Part:BBa_K625002

Pu promoter long version with stop codon

This is an adapted version of the Pu promoter BBa_I723020. The original design contains the RBS and first 81bp of the XylU. To prevent unwanted fusion proteins from emerging (when this BioBrick is cloned without scar), a double stop codon was inserted at the end of the promoter.

Experimental setup

Figure 1: Experimental setup of XylR experiment.

Figure 2: Schematic overview of the testsystem for XylR characterization

E. coli strain JM101 was transformed with two plasmids containing the transcriptional regulator XylR, the degradation cassette xylMABN and a GFP reporter coupled to BBa_K625002 respectively BBa_K625003. For the first two of those we used the plasmid pCK04AxylR according to [1]. The reporter plasmid we constructed ourselves by using BBa_K625005 as a backbone.

We inoculated 50 mL of LB medium with an overnight culture of the co-transformed strains and set the OD₆₀₀ to 0.1. Upon reaching the exponential growth phase, the cultures were induced with m-xylene. Therefore we put a sterile test tube with m-xylene into the flask and sealed it with parafilm in order to get an air-induced response.

The samples were taken 3 hours after induction. OD₆₀₀ and GFP fluorescence was measured then in a 96-well plate.

results

Figure 3: Characterization of BBa_K625002 and BBa_K625003 m-xylene was added by air-induction.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 195
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] [http://aem.asm.org/cgi/content/abstract/64/2/748 S. Panke, J. M. Sanchez Romero, V. de Lorenzo: Engineering of Quasi-Natural Pseudomonas putida Strains for Toluene Metabolism through an ortho-Cleavage Degradation Pathway , Applied and Environmental Microbiology, 1998, Vol. 64, No. 2]