Difference between revisions of "Part:BBa K3989009"

Latest revision as of 08:49, 9 October 2023

Quroum sensing esaS promoter: PesaS

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

Basic information

In contrast to P_esaRC( BBa_K3989008), this promoter will down-regulate the gene expression when EsaR attaches to the esa box on it. Meanwhile, this promoter has only one esa box which is at position -60.(see figure 1)

Figure 1. Mechanism of the EsaR being an activator under the control of P_esaS. The red dots are the specific AHL molecule and in our project it is 3OC6HSL[1]. When interacting with the AHL molecules, the EsaR protein will dislocate from the promoter. Thus, the P_esaS will not recruit the RNA polymerase and down-regulate the transciption.

Characterisation

Together with the EsaR protein and its variants, we characterised this promoter using the same strategy. The strategy we used is to express a GFP in a plasmid(detail of the construct: BBa_K3989025) with different EsaR protein variants. 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 2. 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.

Figure 3. Fluorescence intensity measurement by flow cytometry. The samples are taken from the plate, in which the bacteria has been cultured for 7 hours.

From the characterisation, we reproduced the results from the literature that P_esaS can down-regulate the gene expression when the EsaR protein is dislocated from the esa box.

Characterization contribution made by iGEM23_SDU-CHINA

Author: Suiru Lu PesaS is a natural EsaR-activated promoter. EsaR can act as both transcriptional activator and repressor. PesaR is a natural EsaR-repressed promoter, whereas PesaS is a natural EsaR-activated promoter. At low cell density (low ρ), EsaR binds to its esa box to turn off PesaR and turn on PesaS. In the presence of AHL, EsaR can bind to AHL and release from the DNA. Thus, at high cell density(high ρ), the PesaR is turned on and the PesaS is turned off(3).

Protocols

Our experimental conditions for characterizing this part were as follows:

• E. coli MG1655
• 30^oC, 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-x). 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

We characterized PesaS-RBS-GFP together with PesaRwt-RBS-mkate, PesaRc-RBS-mkate and PesaRp-RBS-mkate. The green curve in the figure shows the results for PesaS. PesaS expression peaks at around 4-6 h and then rapidly declines to 0 at around 8-10 h.

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.

2) 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.

3)Gu, F., Jiang, W., Mu, Y., Huang, H., Su, T., Luo, Y., Liang, Q., & Qi, Q. (2020). Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS synthetic biology, 9(2), 209–217.