Contribution
For our contribution to iGEM, we wanted to present our prototype for our Microbial Fuel Cell (MFC). Indeed, seeing as StarchLight is a two-bacteria system, we had to adapt our MFC to accommodate both of them. Moreover, we have access to a 3D printer at our school, so we had the freedom to design it as we saw fit. We did research in order to build it correctly, and saw that each year, few iGEM teams have projects involving MFCs. Therefore, we wanted to help them save time by explaining how we designed our MFC, how we created the 3D plans, and how to use a 3D printer. We also included the steps on making our very first prototype that we used for preliminary tests on the electricity production of Shewanella oneidensis.
Finally, we also wanted to help future iGEM teams by including a troubleshooting part. We explained the problems we encountered in our Wet Lab throughout the project, and we hope other teams will be able to learn from our mistakes.
StarchLight prototype plans
As explained before, StarchLight designed its MFC with three compartments : the first one with E. coli to produce the lactate, the second one contains the anode and S. oneidensis, and the third one with the cathode.
Each compartment is separated by selective membranes. Between the first and second compartment, there is a lactate selective membrane, and the second and the third compartment are separated by a proton exchange membrane (see Figure 1).
Figure 1: Scheme of the StarchLight MFC. The StarchLight Microbial Fuel Cell has three compartments. The first compartment, on the right of the scheme, is where the bioengineered E. coli degrades the starch from the brewer's spent grain to produce polylactate. When E. coli receives an external signal, here blue light exposition, it degrades the polylactate into lactate. The lactate goes through the Lactate-selective membrane into the second compartment where S. oneidensis is growing on the anode. S. oneidensis uses the lactate to produce electrons that go to the cathode thanks to the electric wire. It also produces protons that go through the proton exchange membrane into the third compartment.
We created the plans of our MFC thanks to the SolidWorks software (https://www.solidworks.com/fr) in order to be able to 3D print it afterwards (see Figure 2). We've made these plans available to download for everyone if they want to use them for their own project, modify them or print them!
Figure 2: Image from SolidWorks of the prototype of the MFC battery. A view of all the different parts of the StarchLight MFC. At the bottom is the main part of the battery, with the three compartments. Above it are the two membrane frames that hold the lactate permeable membrane and the proton permeable membrane. Finally, the lid of the battery has the holes the anode and the cathode, as well as an opening to activate the optogenetic system.
Tutorial on how using a conception 3D site
SolidWorks is a great software to produce 3D plans, but it can be quite complicated. In our team, it was Louis, a member of our Dry Lab team, who used Solidworks to make the 3D plans of the StarchLight prototype, as he was quite experienced with it. However, in order to help over teams either modify our plans or create their own, we decided to help them by creating a little tutorial video, with some of the basic functionalities of the software. The tutorial is based on how to design a screw from our prototype.
Figure 3: Tutorial on how using SolidWorks, a 3D conception software, done by Louis
Tutorial on how using a 3D printer
Moreover, once we had our plans, we had to 3D print them in order to be able to use it and test the StarchLight system. However, we had never used a 3D printer before, so it was quite intimidating to try. Therefore, once we figured out how to do it with the help of Isaline Roy, we filmed a little explanation video that we hope will be very useful to other teams that face the same issues.
Figure 4: Tutorial on how using a 3D-printer
StarchLight's very first prototype
At the very beginning of our project, we wanted to do basic electricity tests of our electricity-producing bacterium S. oneidensis. However, at that point, we hadn't created our prototype yet. Therefore, we decided to use what we had in the lab to create one.
We used two Falcon 50 mL tubes, one Nafion proton exchange membrane (PEM), and a multimeter. The Falcon tubes serve as two electrode compartments.
We first cut a hole of about 4x1 cm on the side of each falcon tube. We also cut holes in the lids of the Falcon tubes. Between the two lids, we put a rectangle of Nafion exchange membrane, and we parafilmed the two Falcon tubes together to seal the tubes and make it waterproof. We finally used sticky tape to make sure everything held together nicely. In the first Falcon tube, we put the anode of the multimeter, and in the second tube, we put the cathode.
Figure 5: Our first prototype with the cut Falcon tubes, and the Nafion proton exchange membrane PEM between the tubes
Figure 6: Our assembled first prototype for Shewanella oneidensis electricity measures
We put 35 mL of LB in both falcon tubes. In the first tube, the anode compartment, we took a 250 uL aliquot of Shewanella oneidensis with a OD of 0.7 at 600nm. We pipetted this aliquot directly on the anode to facilitate the creation of a biofilm on it by Shewanella oneidensis. Then, for the next few hours, we used the multimeter to see the electricity produced by Shewanella oneidensis.
Troubleshooting
We encountered different issues with the sequences that we had made synthesized. For three of our sequences, the sequences were not correct for the enzyme digestion method and the T7 promoter. These sequences were the composite parts for the Light-activated DNA-binding protein (EL222), the alpha-amylase (AmyH), the D-Lactate Dehydrogenase (LDH).
After digesting LDH and EL222 several times, we always obtained the same results, only one band was observed on our migration gel. By checking again the sequences, we noticed the presence of a methylation at the site of cutting of XbaI. To correct it for LDH, we ordered primers and corrected it via Polymerase Chain Reaction (PCR) (Figure 3) and for EL222, we ordered again the all sequence without the methylation.
Figure 7: Gel electrophoresis of the corrected amplified LDH done on 09/06/2022. The DNA ladder used is the 1 kb DNA Ladder from Promega that is defined on the left. LDH was corrected on the methylation issue. Without counting the DNA ladder, each well corresponds to LDH and for the third well, the PCR did not work.
Then, concerning the T7 promoter of LDH and AmyH, its sequence was altered probably when designing the sequence on SnapGene. To fix it, we designed primers and performed a PCR which is the simplest method.
Figure 8: Gel electrophoresis of the corrected amplified AmyH and LDH done on 09/27/2022. The DNA ladder used is the 1 kb Plus DNA Ladder from NEB that is defined on the left. Both T7 promoters of LDH and AmyH were corrected via PCR. Without counting the last well, the first six wells correspond to AmyH and the last three wells correspond to the amplified LDH. The expected size of the AmyH was 1,629 bp. The observed bands are slightly above 1,500bp. These bands correspond to the expected size of the corrected AmyH.The expected size of the LDH was 1,243 bp. The observed bands are slightly above 1,200bp. These bands correspond to the expected size of the corrected LDH.