Proof of concept
To support the results in silico, we followed a protocol that could provide laboratory evidence. The proof of concept consists in comparing the fluorescence levels of induction in E. coli under two different parameters for the BL21 strain with our designed plasmid; one with IPTG alone and the other with both IPTG and EE2.
Right after designing the plasmid in silico, we sent it to be synthesized by GenScript. Once the process was completed, we transformed the plasmid into E.coli from the BL21 strain.
After the transformation, we prepared our pre-inoculum culture, then began growing the inoculums, starting with an optimal density (OD) of 0.1. We kept the incubation and measured the OD until reaching 0.6, indicating that the cells were ready for induction with IPTG, which we carried overnight.
The induction was repeated, but this second time, we induced it with both IPTG and EE2 in the culture in order to observe any difference in the protein expression and functionality as EE2 was expected to interact with the designed protein.
After both induction conditions, the inoculums were centrifuged and lysated to release the expressed proteins, which were analyzed by measuring fluorescence, as this allowed us to quantify the induction levels and compare the effects of the two conditions: IPTG alone vs. IPTG with EE2.This comparison served as a proof of concept for the interaction predicted during the in silico phase of the project and to demonstrate the potential interaction between EE2 and the engineered protein.
The process followed and previously explained can be observed in the following diagram:
Proof of concept diagram