Part:BBa_K1973006

miniTn7BB-Gm-nahR-Psal-lapG

nahR (Pseudomonas putida, see BBa_K1031610) is expressed under the constitutive Pr promoter. It encodes a regulatory protein that activates the Psal promoter (see BBa_J61051) in the presence of salicylate. lapG (see BBa_K1973000) is expressed from the Psal promoter in the presence of this molecule. The miniTn7 tool allows the stable integration of this module in the chromosome of a wide range of bacteria (see https://parts.igem.org/Genome_Integration).

Regarding the biofilm growth, KT2442 lapG- (mutant with no LapG activity) produces 2-3 times more biofilm than the wild type and does not disperse the biofilm due to the fact that KT2442 lapG- is a biofilm superproducer. The complementation of lapG in the lapG mutant under the nahR-Psal induced expression system causes a delay in biofilm formation, a reduction of the maximum amount of biofilm and the biofilm dispersion. The expression of lapG in the wild type does not cause a big change in the behavior of the biofilm development, it only produces a little reduction of the amount of biofilm.

Therefore, we can conclude that the expression of lapG under the nahR-Psal system allows the mutant bacterium to disperse biofilm again, when the inductor (salicylate) is added to the media.

Figure 1. Serial dilution-based growth curves of the expression system nahR-Psal-lapG (20 hours). The image represents the growth curves of the strains wild type in LB (A), wild type in LB+salicylate 2 mM (B), lapG^- in LB (C) and lapG^- in LB+salicylate 2mM (D). We assay the strains wild type Tn7 Ø and lapG^- Tn7 Ø concurrently to compare the induction effect of the expression system. Each blot represent the average of 24 data.

For further information, check our wiki page http://2016.igem.org/Team:UPO-Sevilla/Experiments .

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 4367
  Illegal suffix found in sequence at 1
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4367
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 4373
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4367
  Illegal BglII site found at 3051
  Illegal BglII site found at 3322
  Illegal BglII site found at 3608
  Illegal BglII site found at 4972
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 4367
  Illegal suffix found in sequence at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found in sequence at 4367
  Illegal XbaI site found at 4382
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NgoMIV site found at 4804
  Illegal NgoMIV site found at 5929
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1681
  Illegal SapI.rc site found at 2763