Part:BBa_K3989008

Engineered quorum sensing esaR promoter: PesaR-C

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

Basic information

This part represents a promoter(P_esa) variant with two esa box, which can be bound to EsaR protein(see figure 2). esa box is a 18bps period located at a specific position of the promoter. After binding to the box, the EsaR will repress the recruitment of the RNA polymerase and inhibit the downstream transcription. However, when there are AHL molecules(such as 3OC6HSL) existing in the environment, they will interact with the EsaR and release it from the promoter, thus the transcription can be started.(Figure 1) The transcription level is controlled by the concentration of the AHL molecules.

Figure 1. Mechanism of the EsaR being an repressor under the control of the original P_esaR. The red dots are the specific AHL molecule and in our project it is 3OC6HSL[1]

Below is the figure of the structure of the original promoter and the engineered one.

Figure 2. The structure of P_esaR-C and the original P_esaR.[2]

As we can see in figure 2, the original promoter: P_esaR has only one esa box, which is at position -10. The P_esaR-C has an additional esa box which locates between position -10 and -35. Previous studies has shown that the downstream gene expression level in this system depends on all these three factors: type of promoter, type of EsaR and concentration of the AHL molecule.(see figure 3)

Figure 3.Gene expression controlled by AHL molecules, EsaR regulator and P_esaR promoter. On the left side, the test is based on the P_esaR promoter and on the right side, the test is based on the P_esaRC promoter. The luminescence level represents the gene expression level. It is clear that the regulation is controlled by all three factors which are mentioned above.

From figure 3 we can see that, the promoter P_esaRC generally shows a higher transcription efficiency in comparison to P_esaR. And the same EsaR regulator shows a lower sensitivity to the AHL molecule when it bind to P_esaRC. When there are 10nM of AHL molecules in the environment, EsaR-D91G has already been saturated when it binds to P_esaR but in P_esaRC it doesn’t initiate the downstream gene expression yet.

Characterization and improvement contribution made by iGEM23_SDU-CHINA

The PesaRc was characterized using mkate(Fig. 1) BBa_K4583018. And we used a RBS BBa_B0034.

Fig. 1 . Genetic circuit of PesaRwt-RBS(B0034)-mKate

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-min). 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

Fig. 2 . Characterization results of PesaRc-RBS(B0034)-mKate in L19 and L31

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 281
  Illegal XhoI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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.