Team:NFLS/Design
System 1.0
The general design of our design is to use endoglucanase, exoglucanase and β-glucosidase to break cellulose into glucose that can be utilized by E.coli-based microbial fuel cell to generate electricity. So, first we want to engineering a E.coli with this three enzymes that directly expressed in the cells in order to break cellulose. We use the famous constitutive promoter J23119 as our promoter in the system.
System 2.0
After reading Intracellular cellobiose metabolism and its applications in lignocellulose-based biorefineries(Jay D. Keasling et al.), we found that it’s more efficient if cellobiose is first brought into the cell and then broken down inside the cell. Based on this, we decided to add signal peptide onto endoglucanase and exoglucanase, hence allow them to be secreted outside cell to break cellulose down to cellobiose. Then cellobiose is taken into the genetically-engineered E.coli by transport protein on the membrane. Inside the cell, cellobiose is broken into glucose by β-glucosidase. Once we have glucose, we have the energy source for E.coli to generate electricity inside the microbial fuel cell.
We also designed a hardware that allows a full-developed chain to deal with waste paper. First, waste paper is broken mechanically, then dissolved into organic solvent. Next, the genetically-engineered bacteria will finish the job of breaking it down and generate electricity. (view more in Hardware)
Why we choose E.coli?
Besides its ability to cause diseases in both plants and animals, E.coli is a ideal candidate for our project. Since researchers already have well-developed study on its cell structure, physiology feature and inheritance background, the standard practices on genetically-modifying E.coli is easy and not risky. Moreover, as E.coli has relative loose plasmids that favors large quantity replication in the target cell. Also, the existence of genes like penicillin resistance gene that are easily located makes it easy to locate specific position on the plasmid. Also, in anaerobic respiration condition, hydrogen atom can be produced and grabbed by the electrode, allowing for generation of electricity. The high metabolic rate and short cell cycle also makes E.coli suitable for genetic engineering experiment for the proof of concept. We use strain E.coli K12 in our experiments.
Signal peptide:
Signal peptide are short peptide chain, composed of 5-30 amino acids, that can guide synthesized protein to the secretion pathway. As mentioned before, if exoglucanase and endoglucanase are secreted outside the cell, the efficiency of the whole setup can be improved. We decide to use signal peptide OmpA to mark exoglucanase and endoglucanase, thus the enzymes can be led to exocellular pathway.
Cellobiose Transport and Enzyme Production:
Since cellulose is broken into cellobiose outside the cell, a PTS-dependent cellobiose metabolic system is applied to intake the cellobiose and allow for the next step of breaking down. The system is based on PTS-dependent cellobiose metabolic operons, including ChbB, ChbC and ChbA, as these three elements are enough to be functional. Gene been used include endoglucanase(cen) gene, exoglucanase(cex) gene and β-glucosidase gene are implanted to E.coli plasmids.
System 3.0
After deep communicating with Prof. Yong (human practice). We learned two things that is important to our design. First, we need to add a kill switch to our system in order to ensure safety when it comes to a real application, we choose Supernova which is a gene that can kill the cell by inducing superoxide under the exposure of light. Second, E.coli maybe a good proof of concept, but its efficiency in MFCs is not that high. Following Prof. Yong’s suggestions, we came up with the new system by using Pseudomonas aeruginosa as the chassis in further potential applications. But we didn’t perform experiments on Pseudomonas aeruginosa because of the outbreak of Coronavirus delayed our experiments.
