Difference between revisions of "Part:BBa K2447015"

Revision as of 01:06, 26 October 2017

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

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, pTlpA36 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 pTlpA36. Under low temperature (below 36 degrees), TlpA36 protein dimerises and becomes capable of repressing pTlpA36; 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 pTlpA36 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