iGEM 2023 — Team subcat-gd

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**Day1
8/8/2023**

1. Run
PCR to amplify the DNA sequence of ELOVA5

|  |  |
| --- | --- |
| DNA | 1 ul |
| Primer F | 2.5 ul |
| Primer R | 2.5 ul |
| Max mix | 25 ul |
| ddH2O | 19 ul |

**Procedure**

l 94
degrees Celsius for 5 minutes

l 94
degrees Celsius 30 secs --> 60 degrees Celsius 30 secs --> 72 degrees Celsius 1 min,
cycles 35

l 72
degrees Celsius 10 mins --> 4 degrees Celsius storage

**2.
Sterilization of LB
liquid media**

a)
Place the flask containing LB liquid media into the sterilizer

b)
Set the temperature to 121 degrees Celsius for 20 minutes

**Procedure
in
details:**

-Prepare
for Gel Electrophoresis:

TAE
preparation, 2% agarose gel preparation

-Run
Gel Electrophoresis

**Day2
8/9/2023**

1.
LB media,
Prepare for PCR, Run PCR, Perform DNA purification of genes Fat1 and ELOVL5 yielded from
PCR.

-
Prepare 100 ml LB liquid media

l 0.5g
Yeast Extract into the flask

l 1g
Trypton into the flask

l 1g
NaCl into the flask

l 100mL
ddH2O into the conical flask

2.
Sterilization of LB liquid media

a)
Place the flask containing LB liquid media into the sterilizer

b)
Set the temperature to 121 degrees Celsius for 20 minutes

**Procedure
in details:**

l Gel
electrophoresis of Fat1 and ELOVL5 yielded from PCR done on day 1

l Reuse
gel from the previous step.

3. Plasmid
extract

Procedure
in details:

l Digestion
of Fat1 gene

l Run
gel electrophoresis to verify the presence of digested Fat1 gene

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

**Day3
8/10/2023**

1. Run
gel electrophoresis to determine Fat1 and ELOVL5 gene fragment size, verifying digestion's
success.

2. Prepare
LB liquid media and separate DNA fragments from the agarose gel for purification. Perform
ligation of pET-28a plasmid and target genes.

3.
 Sterilization of LB liquid media

a)
Place the flask containing LB liquid media into the sterilizer

b)
Set the temperature to 121 degrees Celsius for 20 minutes

4.
Purification of digested Fat1, ELOVL5, and corresponding plasmids

5.
Determine the yield and purity of the digested Fat1, ELOVL5, and corresponding plasmids by
comparing the sample’s light absorbance ratio of 260nm and 280nm to that of pure
DNA

6.
 Ligation of the plasmids and target genes (25 microliters)

EVOLA5
and Fat1

7.
Cultivate DH5- E.coli on LB plate (petri dish)

**Day4
8/11/2023**

1. Prepare
300 mL of sterilized liquid LB media for bacterial cultivation.

2. Perform
homologous recombination of pET-28a-fat, GFP, and elovl5. Run PCR to replicate recombined
plasmids. Transform the plasmids into DH5α-E.
coli competent cells. Cultivate E. coli on the petri dish.

3. Test
the yield and purity of GFP

4. Homologous
recombination and PCR

5. Transformation

6. Cultivation
on Petri dishes \*5(done next to an alcohol burner)

**Day5
8/12/2023**

1. Prepare
Kana+ LB
liquid media and transfer E. coli colonies to the LB liquid media.

2. Perform
gel electrophoresis and PCR of plasmids pET-28a-Fat1, pET-28a-ELOVL5,
pET-28a-GFP-Fat1-ELOVL5, and digested Not1. Transform the plasmids into DH5α-E.
coli cells. Extract and purify the DNA fragments from the gel. Conduct Restriction digestion
of NotI.

3. Transform
the plasmids into DH5α-E. coli cells using the heat shock method

4. Purify
the Not1 digested DNA

**Day6
8/13/2023**

1. Perform
plasmid extraction and purification of pET-28a-Fat1.

2. Process
glue recovery, and measure the concentration of DNA. Transform the pET-28a-GFP-Fat1-ELOVL5
plasmids into DH5α-E. coli and BL21(DE3). coli competent cells. Cultivate the
transformed competent cells on the LB plates.

3. Cultivate
the transformed competent cells on LB plates

**Day7
8/14/2023**

1. Check
the growth of E. coli cells. Perform plasmid transformation. Perform OD test, and extract
plasmids.

2. Perform
plasmid extraction

3. Prepare
gel for the SDS page.

4. Gel
at the bottom and the top

**Day8
8/15/2023**

1. Transfer
the bacteria cultures into six tubes to centrifuge. Introducing the crack solution
and
using ultrasonic broken bacteria.
Then centrifuge at 13000 rpm for 30 min, and remove supernatant for further
purification.

2. protein
purification, retrieve the SDS-page, gel electrophoresis

**Day****9****8/1****6****/2023**

1. Plasmid
extract

2. Obtained
the plasmid pET28a-Fat1 and sent it to Genscript for protein expression level
evaluation.

**Day****10****9/11/2023-10/7/2023**

Functional
tests

1.
Purified
Elovl5 to validate its ability to elongate EPA into DPA

2.
GC-FID
analysis (Testing conducted through professional testing organization-Institute of
Agro-products Processing Science and Technology)

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Overview

Here
we used genetic engineering techniques to overexpress enzymes associated with Omega-3 synthesis in E.
coli. Fat-1 and ELOVL5 are two important enzymes in the Omega-3 synthesis process. Our results mainly
consist of two parts. First, we investigated the genes of these two enzymes and performed codon
optimization for chicken codon bias. Subsequently, we designed three plasmids: pET-28a-Fat1,
pET-28a-ELOVL5, and pET-28a-Fat1-ELOVL5. Second, these enzymes were expressed in the prokaryotic system
and performed functional tests.

Goal

The
primary objective of our laboratory is to construct three distinct plasmids, each serving a vital role in
our ambitious project. The initial two plasmids are designed to express the crucial enzymes required to
synthesize omega-3 fatty acids, offering a potential solution to address nutritional deficiencies. These
enzymes play a pivotal role in converting omega-6 fatty acids, which are abundantly available in various
food sources, into omega-3 fatty acids, often lacking in adequate quantities.

The
first two plasmids (pET-28a-Fat1,
pET-28a-ELOVL5),
meticulously engineered, will serve as carriers for the genes responsible for producing these essential
enzymes. By introducing these plasmids into appropriate host organisms, we aim to facilitate the
production of omega-3s in a controlled and efficient manner. Through this innovative approach, we aspire
to revolutionize the nutritional landscape for animals and humans alike. In tandem with the development of
these enzyme-expressing plasmids, our laboratory also endeavors to construct a third plasmid, which holds
a dual purpose.

The
overarching goal of our project is to harness the potential of these genetically modified plasmids and
explore their application in live organisms. By implanting the plasmids into animals, we envision a future
where these organisms can naturally convert omega-6 fatty acids into omega-3s within their bodies. This
transformative process could alleviate nutritional imbalances and contribute to overall health and
well-being. Furthermore, we aim to make this genetically modified product accessible to humans, providing
a reliable and sustainable source of omega-3s to meet their daily dietary requirements. Through meticulous
research, rigorous experimentation, and a dedication to scientific advancement, our laboratory endeavors
to pave the way for a paradigm shift in nutrition. Our efforts are driven by the potential to improve the
lives of both animals and humans, bridging the gap between omega-6 and omega-3 fatty acids and ensuring a
healthier future for all.

Cycle1- BBa\_K4909007-pET-28a-Fat1

**Design**

Construction
of the pET-28a-Fat1 plasmid:
Primer-assisted
codon-optimized fat-1 CDS amplified
by PCR.
Fat-1 CDS and pET-28a were then digested with 5'BamHI/3'NotI, and After that, T4 DNA ligase was used to
ligate and convert the target gene and vector. The following day, recombinant plasmids were extracted, and
enzyme-digested to identify them, and positive clones were selected, amplified, and removed. The
sequencing business received the positive recombinant plasmids for additional sequencing
identification.

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LB Culture Mediums Preparation

**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

**-****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 ![](file:///C:/Users/1/AppData/Local/Temp/ksohtml23108/wps1.jpg)

2) Make
a mixture with 6mL of Kana+ LB and 100 ![](file:///C:/Users/1/AppData/Local/Temp/ksohtml23108/wps2.jpg) 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

PCR

**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

**-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](https://www.thermofisher.cn/document-connect/document-connect.html?url=https://assets.thermofisher.cn/TFS-Assets%2FLSG%2Fmanuals%2FMAN0018667_DualLED_Transilluminator_QR.pdf) | 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.

Purification PCR DNA

**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 µlof
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

Determine the yield and purity

**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

Plasmid Digestion

**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![](file:///C:/Users/1/AppData/Local/Temp/ksohtml23108/wps3.jpg) of
ddH2O and
12.3 ![](file:///C:/Users/1/AppData/Local/Temp/ksohtml23108/wps4.jpg) of
extracted Fat1 containing 500
ng of
pure Fat1 gene

2) Add
5 ![](file:///C:/Users/1/AppData/Local/Temp/ksohtml23108/wps5.jpg) of
10X Cutsmart solution

3) Add
1 ![](file:///C:/Users/1/AppData/Local/Temp/ksohtml23108/wps6.jpg) of
BamH I restriction enzyme and 1 ![](file:///C:/Users/1/AppData/Local/Temp/ksohtml23108/wps7.jpg) of
Not I restrictive enzyme

4) Vortex
the mixture

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

Plasmid Extraction

**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

Ligation of the plasmids and target genes

**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

Homologous recombination and PCR

**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

Transformation

**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

SDS-PAGE

**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

Protein Purification

**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)

Elovl5 Elongase Enzyme Activity Assay

**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%.

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Overview

**Overview**

Here
we used genetic engineering techniques to overexpress enzymes associated with Omega-3 synthesis in
*E. coli*.
Fat-1 and ELOVL5 are two important enzymes in
the Omega-3 synthesis process. Our results mainly consist of two parts. First, we investigated the genes
of these two enzymes and performed codon optimization for chicken codon bias. Subsequently,
we designed three plasmids: pET-28a-Fat1, pET-28a-ELOVL5, and pET-28a-Fat1-ELOVL5. Second, these
enzymes were expressed in the prokaryotic system and performed functional tests.

Results

**1.
Construct****ion of
pET-28a-Fat1 and pET-28a-ELOVL5 plasmids**

Construction
of the pET-28a-Fat1 plasmid:
Primer-assisted
codon-optimized fat-1 CDS amplified
by PCR.
Fat-1 CDS and pET-28a were then digested with 5'BamHI/3'NotI, and After that, T4 DNA ligase was used to
ligate and convert the target gene and vector. The following day, recombinant plasmids were extracted, and
enzyme-digested to identify them, and positive clones were selected, amplified, and removed. The
sequencing business received the positive recombinant plasmids for additional sequencing
identification.

Construction
of the pET-28a-elovl5 plasmid: The company synthesized the elovl5 gene's CDS sequence. Primer-assisted
codon-optimized elovl5 CDS amplified
by PCR.
The target gene and pET-28a were then digested with HindIII/NotI, recovered, and purified. The target gene
and vector were then ligated and transformed with T4 DNA ligase. The following day, recombinant plasmids
were extracted, and enzyme-digested to identify them, and positive clones were selected, amplified, and
removed. The sequencing business received the positive recombinant plasmids for additional sequencing
identification.

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