Difference between revisions of "Part:BBa K3763042"
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==Background== | ==Background== | ||
| − | FadE, which is also called acyl-CoA dehydrogenases , catalyze the first reaction of the b-oxidation cycle. All acyl-CoA dehydrogenases carry noncovalently (but tightly) bound FAD, which is reduced during the oxidation of the fatty acid. As shown in Figure, FADH2 trans- fers its electrons to an electron transfer flavoprotein (ETF). Reduced ETF is reoxidized by a specific oxidoreductase (an iron–sulfur protein), which in turn sends the electrons on to the electron-transport chain at the level of coenzyme Q. The mitochondrial oxidation of FAD in this way eventually results in the net formation of about 1.5 ATPs. The mechanism of the acyl-CoA dehydrogenase involves deprotonation of the fatty acid chain at the a-carbon, followed by hydride transfer from the b-carbon to FAD.<br> | + | FadE, which is also called acyl-CoA dehydrogenases , catalyze the first reaction of the b-oxidation cycle. All acyl-CoA dehydrogenases carry noncovalently (but tightly) bound FAD, which is reduced during the oxidation of the fatty acid. As shown in Figure, FADH2 trans- fers its electrons to an electron transfer flavoprotein (ETF). Reduced ETF is reoxidized by a specific oxidoreductase (an iron–sulfur protein), which in turn sends the electrons on to the electron-transport chain at the level of coenzyme Q. The mitochondrial oxidation of FAD in this way eventually results in the net formation of about 1.5 ATPs. The mechanism of the acyl-CoA dehydrogenase involves deprotonation of the fatty acid chain at the a-carbon, followed by hydride transfer from the b-carbon to FAD.<br><br> |
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| − | == | + | [[File:T--WHU China--42 1 Bacground1.png|500px|Figure 1. '''Figure1. The FadE catalytic principle.''']] |
| − | In our experiment, we hope to improve the | + | |
| − | + | <br> Figure1. The FadE catalytic principle. | |
| − | + | ||
| − | + | ==Design== | |
| − | + | In our experiment, we hope to improve the β-oxidation capacity of our engineered bacteria by | |
| − | + | overexpressing FadE protein . As shown in the figure below, we constructed a recombinant plasmid | |
| − | + | containing FadE gene and introduced it into our engineered bacteria.<br><br> | |
| + | [[File:Schematic diagram of recombinant vector containing FadE.png|500px|Figure 1. '''Figure1. The FadE catalytic principle.''']] | ||
| + | <br><br>Figure2.Schematic diagram of recombinant vector containing FadE | ||
| + | |||
| + | ==Result== | ||
After confirming that we correctly constructed and transferred the recombinant plasmid into the engineering strain E. coli DH5 α, we used arabinose to induce the expression of FadE and tested its improvement on the fatty acid decomposition ability of engineered bacteria. Our experimental results showed that induced overexpression of FadE did not significantly improve the fatty acid decomposition ability of engineered bacteria, and did not reproduce the experimental results in references.<br><br> | After confirming that we correctly constructed and transferred the recombinant plasmid into the engineering strain E. coli DH5 α, we used arabinose to induce the expression of FadE and tested its improvement on the fatty acid decomposition ability of engineered bacteria. Our experimental results showed that induced overexpression of FadE did not significantly improve the fatty acid decomposition ability of engineered bacteria, and did not reproduce the experimental results in references.<br><br> | ||
| − | + | [[File:Changes of fatty acid decomposition ability of engineering bacteria overexpressing FadE protein.png|500px|Figure 1. '''Figure1. The FadE catalytic principle.''']] | |
| − | + | <br><br>Figure3. Changes of fatty acid decomposition ability of engineering | |
| − | + | bacteria overexpressing FadE protein.<br><br> | |
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In order to explore the reasons for the failure of the experiment, we detected the protein expression more carefully. After inducing the expression of FadE proteins, they were purified by Ni2+ affinity chromatography column. After purification, SDS-PAGE results showed that the molecular weight of our target band was about 20 kDa lower than our predicted value. It is speculated that the engineering strain we used is E. coli DH5 α. The endogenous protease system of this strain has not been artificially knocked out, so the overexpressed protein is easy to be degraded. Considering this possibility, we replaced our engineered strain with E. coli BL21 strain. Then we purified the protein by Ni2+ affinity chromatography column and detected it by SDS-PAGE. The results showed that the bands of FadD and FadE protein were in line with our prediction.<br> | In order to explore the reasons for the failure of the experiment, we detected the protein expression more carefully. After inducing the expression of FadE proteins, they were purified by Ni2+ affinity chromatography column. After purification, SDS-PAGE results showed that the molecular weight of our target band was about 20 kDa lower than our predicted value. It is speculated that the engineering strain we used is E. coli DH5 α. The endogenous protease system of this strain has not been artificially knocked out, so the overexpressed protein is easy to be degraded. Considering this possibility, we replaced our engineered strain with E. coli BL21 strain. Then we purified the protein by Ni2+ affinity chromatography column and detected it by SDS-PAGE. The results showed that the bands of FadD and FadE protein were in line with our prediction.<br> | ||
| + | [[File:Fig 3 . The result of Ni-resin purification.png|500px|Figure 1. '''Figure1. The FadE catalytic principle.''']] | ||
| + | <br><br>Fig 3 . The result of Ni-resin purification. (A) Detection of protein expression in E. coli DH5α strain . Lane 1-4. Ni-resin purification result of FadD protein. Lane 4. The purpose bond was about 20 kDa lower than our predicted | ||
| + | value. Lane 5-8 Ni-resin purification result of FadE protein. Lane 8. The purpose bond was about 20 kDa lower | ||
| + | than our predicted value. (C) Detection of FadE protein expression in E. coli BL21 strain. Lane 1-4. Ni-resin | ||
| + | purification result of FadD protein. Lane 4 The FadE protein bond was is basically the same as we expected. | ||
| + | </html> | ||
<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
| − | <partinfo> | + | <partinfo>BBa_K3763042 SequenceAndFeatures</partinfo> |
| − | <!-- Uncomment this to enable Functional Parameter display | + | |
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| + | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
| − | <partinfo> | + | <partinfo>BBa_K3763042 parameters</partinfo> |
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Latest revision as of 17:06, 21 October 2021
FadE, which is also called acyl-CoA dehydrogenases , catalyze the first reaction of β-oxidaion
Background
FadE, which is also called acyl-CoA dehydrogenases , catalyze the first reaction of the b-oxidation cycle. All acyl-CoA dehydrogenases carry noncovalently (but tightly) bound FAD, which is reduced during the oxidation of the fatty acid. As shown in Figure, FADH2 trans- fers its electrons to an electron transfer flavoprotein (ETF). Reduced ETF is reoxidized by a specific oxidoreductase (an iron–sulfur protein), which in turn sends the electrons on to the electron-transport chain at the level of coenzyme Q. The mitochondrial oxidation of FAD in this way eventually results in the net formation of about 1.5 ATPs. The mechanism of the acyl-CoA dehydrogenase involves deprotonation of the fatty acid chain at the a-carbon, followed by hydride transfer from the b-carbon to FAD.
Figure1. The FadE catalytic principle.
Design
In our experiment, we hope to improve the β-oxidation capacity of our engineered bacteria by
overexpressing FadE protein . As shown in the figure below, we constructed a recombinant plasmid
containing FadE gene and introduced it into our engineered bacteria.
Figure2.Schematic diagram of recombinant vector containing FadE
Result
After confirming that we correctly constructed and transferred the recombinant plasmid into the engineering strain E. coli DH5 α, we used arabinose to induce the expression of FadE and tested its improvement on the fatty acid decomposition ability of engineered bacteria. Our experimental results showed that induced overexpression of FadE did not significantly improve the fatty acid decomposition ability of engineered bacteria, and did not reproduce the experimental results in references.
Figure3. Changes of fatty acid decomposition ability of engineering
bacteria overexpressing FadE protein.
In order to explore the reasons for the failure of the experiment, we detected the protein expression more carefully. After inducing the expression of FadE proteins, they were purified by Ni2+ affinity chromatography column. After purification, SDS-PAGE results showed that the molecular weight of our target band was about 20 kDa lower than our predicted value. It is speculated that the engineering strain we used is E. coli DH5 α. The endogenous protease system of this strain has not been artificially knocked out, so the overexpressed protein is easy to be degraded. Considering this possibility, we replaced our engineered strain with E. coli BL21 strain. Then we purified the protein by Ni2+ affinity chromatography column and detected it by SDS-PAGE. The results showed that the bands of FadD and FadE protein were in line with our prediction.
Fig 3 . The result of Ni-resin purification. (A) Detection of protein expression in E. coli DH5α strain . Lane 1-4. Ni-resin purification result of FadD protein. Lane 4. The purpose bond was about 20 kDa lower than our predicted
value. Lane 5-8 Ni-resin purification result of FadE protein. Lane 8. The purpose bond was about 20 kDa lower
than our predicted value. (C) Detection of FadE protein expression in E. coli BL21 strain. Lane 1-4. Ni-resin
purification result of FadD protein. Lane 4 The FadE protein bond was is basically the same as we expected.
</html>
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 584
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 367
Illegal SapI.rc site found at 1240
Illegal SapI.rc site found at 2383