Objectives

To investigate why GFP expression was high in the absence of signal in our engineered BDSF system.

BDSF-Induced Phosphorylation of BCAM0228[P] Mediates GFP Expression

We constructed models¹ similar to those used in the PQS system to investigate the signal-response behaviour of the BDSF system. Our results verified what we had expected to happen^2,3. Phosphorylation of BCAM0228 is induced by BDSF binding to a cell receptor. BCAM0228[P] then activated the expression of GFP through the engineered cbld promoter. In the absence of a BDSF signal, BCAM0228 remained in its unphosphorylated form and GFP expression was repressed. Increasing the signal induced an increased GFP response (Fig 1).

BCAM0228 is Phosphorylated by Unknown Compound

Our experimental results revealed that in the absence of BDSF, the cblD promoter was active in our engineered system and hence GFP expression upregulated.

From our system of equations we know that the expression of GFP is dependant on the levels of BCAM0228[P]. We hypothesised that one possible mechanism by which our cell could be expressing GFP in the absence of signal was by the presence of BCAM0228[P] independent of BDSF-binding mediated phosphorylation. This suggests that an unknown compound could be phosphorylating BCAM0228 in our engineered cells.

Comparing Fig 2 to Fig 1, it is clear that the model predicts that the expression of GFP is higher when BCAM0228 is being constitutively phosphorylated than in the “normal” case. Moreover, we are also able to conclude that the model predicts that the presence of BDSF has very little effect on the overall expression of GFP when the constitutive phosphorylation process is operating.

Conclusions

Our models predict that an as yet unknown agent phosphorylates BCAM0228 in the absence of a BDSF signal and that in fact the level of the BDSF signal has little quantifiable effect on GFP expression in our engineered cells.