Difference between revisions of "Part:BBa K2116001"

Latest revision as of 20:57, 20 October 2021

Transcriptional regulator esaR

esaR is a transcriptional regulator from the plant pathogen Pantoea stewartii . It binds an 18bp sequence on DNA, and can be released by binding to the specific AHL molecule it responds to...

Characterization and improvement by 2021 iGEM UZurich

Our team characterised the sensitivity of EsaR protein and its variants to a certain AHL molecule: 3OC6HSL. The strategy we used is to express a GFP in a plasmid(detail of the construct: BBa_K3989025) using promoter P_esaS. The AHL molecule's concentration are same as people used in the literature[1]. We used plate reader and flow cytometry to analyse the fluorescence generated by GFP, the results are shown below:

Figure 1. 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 2. 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.

@@ Line 5: / Line 5: @@
 esaR is a transcriptional regulator from the plant pathogen <i>Pantoea stewartii </i>. It binds an 18bp sequence on DNA, and can be released by binding to the specific AHL molecule it responds to...
-<!-- Add more about the biology of this part here
-===Usage and Biology===
+===Characterization and improvement by 2021 iGEM UZurich===
+<html>
+Our team characterised the sensitivity of EsaR protein and its variants to a certain AHL molecule: 3OC6HSL. 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>esaS</sub>. The AHL molecule's concentration are same as people used in the literature[1].
+We used plate reader and flow cytometry to analyse the fluorescence generated by GFP, the results are shown below:
+</html>
+[[File:21_UZurich_characterisation_plate_reader.jpeg|700px|]]
+<b>Figure 1.</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.
+<html>
+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.
+</html>
+[[File:21_UZurich_characterisation_facs.png|700px|]]
+<b>Figure 2.</b> Fluorescence intensity measurement by flow cytometry. The samples are taken from the plate, in which the bacteria has been cultured for 7 hours.
+<html>
+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.
+</html>
 <!-- -->
@@ Line 17: / Line 40: @@
 <partinfo>BBa_K2116001 parameters</partinfo>
 <!-- -->
+===References===
+<html>
+) 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.
+</html>