Team:EPFL/Contribution
Our Contribution
We characterized the following parts in our self-made E. Coli lysates : BBa_E0040 and BBa_J64997.
BBa_E0040 : Expression of GFP-mut3b in cell lysate
Cell-free GFP-mut3b synthesis was analyzed in self-made E. Coli lysate from strain BL21(DE3). Fluorescence was measured at 37°C for five hours on a plate reader. For details on how the lysate and the energy solution were made and which components went into the final reaction volume of 10uL, check out our protocols. Shown are three repeats with a negative control as well as a shaded error graph (control was subtracted) summarizing the result. GFP-mut3b expression yields high signals in lysate and thus is a good choice of reporter while working on a cell-free chassis. Saturation occurs after about five hours.
BBa_J64997 : Effect of a promoter on protein synthesis in cell-free expression systems
The T7 promoter was characterized in self-made E.Coli lysate from strain BL21(DE). Cell-free T7-GFP-mut3b versus GFP-mut3b (BBa_E0040) synthesis was analyzed. Fluorescence was measured at 37°C for five hours on a plate reader. For details on how the lysate and the energy solution were made and which components went into the final reaction volume of 10uL, check out our protocols. Shown are two times three repeats and a negative control, a shaded error graph (control was subtracted) and a bar plot summarizing the result.
GFP-mut3b expression cannot be dinstinguished from the no DNA control. T7-GFP-mut3b expression however is high. Saturation occurs after about five hours. Adding the T7 promoter in front of the GFP-mut3b enables transcription by the T7 polymerase, a much more efficient polymerase than the E.Coli polymerase found natively in E.Coli cells.
