Part:BBa_K1973033

nahR-Psal (inducible promoter)

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.

This expression and regulatory system has been tested in all our physiological assays in order to check if the expression is correct and if there is an induction by salicylate. For example, the expression of the lapG gene was tested.

The growth curves test of the strains KT2442-TpMRB128 (nahR-Psal-lapG) and KT2442 lapG--TpMRB128 (nahR-Psal-nasF-lapG), which are lapG mutants, will reveal if the nahR-Psal expression system express correctly the lapG gene at the induction conditions with and without salicylate (Figure 1). After the experiments, we observed that both strains have a similar planktonic growth curves to the control and between them in LB and LB+salicylate. As regards the biofilm growth, KT2442 lapG- produce 2-3 times more biofilm than the wild type and do 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 induced expression system causes a delay of the biofilm formation, a reduction of the maximum amount of biofilm and the biofilm disperses. 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.

Figure 1. Serial dilution-based growth curves of the expression system nahR-Psal-nasF-lapG (30 hours). The image represents the growth curves of the strains lapG^- in LB (A) and lapG^- in LB+salicylate 2 mM (B). We assay the strain lapG^- Tn7 Ø concurrently to compare the induction effect of the expression system. Each blot represent the average of 8 data.

The biofilm growth tendency of the strain, when it is induced by salicylate, shows that the expression of LapG protein promotes the biofilm dispersal like the wild type. The strains without inducing have grown in a different rate, although the tendency of both curves are similar.

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

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 576
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 408
  
• 1000
  COMPATIBLE WITH RFC[1000]