Essential fatty acids need to be obtained from foods because these cannot be synthesized in the human body. There are two families of essential fatty acids, omega-3 (ω-3) and omega-6 (ω-6) polyunsaturated fatty acids (PUFAs). Fish products are major sources of ω-3 fatty acids, and ω-6 fatty acids are mainly from grass-fed meat. However, overfishing has exceeded the sustainable capacity of natural fish stocks, leading to an inadequate uptake of ω-3 fatty acids from traditional sources. Our project aims to design and construct fat-1 and ELOVL5 prokaryotic expression plasmids, as well as express ω-3 desaturase and ELOVL5 in the E. coli cells to synthesize DPA. To achieve this, recombined plasmids, namely pET-28a-fat1, and pET-28a-ELOVL5 will be constructed, transformed, and expressed into DH5-alpha and BL21 competent cells. The presence of these two enzymes could enable the production of ω-3 polyunsaturated fatty acids in E. coli. The success of this initial experiment on E.coli suggest the potential application in oil-producing plants, allowing their seeds to be enriched with omega-3 fatty acids and can be consumed by human, alleviating the shortage of marine sources for ω-3 fatty acids.

 

To convert readily available ω-6 PUFAs into ω-3 PUFAs, which are difficult to synthesize and less likely to be uptake by the human body, we will construct plasmid pET-28a-fat1.The fat1 gene can encode an ω-3 PUFAs desaturase, which dehydrogenates substrate ω-6 PUFAs to produce the corresponding ω-3 PUFAs, leading to alter the intracellular ratio of ω-6/ω-3 PUFAs.

We have made a useful contribution to future iGEM teams. Pet28a-fat1 is a new part created by us.

Construct design

The codon-optimized fat-1 CDS was amplified, and then we used 5'BamHI/3'NotI enzymes to digest fat-1 CDS and pET28a, after which the target genes and vectors were ligated with T4 DNA ligase and transformed in DH5α. On the second day, single clones were selected for amplification; on the third day, the recombinant plasmid was extracted; after that, it was digested and identified, and the positive recombinant plasmid was submitted to a sequencing company for sequencing and identification.

Proof of Contribution 

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 Plasmid pET-28a-Fat1

 

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.

Figure 2. TAE agarose gel electrophoresis to verify the construction of pET-28a-Fat1

 

To ensure that the plasmid construction is 100% correct, we sequenced the target genes that FAT.

Figure 3. The plasmid pET28a-Fat1 sequencing result

 

To test Fat1 proteins, we ran a Western blot using a Supernatant of cell lysate and a Pellet of cell lysate. Fig4 shows that Fat1 protein is found on the Pellet of cell lysate, marker 50KDa, indicating that Fat1 proteins are expressed successfully.

Figure 4. Western Blotting for Fat1 Protein Detection

 

To convert readily available ω-6 PUFAs into ω-3 PUFAs, which are difficult to synthesize and less likely to be uptake by the human body, we will construct plasmid pET-28a-elovl5. The synthesis process of DHA, as the ω-3 PUFAs with the longest carbon chain, also requires the participation of ultra-long-chain fatty acid elongases. It is currently believed that ELOVL5, a member of the ultra-long-chain fatty acid elongase family, is the enzyme involved in the extension of long-chain polyunsaturated fatty acids such as DHA. We have made a useful contribution to future iGEM teams. pET28a- Elovl5 is a new part created by us.

Construct design

ω-3 polyunsaturated fatty acids (ω-3 PUFAs) are a type of unsaturated fatty acids found in animals, mainly including α-linolenic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Among them, DHA is the polyunsaturated fatty acid with the longest carbon chain and the highest degree of unsaturation found so far. Due to its important physiological functions, DHA is considered to be an unsaturated fatty acid with high nutritional value and is widely used in the market. However, mammals lack the enzymes to synthesize the precursors of ω-3 PUFAs, so DHA can only be ingested from food. Given the shortage of supply of deep-sea fish oil, which is the main source of DHA for human beings, the construction of a new DHA-rich food supply has become a social problem that needs to be solved urgently. The synthesis process of DHA, as the ω-3 PUFAs with the longest carbon chain, also requires the participation of ultra-long-chain fatty acid elongases. It is currently believed that ELOVL5, a member of the ultra-long-chain fatty acid elongase family, is the enzyme involved in the extension of long-chain polyunsaturated fatty acids such as DHA.

The codon-optimized ELOVL5 CDS was amplified, and then we used HindIII/NotI enzymes to digest ELOVL5 CDS and pET28a, after which the target genes and vectors were ligated with T4 DNA ligase and transformed in DH5α. On the second day, single clones were selected for amplification; on the third day, the recombinant plasmid was extracted; after that, it was digested and identified, and the positive recombinant plasmid was submitted to a sequencing company for sequencing and identification.

 

Proof of Contribution 

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

 

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

 

Plasmids (pET-28a-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.

Fig. 8 Expression and purification of protein ELOVL5.

                 

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.

Fig. 9 TAE agarose gel electrophoresis to verify the PCR outcomes

 

Eventually, the pET28a-fat1-elovl5 recombinant plasmid is cloned into the pET-28a-fat plasmid. 

Fig.10 Plate transformation for homologous recombinant product

 

Then transformed into E. coli (DH5α). The colonies were selected and identified by colony PCR.

Fig. 11 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.

Fig.12 pET28-Fat1-evovl5 sequencing results

 

[1] KangJX, W. I., & Wu, L. (2004). Transgenic mice: fat-I mice convert n-6 to n-3 fatty acids. Nature, 427(6974), 504.

[2] Lai, L., Kang, J. X., Li, R., Wang, J., Witt, W. T., Yong, H. Y., ... & Dai, Y. (2006). Generation of cloned transgenic pigs rich in omega-3 fatty acids. Nature Biotechnology, 24(4), 435-436. 

[3] Wu, X., Ouyang, H., Duan, B., Pang, D., Zhang, L., Yuan, T., ... & Li, G. P. (2012). Production of cloned transgenic cow expressing omega-3 fatty acids. Transgenic Research, 21, 537-543.