Difference between revisions of "Part:BBa K3989008"

(Characterization and improvement contribution made by iGEM23_SDU-CHINA)
 
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This part represents a promoter(P<sub>esa</sub>) variant with two <i>esa box</i>, which can be bound to EsaR protein(see figure 2). <i>esa box</i> is a 18bps period located at a specific position of the promoter. After binding to the box, the EsaR will repress the recruitment of the RNA polymerase and inhibit the downstream transcription. However, when there are AHL molecules(such as 3OC6HSL) existing in the environment, they will interact with the EsaR and release it from the promoter, thus the transcription can be started.(Figure 1) The transcription level is controlled by the concentration of the AHL molecules.
 
This part represents a promoter(P<sub>esa</sub>) variant with two <i>esa box</i>, which can be bound to EsaR protein(see figure 2). <i>esa box</i> is a 18bps period located at a specific position of the promoter. After binding to the box, the EsaR will repress the recruitment of the RNA polymerase and inhibit the downstream transcription. However, when there are AHL molecules(such as 3OC6HSL) existing in the environment, they will interact with the EsaR and release it from the promoter, thus the transcription can be started.(Figure 1) The transcription level is controlled by the concentration of the AHL molecules.
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<figure>
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[[File: 21 UZurich EsaR repression.jpeg|700px]]
  <img src="https://static.igem.org/mediawiki/parts/b/ba/21_UZurich_EsaR_repression.jpeg">
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  <figcaption><b>Figure 1.</b> Mechanism of the EsaR being an repressor under the control of the original P<sub>esaR</sub>. The red dots are the specific AHL molecule and in our project it is 3OC6HSL[1]</figcaption>
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<b>Figure 1.</b> Mechanism of the EsaR being an repressor under the control of the original P<sub>esaR</sub>. The red dots are the specific AHL molecule and in our project it is 3OC6HSL[1]
</figure>
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Below is the figure of the structure of the original promoter and the engineered one.
 
Below is the figure of the structure of the original promoter and the engineered one.
  
<figure>
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[[File: 21 UZurich PesaRC.jpeg|700px]]
  <img src="https://static.igem.org/mediawiki/parts/a/a1/21_UZurich_PesaRC.jpeg">
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[[File: 21 UZurich PesaR.jpeg|700px]]
  <img src="https://static.igem.org/mediawiki/parts/f/fb/21_UZurich_PesaR.jpeg">
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  <figcaption><b>Figure 2.</b> The structure of P<sub>esaR-C</sub> and the original P<sub>esaR</sub>.[2]</figcaption>
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</figure>
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As we can see in figure 2, the original promoter: P<sub>esaR</sub> has only one <i>esa box</i> located at position -10 and the P<sub>esaR-C</sub> has an additional <i>esa box</i> which locates between position -10 and -35. Previous studies has shown that even with a same amount of AHL molecules and same type of the EsaR regulator, the gene expression level can still be different depending on the type of the promoter.(see figure 3)
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<b>Figure 2.</b> The structure of P<sub>esaR-C</sub> and the original P<sub>esaR</sub>.[2]
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As we can see in figure 2, the original promoter: P<sub>esaR</sub> has only one <i>esa box</i>, which is at position -10. The P<sub>esaR-C</sub> has an additional <i>esa box</i> which locates between position -10 and -35. Previous studies has shown that the downstream gene expression level in this system depends on all these three factors: type of promoter, type of EsaR and concentration of the AHL molecule.(see figure 3)
 
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</html>
  
  
[[File:21 UZurich PeaR charac.jpeg|400px]][[File:21 UZurich PeaRC charac.jpeg|400px]]
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[[File:21 UZurich PeaR charac.jpeg|400px|]][[File:21 UZurich PeaRC charac.jpeg|400px]]
  
 
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<html>
//<b>Figure 3.</b>Gene expression controlled by AHL molecules, EsaR regulator and P<sub>esaR</sub> promoter. On the  
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<b>Figure 3.</b>Gene expression controlled by AHL molecules, EsaR regulator and P<sub>esaR</sub> promoter. On the  
left side, the test is based on the P<sub>esaR</sub> promoter and on the right side, the test is based on the P<sub>esaRC</sub> promoter. If one use 10nM
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left side, the test is based on the P<sub>esaR</sub> promoter and on the right side, the test is based on the P<sub>esaRC</sub> promoter. The luminescence level represents the gene expression level. It is clear that the regulation is controlled by all three factors which are mentioned above.
 
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</html>
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From figure 3 we can see that, the promoter P<sub>esaRC</sub> generally shows a higher transcription efficiency in comparison to P<sub>esaR</sub>. And the same EsaR regulator shows a lower sensitivity to the AHL molecule when it bind to P<sub>esaRC</sub>. When there are 10nM of AHL molecules in the environment, EsaR-D91G has already been saturated when it binds to P<sub>esaR</sub> but in P<sub>esaRC</sub> it doesn’t initiate the downstream gene expression yet.
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</html>
 
</html>
 
<!-- -->
 
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<span class='h3bb'>Sequence and Features</span>
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==Characterization and improvement contribution made by iGEM23_SDU-CHINA==
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The PesaRc was characterized using mkate(Fig. 1) <html><a href="https://parts.igem.org/Part:BBa_K4583018"> BBa_K4583018</a></html>. And we used a RBS <html><a href="https://parts.igem.org/Part:BBa_B0034"> BBa_B0034</a></html>.
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<figure>
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  <img src="https://static.igem.wiki/teams/4583/wiki/pesarc.png"width="410" height="210">
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  <figcaption><b>Fig. 1 </b>. Genetic circuit of PesaRwt-RBS(B0034)-mKate </figcaption>
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</figure>
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</html>
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===Protocols===
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Our experimental conditions for characterizing this part were as follows:
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* <em>E. coli</em> MG1655
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* 30<sup>o</sup>C, 48h,  under vigorous shaking
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* Plasmid Backbone: pCL
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* Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.).
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We used mkate (excitation at 485 nm and emission at 528 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-min). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).
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===Results===
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<html>
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<figure>
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  <img src="https://static.igem.wiki/teams/4583/wiki/src.png"width="540" height="210">
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  <figcaption><b>Fig. 2 </b>. Characterization results of PesaRc-RBS(B0034)-mKate in L19 and L31</figcaption>
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</figure>
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</html>
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===Sequence and Features===
 
<partinfo>BBa_K3989008 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3989008 SequenceAndFeatures</partinfo>
  
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<partinfo>BBa_K3989008 parameters</partinfo>
 
<partinfo>BBa_K3989008 parameters</partinfo>
 
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===Reference===
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<html>
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1) Shong, J., & Collins, C. H. (2013). Engineering the esaR promoter for tunable quorum sensing-dependent gene expression. ACS synthetic biology, 2(10), 568-575.
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</html>

Latest revision as of 09:15, 9 October 2023


Engineered quorum sensing esaR promoter: PesaR-C

A promoter that can interact with EsaR protein and inhibit downstream gene transcription.

Basic information

This part represents a promoter(Pesa) variant with two esa box, which can be bound to EsaR protein(see figure 2). esa box is a 18bps period located at a specific position of the promoter. After binding to the box, the EsaR will repress the recruitment of the RNA polymerase and inhibit the downstream transcription. However, when there are AHL molecules(such as 3OC6HSL) existing in the environment, they will interact with the EsaR and release it from the promoter, thus the transcription can be started.(Figure 1) The transcription level is controlled by the concentration of the AHL molecules.

21 UZurich EsaR repression.jpeg

Figure 1. Mechanism of the EsaR being an repressor under the control of the original PesaR. The red dots are the specific AHL molecule and in our project it is 3OC6HSL[1]

Below is the figure of the structure of the original promoter and the engineered one.

21 UZurich PesaRC.jpeg 21 UZurich PesaR.jpeg

Figure 2. The structure of PesaR-C and the original PesaR.[2]

As we can see in figure 2, the original promoter: PesaR has only one esa box, which is at position -10. The PesaR-C has an additional esa box which locates between position -10 and -35. Previous studies has shown that the downstream gene expression level in this system depends on all these three factors: type of promoter, type of EsaR and concentration of the AHL molecule.(see figure 3)


21 UZurich PeaR charac.jpeg21 UZurich PeaRC charac.jpeg

Figure 3.Gene expression controlled by AHL molecules, EsaR regulator and PesaR promoter. On the left side, the test is based on the PesaR promoter and on the right side, the test is based on the PesaRC promoter. The luminescence level represents the gene expression level. It is clear that the regulation is controlled by all three factors which are mentioned above.

From figure 3 we can see that, the promoter PesaRC generally shows a higher transcription efficiency in comparison to PesaR. And the same EsaR regulator shows a lower sensitivity to the AHL molecule when it bind to PesaRC. When there are 10nM of AHL molecules in the environment, EsaR-D91G has already been saturated when it binds to PesaR but in PesaRC it doesn’t initiate the downstream gene expression yet.

Characterization and improvement contribution made by iGEM23_SDU-CHINA

The PesaRc was characterized using mkate(Fig. 1) BBa_K4583018. And we used a RBS BBa_B0034.

Fig. 1 . Genetic circuit of PesaRwt-RBS(B0034)-mKate

Protocols

Our experimental conditions for characterizing this part were as follows:

  • E. coli MG1655
  • 30oC, 48h, under vigorous shaking
  • Plasmid Backbone: pCL
  • Equipment: Multi-Detection Microplate Reader (Synergy HT, Biotek, U.S.).

We used mkate (excitation at 485 nm and emission at 528 nm) to characterize this part. As our focus was mainly on the expression time, we processed the obtained fluorescence data by means of the following equation: x'=(x-min)/(max-min). This treatment makes all data fall between 0 and 1, which is easier to use for comparisons between different fluorescence data (since our focus is on expression time).

Results

Fig. 2 . Characterization results of PesaRc-RBS(B0034)-mKate in L19 and L31

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 281
    Illegal XhoI site found at 1
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

1) Shong, J., & Collins, C. H. (2013). Engineering the esaR promoter for tunable quorum sensing-dependent gene expression. ACS synthetic biology, 2(10), 568-575.