Difference between revisions of "Part:BBa K5013002"

 
 
Line 5: Line 5:
 
We designed the trans-cinnamic acid biosensor pSenCA. this sensor undergoes a conformational change in the HcaR regulatory factor upon binding to supplemented TCA, allowing it to bind to the target promoter PhcaE and activate downstream gene expression.
 
We designed the trans-cinnamic acid biosensor pSenCA. this sensor undergoes a conformational change in the HcaR regulatory factor upon binding to supplemented TCA, allowing it to bind to the target promoter PhcaE and activate downstream gene expression.
  
−
<!-- 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/5013/wiki/part/basic-part-3-trans-cinnamic-acid-tca-promoter-new-part-successful-project/2023-09-04-19-20-35.png" style="width: 500px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 1  Design of the trans-cinnamic acid biosensor pSenCA.</p >
 +
</div>
 +
</html>
 +
 +
The trans-cinnamic acid biosensor pSenCA. Upon binding to supplemented TCA, the HcaR regulatory factor undergoes a conformational change that allows it to bind to the target promoter PhcaE and activate downstream gene expression.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5013/wiki/part/basic-part-3-trans-cinnamic-acid-tca-promoter-new-part-successful-project/image-27.png" style="width: 300px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;"> Figure2  Gel electrophoresis of the pTCA.</p >
 +
</div>
 +
</html>
 +
 +
===Characterization===
 +
To verify whether environmental TCA can effectively activate PTCA to express downstream genes in an in vitro transformation system.The mRFP gene was constructed downstream of the pTCA promoter. 1mM TCA was added to the M9 medium, and the difference in mRFP fluorescence intensity before and after TCA addition was compared. The experimental results are shown in Figure 3. Before adding TCA, the strain still expressed a small amount of fluorescent protein, with fluorescence intensity ranging from 0A.U to 5A.U. After adding 1mM TCA, there was a significant increase in mRFP fluorescence intensity, reaching around 15A.U. This experiment demonstrates that the presence of TCA can effectively activate the downstream gene expression of pTCA, and that pTCA has baseline expression capability.
 +
<html>
 +
<div style="display:flex; flex-direction: column; align-items: center;">
 +
<img src="https://static.igem.wiki/teams/5013/wiki/part/basic-part-3-trans-cinnamic-acid-tca-promoter-new-part-successful-project/image-28.png" style="width: 300px;margin: 0 auto" />
 +
<p style="font-size: 98%; line-height: 1.4em;">Figure 3  Testing of the trans-cinnamic acid (TCA) promoter.</p >
 +
</div>
 +
</html>
 +
 +
===Potential application directions===
 +
This experimental component involves the use of TCA promoters to regulate the expression levels of specific genes. This technology can be applied in the future to precisely control the biopharmaceutical production process and improve the yield and purity of protein drugs. This is expected to solve the problems of insufficient yield and quality control in the field of biopharmaceuticals, and provide a more reliable method for the development of innovative drugs, which has a promising future.
 +
 +
===References===
 +
Flachbart, Lion Konstantin, Sascha Sokolowsky, and Jan Marienhagen. "Displaced by deceivers: prevention of biosensor cross-talk is pivotal for successful biosensor-based high-throughput screening campaigns." ACS synthetic biology 8.8 (2019): 1847-1857.
 +
Binder, Stephan, et al. "A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level." Genome biology 13.5 (2012): 1-12.
 +
  
 
<!-- -->
 
<!-- -->

Latest revision as of 05:17, 11 October 2023


trans-cinnamic acid biosensor pSenCA

We designed the trans-cinnamic acid biosensor pSenCA. this sensor undergoes a conformational change in the HcaR regulatory factor upon binding to supplemented TCA, allowing it to bind to the target promoter PhcaE and activate downstream gene expression.

Usage and Biology

Figure 1 Design of the trans-cinnamic acid biosensor pSenCA.

The trans-cinnamic acid biosensor pSenCA. Upon binding to supplemented TCA, the HcaR regulatory factor undergoes a conformational change that allows it to bind to the target promoter PhcaE and activate downstream gene expression.

Figure2 Gel electrophoresis of the pTCA.

Characterization

To verify whether environmental TCA can effectively activate PTCA to express downstream genes in an in vitro transformation system.The mRFP gene was constructed downstream of the pTCA promoter. 1mM TCA was added to the M9 medium, and the difference in mRFP fluorescence intensity before and after TCA addition was compared. The experimental results are shown in Figure 3. Before adding TCA, the strain still expressed a small amount of fluorescent protein, with fluorescence intensity ranging from 0A.U to 5A.U. After adding 1mM TCA, there was a significant increase in mRFP fluorescence intensity, reaching around 15A.U. This experiment demonstrates that the presence of TCA can effectively activate the downstream gene expression of pTCA, and that pTCA has baseline expression capability.

Figure 3 Testing of the trans-cinnamic acid (TCA) promoter.

Potential application directions

This experimental component involves the use of TCA promoters to regulate the expression levels of specific genes. This technology can be applied in the future to precisely control the biopharmaceutical production process and improve the yield and purity of protein drugs. This is expected to solve the problems of insufficient yield and quality control in the field of biopharmaceuticals, and provide a more reliable method for the development of innovative drugs, which has a promising future.

References

Flachbart, Lion Konstantin, Sascha Sokolowsky, and Jan Marienhagen. "Displaced by deceivers: prevention of biosensor cross-talk is pivotal for successful biosensor-based high-throughput screening campaigns." ACS synthetic biology 8.8 (2019): 1847-1857. Binder, Stephan, et al. "A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level." Genome biology 13.5 (2012): 1-12.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 112
    Illegal AgeI site found at 139
  • 1000
    COMPATIBLE WITH RFC[1000]