Part:BBa_K2447015

Phosphate-dependent, temperature-sensitive cascaded system with GFP reporter

A phosphate repressible system is attached to downstream temperature system. At high phosphate level, phosphate-sensitive promoter is repressed and leading to no production of tlpa36. As a result, promoter tlpa36 will be constitutive for production of GFP. At low phosphate level, tlpa36 proteins are actively produced. Under high temperature, tlpa36 proteins exist in dimer and having no repressive effect on ptlpa36. Under low temperature (below 36 degree), tlpa36 protein de-dimerises to form tlpa36 monomer, and capable of repressing ptlpa36; and hence repressing downstream expression of GFP.

This construct is characterized in 10β cells in MOPS medium (0.2% casamino acid and 0.2% glucose). Construct is partially functional as seen by the 1-fold increase in GFP expression at the higher temperature (37 degrees) ,comparing to 30 degrees, where the prmoter pTlpa36 is unbounded and able to recruit RNA polymerase for downstream GFP expression. However, the construct is not sensitive to high phosphate concentration. Under high phosphate concentrations, regardless of the temperature conditions, high GFP expression should be expected but was not observed in the results (Figure 1).

Figure 1: GFP/OD of phosphate-temperature sensitive construct at OD = 0.2, under varying phosphate concentrations and temperatures.

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 1026
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1311
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1614
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2579