Part:BBa_K4583018

PesaRwt-RBS(B0034)-mKate

Usage and Biology

QS system

Quorum sensing (QS) is a natural form of cell-cell communication that regulates the metabolic behaviour of bacteria based on changes in their local cell density. As cell density increases, signalling molecules accumulate and are sensed by QS-controlled gene expression regulators, which turn on relevant gene expression.

Esa I/R system

The Esa I/R system is quite special from traditional QS system. The EsaI/R QS system is homologous to the LuxI/R QS system and originates the maize pathogen--Pantoea stewartii subsp. stewartia. 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[2].

Fig. 1 . schematic illustration of Esa I/R system

BBa_K4583009(PesaRwt)

PesaRwt refers to the wild-type PesaR.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 946
  Illegal SapI.rc site found at 328
  

Characterization

The PesaRwt was characterized using mkate(Fig. 2) BBa_K4583018. And we used a RBS BBa_B0034.

Fig. 2 . 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.) and Molecular Devices SpectraMax i3x.

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

The red curve in the figure shows the characterization results for this part, and the green curve shows the characterization results for PesaS.

Fig. 3 . Characterization results of PesaRwt-RBS(B0034)-mKate in L19 and L31

Reference

[1] Gu, F., et al., Quorum Sensing-Based Dual-Function Switch and Its Application in Solving Two Key Metabolic Engineering Problems. ACS Synth Biol, 2020. 9(2): p. 209-217.