Team:Nanjing NFLS/Implementation
Safety and security no wonder are prior things we have to consider when dealing with a synthetic biology project. We consider our safe work in two aspects: The lab we are working have the bio-safety cabinet for the biosafety level 2 organisms. The organism we use is Pseudomonas aeruginosa (PAO-1), (Bio Safety Level 2), an opportunistic pathogen normally may cause slight fever in susceptible population but under control in most situations. Though our project is not involved in human bodies, there are still many other safety rules to consider, not release, anti-microbial resistance, etc. We should not neglect any possible safety risk, while for our team is the Not Release rule. On the left a primary sketch of our hardware, reaction chamber of MFCs. (anode and cathode chamber with same structure, the electrodes and proton exchange membrane (PEM) not shown in the picture) We don’t add new components to our design, both with electrodes, chamber, PEM and several opens help fill in materials and pour out excessive wastes. But we also make some changes: To ensure safety, we should keep all the opens sealed outside a controlled area like lab, so that the bacteria will not release and contaminate the surroundings. Open A will be used for infusing air at start-up stage of bacteria cultivation, because we will carry out the aerobic and anaerobic strategy for higher PYO production and better biofilm formation (mentioned in our design, focus 5). Open C will be open at the same time, and air rich in oxygen will pass through the solution from the bottom to the top, providing enough oxygen for the start-up growth of P. aeruginosa. The procedure should be done in controlled places before the MFCs are put into electricity generation. The process can also be realized by a magnetic stirrer in the lab, but that will be not practical in large scale for commercial use. It’s worth mentioning that current waste treatment applications are mainly in anode, and electrochemically active bacteria like P. aeruginosa should experience acclimation. In acclimation organism adjusting to a change (like carbon source change) in its environment , before they can effectively gain material from the pollution water. For example, P. aeruginosa will be cultivate in a mixture of extremely low concentrate wastewater and glucose (1 g/L) as the carbon source was fed at the first feeding cycle to facilitate initial microorganism growth. During the following cycles, we should gradually increate the concentration of waste water while decrease glucose to 0 g / L, so that P.aeruginosa can consume some recalcitrant pollutions. After the hardware reach its lifetime or the electricity output has dropped far below the expectation or any accident happens, the MFCs will be immediately recycled by us and scrap them. All P. aeruginosa will be inactivated as carefully disposed. We will formulate the protocol for operations in different scenarios, and thoroughly follow the rule “Not Release”. In current stage, we finish the demonstration of overexpressing three gene. Due to limited access to lab under the pandemic of COVID-19, we cannot carry out experiments by incubating our engineered P. aeruginosa in modified MFCs. Although we use math modeling for predicting the performance of modified MFCs, the test in vitro cannot replace the experiments of real operation of MFCs. If we have the change, we will conduct the following experiments and demonstrate the electricity output improvement and finally fulfill the proof of concept. The major bottlenecks of MFCs application include (1) low power density, (2) high capital expense and (3) high operational expense. Even though engineer the bacteria by synthetic method, the math modeling and related article show that the electricity output is still incomparable with real life use. Besides electron transfer and biocatalytic activity, other non-bio imitations in performance due to design factors like electrode material, reactor vessel design, electrical configuration (series or parallel), internal resistance, electron transfer and biocatalytic activity hinder the popularization of MFCs. We hope more progress will be made in future, hasten the application of the promising “Midas finger”. 
Safety
Lab Safety
Teammates who are working with these organisms have to be well trained by our professors and Phds before the experiment, learning about lab access and rules, equipment and technique choosing and operation, emergency procedures, physical and personnel biosecurity, etc. Our PI, Professor Yong is responsible for our safety and security during the experiment. He is expert in MFC researches and he has a very long history of working with P. Aeruginosa and other bacteria.
We will wear lab coat and gloves in the experiments; we will disinfect our hands after the experiments. The wasted bacteria will be diposed in a specific bag and sent to expects of taking bio-waste. The demonstration of overexpressing nadE (NAD synthase gene), rhlA (rhamnosyl transferase gene), and phzM (methyltransferase encoding gene), are all followed by protocols and lab rules.
Specific Biosafety ------ Supernova
Although we don’t introduce any exogenous genes to P. aeruginosa, the added plasmids contain these genes may be easily transfacted to other wild type organisms and may cause unpredictable consequences.
To tackle the safety problems in the anode, we consider which strategy to use. However, we designed the energy source of the P. aeruginosa is from low concentration waste water, where the composition will be complicated and difficult to control. So we exclude strategies involving molecular regulations to contain engineered bacteria in the anode. While we delve more in identical physical conditions in the anode, team Tsinghua inspires us to employ a light kill switch, and we also find a list of candidates in the registry. We finally choose Codon Optimized Supernova (Part:BBa_K1491017) and decide to optimize it for P. aeruginosa in future. (Supernova is the monomeric version of KillerRed (BBa_K1184000). It has been codon optimized for E. coli.)
Like KillerRed, supernova also produces reactive oxygen species (ROS) in the presence of yellow-orange light (540-585 nm). Supernova produces superoxide radical anions by reacting with water. Superoxide (O2•-) is the radical anion of molecular oxygen, which can react with protein side chains and lipids to harm cells.
Codon optimization should be taken into account for uses in P. aeruginosa. We decide to integrate supernova into the plasmid of phzM, and expression of supernova and irradiation with light act a kill-switch for biosafety applications in our MFCs.
Hardware
1) position and number of the opens. Open A for infusing gas which can get full contact with the solution. Open B for infusing liquid to be processed or to provide carbon source. Open C for ventilating gas when necessary, Open D for acquiring supernatant of the solution in the chamber. Open E for drop out all solution
2) opaqueness of the anode and PEM as part of light kill switch system
Safety first
Also, the opaqueness serve to the action of supernova: while P. aeruginosa can thrive on the anode in the dark chamber, they suicide due to supernova gene when exposed in the light outside the chamber. To ensure the effectiveness of supernova, the energy producing workshops should be installed with constant yellow-orange light source.
Enhance electricity output
Open B will be infused with pollution water as energy source for bacteria. We will do more human practices and filter out which kind of waste water meet the criteria: low concentration of certain metal ions and carbon source (mentioned in our design, focus 5).
Use for waste water treatment
We may also use the traditional strategy; Open B will be pumped in low-cost waste water and metal ions. After carbon sources are used up and P. aeruginosa begin to drop in electricity output, we will drop out the solution from Open E, which should be less toxic than before treatment. Remember, the solution should be restrictedly sterilized and then be sent to downstream industry for further treatment. However, our purpose to employ the strategy is mainly to save money and improve electricity output. We interested more in waste water treatment in cathode:
Pollution also come in Open B in cathode, and then interacts sufficiently with the Fenton Reagent produced by novel Fe-Mn/GF composite cathode (mentioned in our design, focus 4). After some time of degrading, we hope to extract the supernatant of the processed solution out of the chamber through Open D. Since no living organisms in cathode, less pretreatment will be conducted for the supernatant.
Recycle
Vision
For our project
We may integrate all the focuses we mentioned in design to figure out how they work together, whether there will be interference between each part, and how much burden are imposed on the integrated system altogether. After that, we can rule out some inapplicable parts and fuel with other new innovations.
We also should do more research and elaborate the complicated pathways of EABs. For example, through overexpression of rhlA and nadE, we find the production of PYO also increase which should be expected for phzM overexpression. More details about quorum sensing, biofilm formation, roles of metal ions, etc. should be clarified by investigating more researches, testing in experiments, and consulting to more experts.
For MFCs