Part:BBa_K3447107

Validation of AHL response system

This part contains a luxR gene, which constitutes the AHL sensing system. By connecting the sfGFP gene downstream, we were able to verify the expression intensity of this part.

The combination of AHL with the luxR protein synthesized by the luxR gene in bacteria forms the Luxl-LuxR complex, which activates the strong expression of the Prlux promoter and thus regulates the expression of a series of downstream genes.

Characterization

LuxR can bind to AHL specifically, which is encoded by luxI, thereby activating the expression of genes after Prlux. In our project, to verify the inducing effect of AHL, the digestion and agarose gel electrophoresis of luxI and luxR-Prlux-sfGFP were performed by a standard protocol (Fig. 1A). As shown in Fig. 1B, the fluorescence of the group with AHL standards shows an obvious dose-dependent manner at a series of AHL concentrations, and the fluorescence intensity induced by our AHL-containing bacteria supernatant was equivalent to that of 0.633 μmol/L standard AHL solution.

Fig. 1 AHL can induce the downstream expression. (A) Digestion and electrophoresis of luxI and luxR-Prlux-sfGFP. (B) Fluorescence intensity of sfGFP under different concentration of AHL. E. coli DH5α was transformed with the designed plasmid, cultured for 8 hrs, and diluted OD600 to 0.1. Then the standards of gradient concentration and AHL-containing bacteria supernatant (which had been pre-cultured for 12 hrs) were added into the medium. The emission intensity at 528 nm was measured at the excitation wavelength of 485 nm. The same measurement has been done 12 hrs later. The experiment was performed three times in triplicate. ***, P < 0.001 from respective control using Student’s t test.

Design

Design Notes

We added some synonymous mutations to avoid part rules.

Source

We found this sequence data in GenBank.

Usage and Biology

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1489
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 985
  

Jilin_China 2021

Charaterization of Pasr promoter with fluorescence intensity measurement

This year, based on the previous part BBa_K3447107(Jilin_China,2020), we characterized this part again and added new documentation to it.

We obtained the fluorescence data by measuring the fluorescence level for 11 hours. As shown in the Fig1, after the induction of AHL, the response effect reached the maximum at about 5 hrs, and reached a plateau after 7 hrs from the beginning. The concentration of AHL in our supernatant were between that of 10 and 100 nM standard solutions.

Figure 1.Fluorescence intensity of sfGFP under different concentration of AHL. The designed construct was transformed into E.coli BL21(DE3). When the OD600 value of the bacteria reached 0.2, the standards of gradient concentration and supernatant were added into the medium. The fluorescence intensity of sfGFP was detected at the indicated time and normalized by the OD600 value. The experiment was performed three times in triplicate. ***, P < 0.01 from respective control using Student’s t test.