In the short time you have to work at your project it is very difficult to express a conductive pilus in E. coli. Especially, since the structural proteins have not yet been clearly analyzed which genes are responsible for the pilus and his conductivity so it isn’t easy to work with it. If you reach your goal it would be a great scientific success. It is very interesting to express this pilus of G. sulfurreducens in E. coli because it has got the highest electron-transfer-constant so it would be very effective.

It is commonly known that pili of E. coli cause human disease. The strain which you are working with is a safety strain without these dangerous pili. In my opinion there doesn’t exist the problem of changing the E. coli S1 strain into a S2 strain because of the fact that this pilus of G. sulfurreducens isn’t associated with diseases rather it is for transferring electrons.

Generally it is a good one. The advantage of the mtrCAB cluster from S. oneidensis is better related to E. coli then the cluster from Geobacter sulfurreducens, but the negative aspect of this idea is that the mtrCAB cluster only has a low electron transfer constant. This means that in comparison to the transfer via mediators or direct transfer via pili the electrons need more time to get through this tunnel-like protein. It could be problematic to get the proteins in correct orientation in the membrane. This problem can be solved by anaerobe coexpression of the ccm-cluster. The idea is challenging but practicable.

It is a good idea to use self-producing mediators for your electron transfer. Phenazine is a protein from Pseudomonas chlororaphes pcl1391 an S1 organism. A problem could be that derivates of phenazine can act as vegetable antibiotic, so it might be better to express different mediator. Maybe you can use riboflavin, which is produced by S. oneidensis MR-1. If you overexpress these genes you could express an efficient mediator. But as there exist no experience with this approach you might have to find a way how to secrete the riboflavin, if this isn't secreted naturally. The combination of the riboflavin from S. oneidensis and the porines would be a good idea.

This is also an interesting approach. I am very interested if this will work, because it could be a great challenge to improve the reduction of the mediator in the bacteria. But it is very important to pay attention that the potential of the membrane is not too low. This is the most important aspect that you have to look at.

You have to minimize all the distance between the different parts in your MFC. Every distance is a resistor and decrease your demand. The optimal size of your MFC depends on the application you want to choose. For testing the principal of your system is the actual size of your MFC good. If you want to get it smaller other problems will appear which you would have to solve. When you want to integrate your system you have to conform it correspondingly.

The system what you want to use is a good one. In my opinion it is save and I don’t think that this will be a problem to use it. There exist another good biosafety idea but for Geobacter sulfurreducens. If you would work with this bacteria you could use the aspect that this organism use only one electron acceptor when it is under anaerobe conditions. When this organism gets out of the MFC it will die because there wouldn’t be the one electron acceptor which is needed. But as E. coli is facultative aerobe this system won’t work here, because E. coli has several possibilities to oxidize the substrate.

It is a very interesting new idea with a high potential for producing compounds/energy or degradation harmful substances. Many scientists of different fields of knowledge research together. There is much research necessary but in the future it will take a part in different fields. For remediation it would be the best to use mixed culture, while Bacteria as E. coli have the most potential to combine synthesis and energy production. Another challenging aspect would also be to introduce biosynthesis specific with electrons. As you see there is a lot of interesting work to be done and I hope that your project can help to develop the MFC a liitle bit further.