Difference between revisions of "Part:BBa K3989003"
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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> | <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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Revision as of 16:02, 19 October 2021
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)
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:
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 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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.