Part:BBa_K5335023

A part (including a reporter gene) that induces and regulates downstream gene expression by salicyli

The part is composed of BBa_K5335024, BBa_K5335001, promoter J61051 and ribosome binding site B0034. The whole circuit can play the role of induce and regulate downstream gene expression by salicyli.

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 786
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 77
  Illegal NgoMIV site found at 618
  Illegal AgeI site found at 1855
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

To sense the salicylic acid signal produced by plants due to disease, we designd salicylic acid sensors. The LysR-type regulator NahR senses salicylic acid signals in the environment.When the salicylic acid concentration reaches a threshold, the Psal promoter is activated to express the HrpR as well as some shRNAs.

Figure 1. Salicylic acid biosensor circuit
To verify the function of NahR and Psal, we designed a genetic circuit that uses the cyan fluorescent protein amCyan1 as a reporter protein. The constructed plasmid vector is illustrated in Figure 2.
Figure 2. Plasmid Vector

Experimental Verification

SDS-PAGE

We transformed the recombinant plasmid into E. coli BL21 (DE3) for subsequent functional verification. Before induction with salicylic acid, we verified that NahR could be expressed normally in E. coli BL21(DE3) using using 10% SDS-PAGE electrophoresis.

Figure 2. 10% SDS-PAGE results of NahR expression validation.
M: protein ladder. control:Total protein sample extracted from E. coli BL21(DE3) without plasmid introduction.
1-4: Total protein samples extracted from expanded cultures of four different single colonies that were successfully transformed.

Phenotypic verification

     After the expression of NahR was verified, in order to verify the function of NahR and sal promoter, we set up a series of salicylic acid concentration gradients for induction (0 μM,0.1 μM,1 μM,10 μM,10 μM,1000 μM). E. coli BL21(DE3) strain without sal promoter was used as a blank control to explore the leakage expression of sal promoter.

     We found that the expression of fluorescent proteins increased rapidly when SA concentration was between 1-10 μM. We added 200 μL of the induced culture medium inoculated with engineered bacteria to a 96-well plate, and detected the emission light intensity at 486nm using a microplate reader. Five groups of parallel replicates were set for each induced concentration.

Figure 3. Results of fluorescence intensity measured by microplate reader.
A. Changes in fluorescence intensity over time for the salicylic acid-induced groups at different concentrations and the control group.
B. Maximum fluorescence intensity for the salicylic acid-induced groups at different concentrations and the control group.
Figure 4. Comparison of bacterial phenotypes induced by different concentrations of salicylic acid.

     From the above two pictures, it can be seen that sal promoter activity is extremely dependent on salicylic acid concentration, and at the same time sal has low background expression in the absence of salicylic acid.

Protocol

    1. Inoculate single colonies with successful plasmid construction into LB medium.

    2. After 12 hours of cultivation, inoculate the bacterial liquid into fresh LB liquid medium at a ratio of 100:1, and add SA to make the concentration of SA in the medium 0, 0.1, 1, 10, 100, and 1000 respectively. Use the engineered bacteria without the Psal promoter as a blank control.

    3. Take 200 µL and add it to a 96-well plate, set up five parallel replicates for each concentration, and run the plate reader under conditions of 37°C and 200 rpm for 4 hours, measuring the fluorescence intensity at 486 nm every 10 minutes.