Part:BBa_K1946006

pu

pu promoter drives transcription of the upper operon of Pseudomonas putida mt-2 TOL plasmid for degradation of toluene and xylenes. pu is activated by the activator protein XylR (BBa_K1946005) in the presence of toluene and xylenes.

We cloned XylR after pL(TetO) promoter which is operated by TetR and sfGFP after pu promoter into pet22b and pZa vectors. We confirmed our cloning with restriction digestion with BglII and NotI. (Fig. 1)

Figure 1: Results of restriction enzyme digestion with BglII and NotI for verification of XylR cloning.

We expect that in the presence of ATC, XylR will be expressed and after XylR expression, in the presence of xylenes or toluene sfGFP will be expressed. We induced our cells ATC and, after 2 hours of incubation, with xylene at different concentrations. SDS-PAGE results showed no over-expression of a protein near 67kDa which is the mass of XylR. (Fig. 2) Unfortunately, we have not seen any difference in sfGFP signal. (Fig. 3) Considering SDS-PAGE and fluorescence measurement result, the reason our construct failing at xylene recognition can be a failure at cloning or design.

Figure 2: SDS Page results of empty control, XylR induced with ATC only, XylR induced with ATC and 0.18x xylene, ATC and 0.37x xylene, ATC and 0.75x xylene, ATC and 1.50x xylene, ATC and 3.00x xylene and with ATC and 6.00x xylene in order. The expected band around 67kDa (corresponding to XylR) was not observed in any of the samples.

Figure 3:GFP signal levels in empty or samples transformed with XylR induced with increasing concentrations of xylene after 8 hours of induction. Y axis is fluorescence intensity (A.U.), X axis is amount of xylene added on 3mL culture(uL).Data normalized with respect to OD600 obsorbance.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 196
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]