Team:Harvard BioDesign/Attributions

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Attributions

What we did:

The iGEM team was responsible for:

• Conducting all wet lab experiments
• Wiki adaptation and content
• Graphic Design, photography, and figures
• Contacting experts and conducting interviews

Special thanks to our advisor Neel Joshi, Ph.D., as well as to our host lab-- the Joshi lab-- at the Wyss Institute for Biologically Inspired Engineering and to our mentors, who guided us through the experimental design of our project and dedicated their time to helping us analyze and interpret our results and data. We would also like to thank the John A. Paulson School of Engineering and Applied Sciences, which generously supported our summer research.

Mentorship and Support:

We would like to thank:

• Pichet (Bom) Praveschotinunt
• David Lips
• Kevin Hof
• Ethan Alley
• Harvard iGEM Club for their administrative and logistical support
• NEGEM for valuable feedback on our ideas and presentations

Our Inspiration:

We would like to acknowledge previous researchers both within and outside iGEM, whose work informed our design and experiments:

• Yoshida et. al. Their 2016 Science paper A bacterium that degrades and assimilates poly(ethylene terephthalate) discovered and characterized the enzyme that forms the basis of our project design. The paper also documents the catalytic activity of PETase expressed in an E. Coli chassis.
• UC Davis iGEM 2012 We adapted their pnpb assay protocol for our own experiments to determine the catalytic activity of PETase. They showed that pelB worked as a signal sequence for sending LC Cutinase (which is similar in size to PETase) to the extracellular medium, which gave us evidence that it might work for PETase. Read more on their work here.
• Oxford iGEM 2015 They showed that YebF, a protein naturally secreted to the extracellular medium in E. Coli lab strains, when fused to their protein, successfully secreted the protein to the outer membrane.
• Bielefeld iGEM 2013 Their work on blueprints for a 3D-printed microbial fuel cell and with exogenous mediators provided a useful foundation on which we built our project.
• Northeastern iGEM 2016 Their rigorous collaboration assays helped inform our holistic view of our project design.
• Shigematsu, Department of Materials and Life Science, Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami, Kumamoto-City, Kumamoto This is the lab that discovered the D. tsuruhatensis bacteria from a domestic wastewater treatment plant and published Delftia tsuruhatensis sp. nov., a terephthalate-assimilating bacterium isolated from activated sludge.

Human Practices Support:

We would like to thank:

• The Seabin Project for taking the time to speak with us and for discussing potential collaborations between our projects.


• Parley for the Oceans for sending us samples of ocean plastic to use in our research.


• BosLab for inviting Harvard BioDesign to volunteer at a public outreach event, 'Building with Biology.'


• Prof. Peter R. Girguis: Professor Joshi suggested reaching out to Professor Girguis, a member of Harvard's faculty who has previously led an iGEM team. His work was of particular interest to our team because it entails, among other things, developing microbial fuel cells for marine environments. The professor provided a number of technical suggestions: he advised the use of titanium wires instead of copper as they release ions and disturb the potential results we will get. He also suggested using a Standalone voltage data logger for voltage and current measurements and data computing. Girguis also encouraged us to consider the economic viability of our fuel cell project. To help illustrate his point, he discussed his own research and explained that in the case of electronic devices in remote locations at the bottom of the ocean, microbial fuel cells can be more cost-effective than alternatives such as batteries that need to be constantly replaced.


• Erika Parra, Ph.D. Parra is an associate of Harvard's Department of Organismic and Evolutionary Biology. She has extensive experience working with fuel cells and was kind enough to visit our lab space at the Wyss to help us assemble our cell.


• Dr. James Weaver: We initially visited the machine shop and they directed us to Dr. Weaver to help us optimize Bielefeld’s MFC design. He noted some design problems in the blueprints for the microbial fuel cell parts. He therefore suggested three changes: extension of the “ear-like” structure, where the bolts go, in the frames as well, besides the shells, so that the frames are better centered; remodeling of the channel that the fluid flows through so that better mixing is induced; and elimination of the “lips” in both the frames and the shells so that the silicone sealing works better and fluid leakage is prevented. Dr. Weaver also helped with the 3D printer that works with UV light, located in 60 Oxford Street, part of Harvard's School John A. Paulson School of Engineering and Applied Sciences (SEAS).


• Prof. Derek Lovley: Prof. Derek Lovely is one of the leading experts of microbial fuel cell (MFC) technology and has conducted numerous lectures on the economic viability of MFCs. We had a conversation over the phone with Prof. Lovley. on June 21. He suggested looking at specific literature for the building of a microbial fuel cell. SInce platinum is expensive, he thought that the use of ferricyanide as a catalyzer would work as well. He advised to make the chamber anaerobic, because the organisms would otherwise transfer electrons to oxygen instead of the anode itself. He suggested to start off with finding an existing anaerobic digester, such that engineering one might not be necessary. Prof. Lovley noted that Geobacter and Shewanella are usually used in microbial fuel cells but that we may need to use another species if we want our bacteria to metabolize terephthalic acid.”


• Buz Barstow, Ph.D.: Professor Joshi also directed us towards Dr. Barstow because he has worked closely with MFCs and could help us with the technical aspects of assembling a cell. We consulted Dr. Barstow regarding the use Bacillus and Arthrobacter in a microbial fuel cell, the efficiency of a MFC, and its potential uses. He also provided us with some insightful questions, ranging from fields as diverse as economics to safety. What follows is a message Barstow addressed to our team:

“That’s a really good question. I’m not too sure about Arthrobacter or Bacillus sp. Protocatechuate, and my guess is as good as yours on whether or not they can be used in a microbial fuel cell.

Also, my guess is as good as yours on whether E. coli can degrade (or be engineered to degrade) terephthalic acid. I would do some google scholaring and see what you can find. How many genes are involved in terephthalic acid degradation?

You might want to ask yourselves the following questions: how much do you care about degrading terephthalic acid and how much do you care about making electricity? Also, does the ability to degrade terephthalic acid translate into an ability to degrade PET.

Typically, the yield of electricity from a fuel cell is fairly small, so even a mountain of PET bottles is not going to be a solution to the world’s energy problems. However, degrading a stack of bottles is a solution the plastic pollution problem. My feeling is that a microbial fuel cell is only useful here if it allows you to get rid of reducing equivalents from the cell that you wouldn’t be able to get rid of otherwise (say if you can’t access oxygen due to the need for oxygen sensitive enzymes in terephthalic acid degradation).

Ask yourself the following question: given that Arthrobacter can already degrade terephthalic acid, what bottleneck is preventing widespread deployment of this microbe in the real world? Then try to come up with a solution to that problem.

Also: be aware that Arthrobacter and Bacillus sp. Protocatechuate could also be BSL2.

All the best, and good luck!”

• Prof. Eiji Masai: He is the corresponding author of papers where researchers attempt to sequence the TPA degradation and uptake genes of the Comamonas sp. Strain E6. He directed us to some bacteria that potentially degrade TPA on ATCC (bacillus species) and informed us how to obtain the Comamonas species that his lab deposited.


• Sara Hamel Ph.D.: We obtained her contact information through one of our mentors, Bom Pichet Praveschotinunt, who said that she could help us with the printing and building of the MFC. Sara assisted in 3D printing the shells and the frames for our initial microbial fuel cell based on the design we provided from the Bielefeld iGem Team 2013. The printer used was MakerBot, located in Pierce Hall, part of Harvard's School John A. Paulson School of Engineering and Applied Sciences. She was also responsible for coordinating access to lab space on campus during the school year.

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