Part:BBa_K4284031
pETD-fadB-ter
pETD-fadB-ter
Contribution
Medium-chain fatty acids (MCFAs) have a favorable safety profile and can be used to treat a variety of conditions, such as benefits for skin care, weight loss, and cardiovascular use. However, most of the medium-chain fatty acids in use today are extracted from tropical plants, which is time-consuming and limits the yield of medium-chain triglycerides. As a result, its yield is much lower than that of long-chain fatty acids. In order to overcome this limitation, we used synthetic biology strategies to conduct experiments on the four core enzymes of the reverse β-oxidation cycle, namely fadB, ydiI, ter, and bktB. This was then applied to the MCFAs synthesis pathway in E. coli to increase the production of MCFAs. The production of medium-chain fatty acids (MCFAs) can be increased by overexpressing key enzymes in the reversal of the β-oxidation cycle. We use the four key enzymes, bktB, fadB, ter, and ydiI to construct the reverse β-oxidation cycle and form the MCFAs synthesis pathway in E. coli. In this way, we can improve the yield of the production of MCFAs. Because the T7 promoter has a strong ability in translation and usually be used as protein expression, we choose the pETDuet-1 vector and pCDFDuet-1 vector, with T7 promoter respectively, to express our target enzymes bktB, fadB, ter, and ydiI to develop r-BOX cycle. To achieve this, we inserted genes fadB and ter into pCDFDuet-1vector, transferred the recombinant plasmid into E. coli BL21(DE3) for protein expression (Figure 1).
Engineering Success
a) Construction of transcriptional activation screening platform In order to construct our plasmids, we amplify all four enzymes with corresponding templates by PCR. Salmonella enterica genome was used as the template to amplify gene fadB, the plasmid pUC57-ter was used as the template to amplify gene ter(Figure 2).
In Figure 2 we can find that there were four clear DNA bands, indicating that genes fadB and ter, were successfully amplified by PCR. Then we used a one-step cloning method to ligate the gene fadB into the NcoI and BamHI sites of the pETDuet-1 vector. Then we transformed the recombinant plasmid into E. coli DH5α competent cells. We inoculated the correct strain and extracted the plasmid pETDuet1-fadB. Then we inserted gene ter into the NdeI and XhoI sites of pETDuet1-fadB through a one-step cloning method. The recombinant plasmid pETDuet1-fadB-ter was verified by NcoI/HindIII and NdeI/XhoI, respectively (Figure3 line2, 4).
We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 5.), and the plasmids were successfully constructed. So far, we have successfully obtained the recombinant plasmids.
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a) Protein expression and purification In order to obtain the two enzymes, we transferred the recombinant plasmid, pETDuet1-fadB-ter, into E. coli BL21(DE3), inoculated the correct colony in the LB culture medium and added IPTG to induce protein expression when the OD600 reached 0.5. After overnight induction and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we used nickel column purification to purify the enzymes we wanted, and detected the proteins by SDS-PAGE (Figure 6).
Two clear protein bands can be seen in figure 6, while there is no corresponding band in the control, indicating the successful expression of fadB and ter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 4337
Illegal XhoI site found at 4534 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 4851
Illegal NgoMIV site found at 8667
Illegal AgeI site found at 1011
Illegal AgeI site found at 2356
Illegal AgeI site found at 2650 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3540
Illegal SapI.rc site found at 241
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