Part:BBa_K2447015

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

For more detailed experimental results, do visit our [http://2017.igem.org/Team:NUS_Singapore/Experiments experimental page].

A phosphate repressible system is combined with a temperature sensitive system. At high phosphate level, phosphate-sensitive promoter is repressed, which leads to stopping of TlpA36 production. As a result, pTlpA promoter will be expressing GFP. At low phosphate concentrations, TlpA36 protein is actively produced. Under high temperature, TlpA36 protein cannot dimerize and has no repressive effect on pTlpA. Under low temperature (below 36 degrees), TlpA36 protein dimerises and becomes capable of repressing pTlpA; and hence starts repressing 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) compared to 30 degrees, where the promoter pTlpA is unbounded and able to recruit RNA polymerase for 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/OD600 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