Difference between revisions of "Part:BBa K4965001"
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The accumulation of long chain fatty acid (LCFA) is one of the main factors leading to acne, we decided to break down LCFA. Therefore, we decided to bioengineer both FadD and FadL gene into bacteria for expression. FadL can bind to LCFA and allow it to enter the periplasmic space of EcN (the space between the cell wall and the cell membrane), while FadD can activate β-oxidation of exogenous LCFA and regulate the expression of the transcription factor FadR. The cooperative function of the two genes is believed to complete the function of metabolizing LCFA. | The accumulation of long chain fatty acid (LCFA) is one of the main factors leading to acne, we decided to break down LCFA. Therefore, we decided to bioengineer both FadD and FadL gene into bacteria for expression. FadL can bind to LCFA and allow it to enter the periplasmic space of EcN (the space between the cell wall and the cell membrane), while FadD can activate β-oxidation of exogenous LCFA and regulate the expression of the transcription factor FadR. The cooperative function of the two genes is believed to complete the function of metabolizing LCFA. | ||
| − | <!-- Add more about the biology of this part here | + | <!-- Add more about the biology of this part here--> |
===Usage and Biology=== | ===Usage and Biology=== | ||
| + | <html> | ||
| + | <div style="display:flex; flex-direction: column; align-items: center;"> | ||
| + | <img src="https://static.igem.wiki/teams/4965/wiki/part/composite-parts-1-fadl-and-fadd/image-2.png" style="width: 500px;margin: 0 auto" /> | ||
| + | <p style="font-size: 98%; line-height: 1.4em;">Figure 1 Design of the fadL and fadD.</p > | ||
| + | </div> | ||
| + | </html> | ||
| + | |||
| + | We used the RBS (BBa_B0034) as the basis for expressing FadD and FadL gene to allow the gene successfully combined with ribosome. (Figure 1) We used promoter T7 (BBa_L719005) and terminator part BBa_B0015 to express FadD and FadL gene and transformed the plasmids into E. coli Rossetta Strain. | ||
| + | ===Characterization=== | ||
| + | We expressed FadD and FadL gene in E.coli Rossetta strain with an none-bioengineered bacteria as control. By comparing the endocellular and extracellular palmitic acid amount with expressing either FadL or FadD, we examined the successful expression and function of this complex part. However, based on the experiment our team did last year, we made some ameliorations such as achieveing more accurate data through extraction step and one-way analysis of variance and having a more strict quantitative result and analysis. From figure 3B, the total concentration of Palmitic acid the treatment group with both FadL and FadD genes is significantly lower than that of Negative control group indicating that the LCFA intake and metabolization are achieved. The three experiments indicates that the system can achieve the goal of Palmitic acid intake and metabolization in vitro, and both synthesized proteins involved in the process, FadL and FadD, are functional and effective. | ||
| + | <html> | ||
| + | <div style="display:flex; flex-direction: column; align-items: center;"> | ||
| + | <img src="https://static.igem.wiki/teams/4965/wiki/part/composite-parts-1-fadl-and-fadd/image-3.png" style="width: 500px;margin: 0 auto" /> | ||
| + | <p style="font-size: 98%; line-height: 1.4em;">Figure2 Gel image of the fadL and fadD.</p > | ||
| + | </div> | ||
| + | </html> | ||
| + | |||
| + | <html> | ||
| + | <div style="display:flex; flex-direction: column; align-items: center;"> | ||
| + | <img src="https://static.igem.wiki/teams/4965/wiki/part/composite-parts-1-fadl-and-fadd/image-4.png" style="width: 700px;margin: 0 auto" /> | ||
| + | <p style="font-size: 98%; line-height: 1.4em;">Fig.3 A. The briefed protocol of the experiment B. The resulting picture of comparing FadL/FadD only expression and FadL and FadD co-expression.</p > | ||
| + | </div> | ||
| + | </html> | ||
| + | |||
| + | ====Potential application directions=== | ||
| + | This experiment validates that FadD and FadL has the function to metabolize LCFA. This complex part thus could involve as cure of many diseases such as diabetes which involves the overaccumulation of LCFA, making it having a bright future of applications. | ||
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Latest revision as of 10:50, 10 October 2023
FadL+FadD
The accumulation of long chain fatty acid (LCFA) is one of the main factors leading to acne, we decided to break down LCFA. Therefore, we decided to bioengineer both FadD and FadL gene into bacteria for expression. FadL can bind to LCFA and allow it to enter the periplasmic space of EcN (the space between the cell wall and the cell membrane), while FadD can activate β-oxidation of exogenous LCFA and regulate the expression of the transcription factor FadR. The cooperative function of the two genes is believed to complete the function of metabolizing LCFA.
Usage and Biology
Figure 1 Design of the fadL and fadD.
We used the RBS (BBa_B0034) as the basis for expressing FadD and FadL gene to allow the gene successfully combined with ribosome. (Figure 1) We used promoter T7 (BBa_L719005) and terminator part BBa_B0015 to express FadD and FadL gene and transformed the plasmids into E. coli Rossetta Strain.
Characterization
We expressed FadD and FadL gene in E.coli Rossetta strain with an none-bioengineered bacteria as control. By comparing the endocellular and extracellular palmitic acid amount with expressing either FadL or FadD, we examined the successful expression and function of this complex part. However, based on the experiment our team did last year, we made some ameliorations such as achieveing more accurate data through extraction step and one-way analysis of variance and having a more strict quantitative result and analysis. From figure 3B, the total concentration of Palmitic acid the treatment group with both FadL and FadD genes is significantly lower than that of Negative control group indicating that the LCFA intake and metabolization are achieved. The three experiments indicates that the system can achieve the goal of Palmitic acid intake and metabolization in vitro, and both synthesized proteins involved in the process, FadL and FadD, are functional and effective.
Figure2 Gel image of the fadL and fadD.
Fig.3 A. The briefed protocol of the experiment B. The resulting picture of comparing FadL/FadD only expression and FadL and FadD co-expression.
=Potential application directions
This experiment validates that FadD and FadL has the function to metabolize LCFA. This complex part thus could involve as cure of many diseases such as diabetes which involves the overaccumulation of LCFA, making it having a bright future of applications.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 716
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]