Part:BBa_K3989003

EsaR I70V variant

A variant of the Quorum Sensing regulator protein EsaR BBa_K2116001 with an isoleucine substituted by valine.

Basic information

EsaR BBa_K2116001 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, we use 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. Besides this wild-type, there are some other variants in which one or more amino acids residues are substituted. Consequently, the sensitivity of these variants to 3OC6HSL are different. There are mainly four variants except the wild-type EsaR: EsaR-D91G( BBa_K3989004 ), EsaR-V220A( BBa_K3989005 ), EsaR-I70V(this part) and EsaR-I70V/V220A. From previous studies, both the variant D91G and V220A show a higher sensitivity to 3OC6HSL compared to the wild-type and the I70V variant shows a similar sensitivity but a lower gene expression level.(See figure 1)

Figure 1. AHL-sensitivity of EsaR and its variant of promoter P_esaS. Different AHL concentrations are used: 0, 1nM, 10nM, 100nM, 1000nM and 10000nM.[1]

How it works

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

Figure 2. 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 P_esaR and P_esaS 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

In our project, we 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 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 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.

Structural information contribution made by iGEM23_SDU-CHINA

Written by Suiru Lu and Xiaoting Wang

Summary

In the EsaI/R Quorum Sensing (QS) system, the interaction between EsaR protein and AHL (3OC6HSL) directly influences the sensitivity of the promoter to AHL, thereby determining the response timing of the QS switch. For enhanced precision control, we introduced 19 point mutations (more detail can be found on our wiki)in EsaR and predicted the subsequent three-dimensional structures using the AlphaFold2 algorithm. Using 3 online tools, we assessed the quality of our model. Also, a comprehensive quantitative analysis of binding affinity was conducted using the AutoDocksoftware. Here, we add structural information for EsaR170V.

3D structure of EsaR 170V

Fig. 1 . 3D structure of EsaR170V

The interaction between EsaR170V and AHL

• The binding energy with AHL is -7.5 kcal/mol.

Fig. 2. The results of the docking between EsaR and AHL molecules.

Fig. 3 . The results of the docking between EsaR and AHL molecules.

Conclusion

Based on the information we provided, future iGEM teams can select appropriate mutants to obtain QS systems with different strengths.

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.