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.

 

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.

 

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.

Figure 1. The structure of the designed plasmid: pET-28a-Fat1

 

Build

Use primers to amplify pET28a-Fat using the primers we developed with NotI, recover and purify, and perform homologous recombination. Then transformed into E. coli (DH5α) .The colonies were selected and identified by colony PCR.

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 that FAT.

 

Figure 3. The plasmid pET28a-Fat1 sequencing results

 

Learn

Gel electrophoresis has provided us with a means to separate and characterize DNA and proteins based on their size and charge. By subjecting samples to an electric field within a gel matrix, we have observed the migration of these biomolecules, allowing us to determine their relative sizes and compare their mobility. This technique has granted us invaluable insights into the nature and composition of the genetic material and proteins we have been studying.

Design

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.

Figure 4. The structure of the designed plasmid: pET-28a-ELOVL5

 

Build

Use primers to amplify pET28a-Fat using the primers we developed with NotI, recover and purify, and perform homologous recombination. Then transformed into E. coli (DH5α) .The colonies were selected and identified by colony PCR.

Fig. 5 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 that elovl5.

Figure 6. pET28-evovl5 sequencing results show it is successful.

 

Learn

When the target plasmid was successfully transferred into Escherichia coli and grew out, we had to extract the plasmid, and the concentration was found to be low after extraction. On reflection, we concluded that the bacteria didn't break the walls enough to let the DNA out. So, we re-conducted the experiment and extracted the plasmid again, and the final concentration reached the standard.

Test

1. Protein expression and purification

Plasmids (pET-28a-Fat1, pET-28a-ELOVL) 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.

Fig. 7  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. The Fig7c shows that 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

 

 

2. Functional 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 1, the concentration of the standard is 0.5 mg / mL, and the area of the standard map is 633614752.

Figure 8. 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 2. Peak area and concentration of samples and controls

 

It can be seen that Figure 9 A does not have the peak area of the docosapentaenoic acid ( DPA ), Figure 9B does not have the peak area of the docosapentaenoic acid ( DPA ), Figure 9C has a clear peak of the docosapentaenoic acid ( DPA ), with an area of 1425052, and Figure 9D 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.

Figure 9.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

 

Through a series of precise techniques, including enzyme digestion, homologous recombination, heat shock transformation, and kana resistance selection, we aim to successfully integrate these genes into plasmids and E. coli cells. Ultimately, our journey culminates in the evaluation of protein purity and expression through the page, providing us with crucial insights into the success of our experimental endeavors.

 

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