Team:NFLS/Proof Of Concept
In our system, we use endoglucanase(cen), exoglucanase(cex) and β-glucosidase to break cellulose into glucose, and we also expecting to use transport proteins to input cellobiose to increase the efficiency of the system. But due to the out break of the coronavirus, our experiments were delayed, and we were only able to test the efficiency of endoglucanase(cen) and exoglucanase(cex).
First, we performed SDS-PAGE to ensure the protein was successfully expressed. Cen protein is in Lane 2-4, and Cex protein is in Lane 5-6. Since endoglucanase is 58.7kDa and exoglucanase is 63.3kDa, our results suggest that these two proteins have already been successfully expressed.
Moreover, we used Congo Red assay to test the activity of the enzyme. Congo red forms red complex with interacted with cellulose. Therefore, when Congo red and cellulose were both added into the plate, the plate is red. When the cellulose in the plate is degraded into oligosaccharide or monosaccharide, the red color diminished and forming orange or transparent circle around the cell colony. Therefore, Conge test is usually used for the detection of cellulose degradation. In our experiment, the Congo test was conducted to demonstrate that our engineered strain which contains both of endoglucanase(cen) and exoglucanase(cex) is capable of degrading cellulose.
Three parallel experiments were conducted simultaneously on one plate. Our engineered strain was incubated on the plate at three different spots. After being incubated for 24 hours under 30 C, all the three clearly showed a destained transparent circle around the colonies of around 2.5 cm each (Figure 2). This clearly indicated that the surrounding cellulose was decomposed by the cell. Thus, the result illustrated that our engineered strain is able to successfully express the desired enzymes and to degrade the cellulose.
Above all, we give a good proof of concept that our system can degrade the cellulose.
What we are going to do in the future
This year, our plan is totally messed up by the coronavirus. We designed many parts that we hope we can characterize this year at the beginning. But we ended up have no lab place to do this. So we would like to continue our project in iGEM next year if possible.
We would like to first characterize the fusion protein of cen and cex with signal peptide, and the transport system. Furthermore, we would like to engineer transport protein cdtC protein in E.coli which no one has ever done before in iGEM.
Lastly, we would like to test our whole system in Pseudomonas aeruginosa which will have better efficiency in MFCs.
