer	6ml
Coagulant	120 µl

Apparatus

	Amount
Glass	8 pieces
Gel board	8 pieces
Test tube	1
Gel box	1
Beaker	1
Pipette(20-200µl)	1

Procedure

-Prepare protein gel for SDS-page:

Clean the 8 pieces of glasses and gel boards needed for the SDS page

Stack 2 pieces of glasses together so it forms a well in between, and settle it in a gel box

-Gel at the bottom:

1) Add 18 mL of 2*15% bottom gel solution into an empty test tube

2) Add 18 mL of bottom gel buffer into the test tube (Tip: Don’t submerge the pipette tip into the solution that has already been added)

3) Mix the solution

4) Add the mixed solution in between the glasses until it touches the line

5) Add Isopropanol

-Gel at the top:

1) Add 6 mL top gel solution

2) Add 6 mL 2* top gel buffer

3) Add 120 µl coagulant

4) Add the top gel onto the bottom gel (the bottom gel must be coagulated)

5) Insert the comb from one side

6) Wait for the top gel to solidify

7) Pull out the comb when the gel solidifies

8) Pull out the gel and store it in the fridge

 

Materials

Reagent	Volume/mass
His-tag purification kit	1
IPTG-induced e. coli media	100 ml

Apparatus

	Amount
50 ml tube	8
1.5 ml tube	10
Purification column	1
Centrifugal machine	1
Incubator	1
Ultrasonic crushing apparatus	1
Pipette(20-200µl)	1

Procedure

-E. coli culture procedure

1) Prepare a starting culture as follows: inoculate 5 mL of Kana⁺ LB medium with a single colony from the freshly streaked plate. Incubate at 37°C with shaking at 220 rpm overnight.

2) Prepare and sterilize 100 ml LB medium. Add the overnight LB medium to it. And Check OD600 every hour. When the OD reaches 0.8 (about in 4 hours), induce the culture with 1 mM IPTG. Stop the fermentation 16 hours after induction.

3) Harvest the cells by centrifugation at 4000 rpm for 10 min.

-Proteins are purified following the general protocol for purification of poly His-tagged proteins from the Beyotime kit.

1) Resuspend cell pellet collected from 100 ml fermentation in 20 mL of lysis buffer.

2) After the cells are completely resuspended, lyse the cells by sonication (three one-minute rounds).

3) Clarify the lysate by centrifugation at 14000 rpm for 30 min.

4) Load the lysate on the column and incubate at 4°C for 2 hours

5) Wash the column with 20 mL of wash buffer

6) Elute protein with elution buffer. Collect 3 mL fractions, and check for protein with Nanodrop.

7) Check protein purity by running SDS-PAGE (120 V; 90 min)

 

Methodology:

Pet28a-BL21 was used as a negative control, with three parallel groups. Bacterial cultures were grown until OD600 reached 0.5.

EPA substrate was added to the culture medium at a concentration of 120 μg/ml.

Incubation was carried out on a shaker at 37°C. Reaction times were 0h, 8h, 24h, 48h, and 72h. The reaction was terminated by adding 0.5% methanol to the system, and cell lysis was performed to collect the supernatant.

GC-FID analysis: Samples were analyzed using an appropriate gas chromatography-mass spectrometry (GC-MS) system. Petroleum ether extraction was performed, and an HP-FFAP capillary column (30m250um0.25um) was used. Helium was used as the carrier gas at a flow rate of 1 ml/min. The ionization voltage was set at 70 eV, and the scanning range was 50-550 Da. The column temperature was initially held at 180°C for 2 minutes and then increased at a rate of 2°C/min to 220°C.

Quantitative analysis: Conversion rate (%) was calculated based on peak areas to determine the optimal enzyme reaction time under different volumes of crude enzyme solution. Conversion rate (%) = Product peak area / (Substrate peak area + Product peak area) * 100%.