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.
1. Construction 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.
![](https://static.igem.wiki/teams/4909/wiki/results/1.jpg)
Fig. 1 The structure of designed plasmids (pET-28a-Fat1, pET-28a-ELOVL5, and pET-28a-Fat1-ELOVL5)
Two recombinant plasmids, pET-28a-Fat1, and pET-28a-ELOVL5were constructed and transformed into E. coli (DH5α). Each of them was identified by colony PCR. (Fig.2)
![](https://static.igem.wiki/teams/4909/wiki/results/2.jpg)
Fig. 2 TAE agarose gel electrophoresis to verify the construction of designed plasmids. A plasmid: pET-28a-ELOVL5; B plasmid: pET-28a-Fat1; C colony PCR outcome to verify the transformation.
To ensure that the plasmid construction is 100% correct, we sequenced the target genes FAT and evovl5.
![](https://static.igem.wiki/teams/4909/wiki/results/3.jpg)
Figure 3. The plasmid pET28a-Fat1 and pET28-evovl5 sequencing results
2. Construction of pET-28a-Fat1-ELOVL5 plasmid
Use primers to amplify pET28a-Fat, amplify elovl5 using the primers we developed with restriction site NotI, digest pET28a-Fat with NotI, recover and purify, and perform homologous recombination.
![](https://static.igem.wiki/teams/4909/wiki/results/4.jpg)
Fig. 4 TAE agarose gel electrophoresis to verify the PCR outcomes
Eventually, the pET28a-fat1-elovl5 recombinant plasmid is cloned into the pET-28a-fat plasmid.
![](https://static.igem.wiki/teams/4909/wiki/results/5.jpg)
Fig.5 Plate transformation for homologous recombinant product
Then transformed into E. coli (DH5α). The colonies were selected and identified by colony PCR.
![](https://static.igem.wiki/teams/4909/wiki/results/6.jpg)
Fig. 6 TAE agarose gel electrophoresis to verify the construction of plasmids pET-28a-Fat1-ELOVL5.
To ensure that the plasmid construction is 100% correct, we sequenced the target genes FAT and evovl5.
![](https://static.igem.wiki/teams/4909/wiki/results/7.jpg)
Fig.7 pET28-Fat1-evovl5 sequencing results
3. protein expression and purification & Western blot
Plasmids (pET-28a-Fat1, pET-28a-ELOVL5, and pET-28a-Fat1-ELOVL5) were all transformed into E.coli BL21 strain which is commonly used in plasmids transformation, and then to verify the protein expression in it. We cultured each group. We prepared 50 ml LB for each group and monitored the bacterial growth. We induced the expression of proteins with IPTG when the OD600 was around 0.6-1.0, and cultured at 16℃ for 12h. Subsequently, we used nickel affinity purification to purify the acquired proteins from other proteins in E. coli (Figure 8).
![](https://static.igem.wiki/teams/4909/wiki/results/8.jpg)
Fig. 8 Expression and purification of protein Fat1 and ELOVL5. A. Incubate the plasmid pET28a-Fat1 containing BL21(DE3). B. SDS-PAGE electrophoresis gel of ELOVL5 protein. C. Western blot of Fat1 protein. Lane 3: Supernatant of cell lysate with induction for 16 h at 15 ℃, Lane 4: Supernatant of cell lysate with induction for 4 h at 37 ℃, Lane 5: Pellet of cell lysate with induction for 16h at 15 ℃, Lane 6: Pellet of cell lysate with induction for 4 h at 37 ℃.
Table 1 is the protein yield calculated from nanodrop. Fat1 protein expression level is less than 1mg/L, and ELOVL5 protein expression level is 3 mg/L. To test ELOVL5 proteins, we ran an SDS-PAGE electrophoresis gel using FT, wash, and elution. The Fig7b showed that ELOV5 protein is found on elution1-3, marker 55KDa, indicating pET-28a-ELOVL5 is purified from E.coli BL21. To test Fat1 proteins, we ran a Western blot using a Supernatant of cell lysate and a Pellet of cell lysate. Fig 8c shows that the Fat1 protein is found on the Pellet of cell lysate, marker 50KDa. These data indicate that ELOV5 protein and Fat1 proteins are expressed successfully.
Table 1 the protein yield of Fat1, ELOVL5
Protein |
Fat1 |
ELOVL5 |
Expression level (mg/L) |
<1 |
3 |
4. Test
We conducted an enzyme activity assay on the purified Elovl5 to validate its ability to elongate EPA into DPA. The experimental design was as follows: we established experimental and control groups, with the experimental group containing the strain Pet28a-Elovl5-BL21 for Elovl5 expression and purification, while the control group did not contain Elovl5. Samples were taken at various time points, including 0h, 8h, 24h, 48h, and 72h. According to the national standard method (GB5009.168-2016), the docosapentaenoic acid (DPA) in the fermentation broth was extracted and determined. The standard sample was dissolved in n-hexane (Sigma) and then directly loaded.
(1) Determination of standard substance
According to Figure 9, the concentration of the standard is 0.5 mg / mL, and the area of the standard map is 633614752.
![](https://static.igem.wiki/teams/4909/wiki/results/1.jpg)
Fig 9. Determination of standard substance
(2) Sample data :
The docosapentaenoic acid (DPA) content was detected by adding pET28a-elovl5 (BL21) bacteria at 37 °C to detect the activity of elovl5. The pET28a (BL21) was used as a blank control. Table 1 shows the peak area and concentration of samples and controls.
Table 1. Peak area and concentration of samples and controls
Number |
Area |
Relative concentration ( μg / mL ) |
pET28a-elovl5(BL21)-0h |
0 |
0 |
pET28a-elovl5(BL21)-12h |
1425052 |
2.2490827 |
pET28a-elovl5(BL21)-24h |
2264369 |
3.5737311 |
pET28a-elovl5(BL21)-CK |
0 |
0 |
pET28a-elovl5(BL21)-CK |
0 |
0 |
pET28a-elovl5(BL21)-CK |
0 |
0 |
It can be seen that Figure 10 A does not have the peak area of the docosapentaenoic acid (DPA), Figure 10B does not have the peak area of the docosapentaenoic acid (DPA), Figure 10C has a clear peak of the docosapentaenoic acid (DPA), with an area of 1425052, and Figure 10D has a clear peak of the docosapentaenoic acid (DPA), with a peak area of 2264369. Therefore, it can be inferred that elovl5 catalyzes the formation of docosapentaenoic acid (DPA). The enzyme elovl5 has activity.
![](https://static.igem.wiki/teams/4909/wiki/results/10.jpg)
Fig 10. The peak area of the docosapentaenoic acid ( DPA )
Note: A: the docosapentaenoic acid (DPA) of pET28a-elovl5(BL21)-0h. B: the docosapentaenoic acid (DPA) of pET28a(BL21). C: the docosapentaenoic acid (DPA) of pET28a-elovl5(BL21)-12h
D: the docosapentaenoic acid (DPA) of pET28a-elovl5(BL21)-24h
We constructed pET-28a-Fat1, pET-28a-ELOVL5, and pET-28a-Fat1-ELOVL5 plasmids, and used E.coli to express the proteins and identified these proteins have enzyme activity.
We also attempted to construct the pET-28a-Fat1-ELOVL5-GFP plasmid in the experiment for better and more convenient detection. Unfortunately, it was not successful (there is a description in the notebook). In our plans, we will continue the experiments to identify the issue and seek solutions.
Therefore, these results provide a possibility that we can use plants and animals to produce DPA in the future. Plans are as follows:
l Optimize expression and purification procedure for enzyme Fat1 and ELOVL5 large-scale production
l We need to learn more about transgenic animals to transfer Fat1 and ELOVL5 genes into animals for expression OMG3
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