Difference between revisions of "Part:BBa K3989003"

 
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Shong, J., Huang, Y. M., Bystroff, C., & Collins, C. H. (2013). Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity. ACS chemical biology, 8(4), 789-795.
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EsaR is a regulator protein which regulates the promoter of a bacterial Quorum Sensing system. By interacting with the Quorum Sensing molecules(a class of molecules called AHL.In our project, the molecule is called 3OC6HSL since it shows a higher sensitivity), it can either act as a repressor or an activator depending on the location of the promoter it binds.
  
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===How it works===
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===Usage and Biology===
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<html>
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In the P<sub>esa</sub> promoter controlled Quorum Sensing system, EsaR acts as a regulator that can bind to a specific site of the promoter P<sub>esa</sub>. Depending on the different types of the P<sub>esa</sub>(P<sub>esaR</sub> and P<sub>esaS</sub>), EsaR can be either a repressor(for P<sub>esaR</sub>) or an activator(for P<sub>esaR</sub>). The detailed mechanism is shown below:
  
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<figure>
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<span class='h3bb'>Sequence and Features</span>
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  <img src="https://static.igem.org/mediawiki/parts/3/37/21_UZurich_EsaR_activation_repression.jpeg">
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<partinfo>BBa_K3989003 SequenceAndFeatures</partinfo>
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  <figcaption><b>Figure 1</b>. EsaR acts as either an activator or a repressor.[1]</figcaption>
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</figure>
  
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From the figure we can see that, the EsaR protein binds to a specific site called the <i>esa box</i>. This binding site has a different location on P<sub>esaR</sub> and P<sub>esaS</sub> and the EsaR binds to it when there is no AHL
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molecules exist. However, when interacting with AHL, the EsaR will perform an allocation change. Subsequently, it will dislocate from the binding site and will either recruit or interfere the RNA polymerase for the transcription.
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===Functional Parameters===
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===Characterisation===
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<partinfo>BBa_K3989003 parameters</partinfo>
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Previous studies has already characterised the 3OC6HSL sensitivity of the EsaR protein and its variants using a promoter activation assay. The result is shown below:
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<figure>
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  <img src="https://static.igem.org/mediawiki/parts/0/0f/21_UZurich_AHL_sensitivity.jpeg">
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  <figcaption><b>Figure 2.</b> AHL-sensitivity of EsaR and its variant of promoter P<sub>esa</sub>S. Different AHL concentrations are used: 0, 1nM, 10nM, 100nM, 1000nM and 10000nM.[1]</figcaption>
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There are mainly four variants except the wild-type EsaR: EsaR-D91G, EsaR-V220A, EsaR-I70V and EsaR-I70V/V220A. And both the variant D91G and V220A show a higher sensitivity to this AHL molecule compared to the wild-type. The I70V variant test shows a similar sensitivity but a lower gene expression level.
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In our project, we also performed a characterisation specifically to the wild-type, variant D91G, V220A and I70V. The strategy we used is to express a GFP in a plasmid(detail of the construct: <a href="https://parts.igem.org/Part:BBa_K3989025"> BBa_K3989025</a>) using promoter P<sub>esa</sub>S. The AHL molecule's concentration are same as people used in the literature.
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We used plate reader and flow cytometry to analyse the fluorescence generated by GFP, the result is shown in figure 3 and 4.
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<figure>
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  <img src="https://static.igem.org/mediawiki/parts/e/e6/21_UZurich_characterisation_plate_reader.jpeg">
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  <figcaption><b>Figure 3.</b> Fluorescence intensity measurement by plate reader(96-well plate). The measurements were done every one hour and this is the curve of the last test.</figcaption>
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</figure>
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From the plate reader result, it is clear that we reproduce the response trend of these variants: D91G and V220A are more sensitive and I70V shows a similar sensitivity compared to wild-type. D91G variant cells shows almost no fluorescence when the AHL concentration is 100nM, where other variants or wild-type are just start to have a lower GFP expression level. However, the initial gene expression level controlled by these systems are not reproducible. D91G and V220A show a lower expression level and I70V is much higher than what we expected.
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<figure>
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  <img src="https://static.igem.org/mediawiki/parts/9/9f/21_UZurich_characterisation_facs.jpeg">
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  <figcaption><b>Figure 4.</b> Fluorescence intensity measurement by flow cytometry. The samples are taken from the plate, in which the bacteria has been cultured for 7 hours.</figcaption>
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</figure>
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In this figure, we mainly show the distribution of the bacterium cells with a different fluorescence intensity. And it shows that except for I70V, other variants and wild-type have the same performance compared to our plate reader analysis. In I70V sample, the cell population is concentrated at a very low fluorescence intensity level and only few of the cells show a high intensity. After increasing the AHL concentration to 100nM, the population that have a high fluorescence intensity shift back to normal and when the concentration raises up to 1000nM, we can barely see any cells with high intensity. This performance might be caused by the quality of our sample because even there is no AHL molecule, there are only a few of cells show a high fluorescence intensity. Further reproduce experiment need to be performed.
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</html>
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===Sequence and Features===
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<partinfo>BBa_K3989012 SequenceAndFeatures</partinfo>
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===References===
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1) Shong, J., Huang, Y. M., Bystroff, C., & Collins, C. H. (2013). Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity. ACS chemical biology, 8(4), 789-795.
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</html>

Revision as of 16:53, 17 October 2021


EsaR regulator

EsaR is a regulator protein which regulates the promoter of a bacterial Quorum Sensing system. By interacting with the Quorum Sensing molecules(a class of molecules called AHL.In our project, the molecule is called 3OC6HSL since it shows a higher sensitivity), it can either act as a repressor or an activator depending on the location of the promoter it binds.

How it works

In the Pesa promoter controlled Quorum Sensing system, EsaR acts as a regulator that can bind to a specific site of the promoter Pesa. Depending on the different types of the Pesa(PesaR and PesaS), EsaR can be either a repressor(for PesaR) or an activator(for PesaR). The detailed mechanism is shown below:

Figure 1. EsaR acts as either an activator or a repressor.[1]
From the figure we can see that, the EsaR protein binds to a specific site called the esa box. This binding site has a different location on PesaR and PesaS and the EsaR binds to it when there is no AHL molecules exist. However, when interacting with AHL, the EsaR will perform an allocation change. Subsequently, it will dislocate from the binding site and will either recruit or interfere the RNA polymerase for the transcription.


Characterisation

Previous studies has already characterised the 3OC6HSL sensitivity of the EsaR protein and its variants using a promoter activation assay. The result is shown below:

Figure 2. AHL-sensitivity of EsaR and its variant of promoter PesaS. Different AHL concentrations are used: 0, 1nM, 10nM, 100nM, 1000nM and 10000nM.[1]
There are mainly four variants except the wild-type EsaR: EsaR-D91G, EsaR-V220A, EsaR-I70V and EsaR-I70V/V220A. And both the variant D91G and V220A show a higher sensitivity to this AHL molecule compared to the wild-type. The I70V variant test shows a similar sensitivity but a lower gene expression level. In our project, we also performed a characterisation specifically to the wild-type, variant D91G, V220A and I70V. The strategy we used is to express a GFP in a plasmid(detail of the construct: BBa_K3989025) using promoter PesaS. The AHL molecule's concentration are same as people used in the literature. We used plate reader and flow cytometry to analyse the fluorescence generated by GFP, the result is shown in figure 3 and 4.
Figure 3. Fluorescence intensity measurement by plate reader(96-well plate). The measurements were done every one hour and this is the curve of the last test.
From the plate reader result, it is clear that we reproduce the response trend of these variants: D91G and V220A are more sensitive and I70V shows a similar sensitivity compared to wild-type. D91G variant cells shows almost no fluorescence when the AHL concentration is 100nM, where other variants or wild-type are just start to have a lower GFP expression level. However, the initial gene expression level controlled by these systems are not reproducible. D91G and V220A show a lower expression level and I70V is much higher than what we expected.
Figure 4. Fluorescence intensity measurement by flow cytometry. The samples are taken from the plate, in which the bacteria has been cultured for 7 hours.
In this figure, we mainly show the distribution of the bacterium cells with a different fluorescence intensity. And it shows that except for I70V, other variants and wild-type have the same performance compared to our plate reader analysis. In I70V sample, the cell population is concentrated at a very low fluorescence intensity level and only few of the cells show a high intensity. After increasing the AHL concentration to 100nM, the population that have a high fluorescence intensity shift back to normal and when the concentration raises up to 1000nM, we can barely see any cells with high intensity. This performance might be caused by the quality of our sample because even there is no AHL molecule, there are only a few of cells show a high fluorescence intensity. Further reproduce experiment need to be performed.

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
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

1) Shong, J., Huang, Y. M., Bystroff, C., & Collins, C. H. (2013). Directed evolution of the quorum-sensing regulator EsaR for increased signal sensitivity. ACS chemical biology, 8(4), 789-795.