Difference between revisions of "Part:BBa K5378019"
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<h1>Functional Verification</h1> | <h1>Functional Verification</h1> | ||
| − | <p>To demonstrate that PEA, a reliable risk factor of HE identified by the current work of our secondary PI (see details in our Design page) , could initiate the downstream gene circuit, we first engineered Escherichia coli Nissle 1917(EcN) to produce FeaR and TynA constantly by transforming EcN with plasmid Pcon-tynA-Pcon-feaR. Thereby, PEA could be degraded by the enzyme TynA into PAG and PAG could bind with FeaR as a transcriptional factor, which could activate the inducible promoter PTynA. Then we transformed the engineered EcN with plasmid PTynA-GFP to demonstrate the feasibility and efficiency of sensing module via fluorensence (Figure | + | <p>To demonstrate that PEA, a reliable risk factor of HE identified by the current work of our secondary PI (see details in our Design page) , could initiate the downstream gene circuit, we first engineered Escherichia coli Nissle 1917(EcN) to produce FeaR and TynA constantly by transforming EcN with plasmid Pcon-tynA-Pcon-feaR. Thereby, PEA could be degraded by the enzyme TynA into PAG and PAG could bind with FeaR as a transcriptional factor, which could activate the inducible promoter PTynA. Then we transformed the engineered EcN with plasmid PTynA-GFP to demonstrate the feasibility and efficiency of sensing module via fluorensence (Figure 2).</p> |
| − | + | ||
<div style="text-align:center;"> | <div style="text-align:center;"> | ||
| − | <img id="image" src="https://static.igem.wiki/teams/5378/ | + | <img id="image" src="https://static.igem.wiki/teams/5378/safety/gsss.webp" width="50%" style="display:block; margin:auto;" alt="example" /> |
<div style="text-align:center;"> | <div style="text-align:center;"> | ||
<caption> | <caption> | ||
| − | <b>Figure 2. </b> CAPTION_HERE | + | <b>Figure 2.Schematic representation of the construction and mechanism of engineered EcN with sensing module. </b> CAPTION_HERE |
</caption> | </caption> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | < | + | <p>After coculturing with 0, 5, 25, 50 and 100ng/ml PEA for 12 hours, results showed a significant increase in fluorensence under microscopy, along with the the increased level in PEA concentration (Figure 3), suggesting a successful expression and high feasibility of the sensing module. Moreover, the fluorescent intensity under different concentrations of PEA throughout 24 hours also verified that our engineered EcN could indeed be more sensitive to the increase in PEA concentration (Figure 4).</p> |
| − | < | + | |
| + | <div style="text-align:center;"> | ||
| + | <img id="image" src="https://static.igem.wiki/teams/5378/safety/gsss.webp" width="50%" style="display:block; margin:auto;" alt="example" /> | ||
| + | <div style="text-align:center;"> | ||
| + | <caption> | ||
| + | <b>Figure 3.Fluorescence Intensity with 100ng/ml, 50ng/ml, 25ng/ml, 5ng/mland 0ng/ml PEA cocultured in engineered EcN. </b> CAPTION_HERE | ||
| + | </caption> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div style="text-align:center;"> | ||
| + | <img id="image" src="https://static.igem.wiki/teams/5378/part/2gs.webp" width="50%" style="display:block; margin:auto;" alt="example" /> | ||
| + | <div style="text-align:center;"> | ||
| + | <caption> | ||
| + | <b>Figure 4.Fluorescence observation of the Pcon-FeaR-Pcon-TynA and PTynA-GFP engineered bacteria fluid cocultured with different concentrations of PEA. </b> CAPTION_HERE | ||
| + | </caption> | ||
| + | </div> | ||
| + | </div> | ||
</body> | </body> | ||
</html> | </html> | ||
Revision as of 06:53, 2 October 2024
PtynA-RBS-GFP
This part adds GFP to BBa_K5378009, which is used to determine whether or not the sensing module will operate effectively.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
This part is used to determine whether or not the sensing module will operate effectively.
Functional Verification
To demonstrate that PEA, a reliable risk factor of HE identified by the current work of our secondary PI (see details in our Design page) , could initiate the downstream gene circuit, we first engineered Escherichia coli Nissle 1917(EcN) to produce FeaR and TynA constantly by transforming EcN with plasmid Pcon-tynA-Pcon-feaR. Thereby, PEA could be degraded by the enzyme TynA into PAG and PAG could bind with FeaR as a transcriptional factor, which could activate the inducible promoter PTynA. Then we transformed the engineered EcN with plasmid PTynA-GFP to demonstrate the feasibility and efficiency of sensing module via fluorensence (Figure 2).
After coculturing with 0, 5, 25, 50 and 100ng/ml PEA for 12 hours, results showed a significant increase in fluorensence under microscopy, along with the the increased level in PEA concentration (Figure 3), suggesting a successful expression and high feasibility of the sensing module. Moreover, the fluorescent intensity under different concentrations of PEA throughout 24 hours also verified that our engineered EcN could indeed be more sensitive to the increase in PEA concentration (Figure 4).
