Difference between revisions of "Team:OLS Canmore AB CA/Collaborations"

This year we have participated in 3 collaborations. All three of these were focused on seeing what other teams thought about our project, soliciting advice about the best usage for our construct and getting insights into ethical concerns regarding implementation.

Two collaborations were with the two universities that have worked on this project in the past. The University of Chicago 2013 iGEM team worked on a project aimed at breaking down feathers with the goal of turning the products produced by the keratin waste into useful products. The University of Sheffield 2014 iGEM team worked on a construct that will break down hair as a side project to their main “fatburger” project. Our collaborations with these teams was centered around the sharing of information regarding their past constructs, as our project is based on troubleshooting and debugging those issues. Further, we hoped to characterize these parts, get input on ethical and safety concerns, and plan the future tests and implementation of our project.

Our third collaboration was with a high school iGEM team based out of Consort, Alberta. Their project they've been working on for the past three seasons has been working to detect oil spills in soil by first detecting xylene, as xylene is a good early biological indicator. We collaborated with them in order to receive feedback on what they thought about our project as well as to give them feedback on their project. As geographically close high school teams, it was nice to have some face time at the mini Alberta jamboree we hosted in May 2015 at our school. We shared a lot of initial feedback about the pros and cons of our presentations, and continued to be in close contact with the Consort team as we prepared for the Giant Jamboree!

The University of Sheffield 2014 Collaboration.

Our first collaboration was made with the the university of Sheffield, we connected with them with the goal of hearing their thoughts on our project and getting any advice that they were willing to give concerning our project, as one of our constructs is based off their construct that was created as a side project to there “Fatburger” project, a project which was started as a solution to fats and oils build ups, which are caused when fats and oils solidified in the drains. The side project to break down hair was intended “To solve the build up of hair that becomes trapped in plugholes in basins, baths and showers. Hair attracts grease and dirt particulates which build up in drainage systems and restrict the flow of water through pipes. Our project has focused on development of constructs that produce lipase and Keratinase enzymes to degrade FOGs and hair. These constructs are designed to sit within a small, under-sink bioreactor that will produce and feed them into the waste system as the sink is drained.”(https://2014.igem.org/Team:Sheffield/Project) Their construct was designed to break down hair by using a Keratinase that would break down the Keratin protein, however there project was unsuccessful. We started by using the University of Sheffield's construct and then optimized it by fixing the known cleaving issue, and secretion of the protein. Our collaboration with the University of Sheffield 2014 iGEM team allowed us to receive some input into our project and what we could do to make it even better.

University of Sheffield Correspondence

To whom it may concern: My name is Freya Morgan, and I am a member of the Our Lady Of the Snows Catholic Academy iGEM team. We are a small high school based based out of Canmore, Alberta, Canada. We are contacting you because we have chosen to tackle build ups of keratin in wastewater treatment, and well as the poultry industry as this year’s project, and you tackled this issue in your 2014 project. When talking about wastewater treatment, these build ups of hair can lead to clogs causing flooding and the breakage of water pumps and pipes, something very costly to repair. The only current way to deal with this issue is to have a worker manually remove the blockage, which is slow, unpleasant for the worker and inefficient. Upon further research our team discovered that the reason hair is such a huge issue for waste treatment facilities is because of the protein keratin, which as you probably know is a very hard protein to break down due the tightly wrapped helixes and hydrogen bonds. Once we discovered this pressing issue we looked into keratin more and realized it is also a component of the basic construct of feathers. We learned that they too build up and are hard to break down. With more searching on the iGEM parts registry we discovered your project, as well as one other project, both of whom attempted to solve the keratin issue. With this research and the exploration of previous iGEM projects, we decided to create a construct that would express a Keratinase enzyme to break down the keratin in the hair and feathers, thus solving the issue. Because iGEM requires collaboration between teams, we decided to try contact you due to your decision to tackle this problem last year. As much of our project is based on building on, and optimizing previous constructs, we are looking for some more in-depth information concerning the cleaving process, and some issues you tackled there, and how you have dealt with them. We know that Keratinases are difficult to express in E. Coli, due to it getting stuck in the periplasm when expressed, because the Keratinase folds, and because when expressed, the export tag does not always get cleaved properly, so if you could give us any insight upon this, as that is an issue you have faced, that would be appreciated. We have worked with our mentors to optimize the Keratinase cleaving issue, but haven't yet managed to express it, due to problems with our lab work. We think that the cleaving issue should be fixed, but are not sure yet. We would also like to get some of your current opinions on the issues, if you have any advice for us that could make us more successful at the competition and if you had devised a way in which you would have deployed your construct outside of the lab, if you had been successful. We also were wondering what your plans were for implementation of the biobrick into the real-world, and safety precautions to be taken outside the lab through implementation. We have been thinking bioreactor implementation for wastewater treatment plants, but regarding poultry farms, it is not a very applicable implementation. Looking forward to hearing back from you soon and we understand if you can't answer some of our questions. Thank You, Sincerely, Our Lady Of The Snows Catholic Academy 2015 iGEM Team

Hi Freya (and team), In regards to the Keratinase results we saw - once we had colonies that we'd confirmed contained the Keratinase via PCR, we went straight to assay via incubation of feathers/nails (grim I know) into cell culture during growth. The paper we'd taken the construct sequence from had a great set of images of feathers being degraded over a period of 24h or a few days. We had no luck there which is why we chose to use SDS-PAGE to see if we had any product at all. If i remember correctly (though you might want to check our wiki) we had a band of 'protein' that corresponded neither to the pre- or post- cleavage kDA size for the enzyme. I think we found a paper indicating that the secretion step may occur in 2 parts, but I don't think we could confirm sizes. We tried out construct in a number of E. coli strains, (the paper we based it on used BL21, but we also tried MC100 and DH5alpha) but we didn't get a chance to look at protein from all of them, we just had breakdown assays to confirm we weren't making an active product. In terms of implementation, we aimed to make an under-sink bioreactor which would tackle the problem close to the source, as well as releasing enzyme into the water supply to help further downstream. We aimed to use filtration systems to allow for enzyme release whilst keeping our cells contained within the device - no worry about water contamination! We did look at kill switches based on time, nutrient depletion etc, but decided it was too risky for what we wanted. There should be some images of the device on the wiki too. We hadn't really looked at implementation of the Keratinase too much, our main focus being the lipase. Interestingly, a few companies are now making hair conditioner that contains peptides - something you would be producing as a byproduct. Perhaps something to look at? Competition-wise, keep everything really simple. Don't make big bold claims - the judges will see through it in a heartbeat. If you don't know something, admit it, but be ready to explain how you would find it out in future. Have your presentation ready before you go!!! We spent 4 days in our hotel lobby putting it together before the jamboree started and so didn't get to see much of Boston while we were there. Well done on making it this far! Let me know how it goes and if the Keratinase does work, I'm sure the rest of my team would like to hear about it too. If you have any more questions, don't hesitate to ask. Lara

Hi again. Thank you very much for your email, it is greatly appreciated that you could give us some insight from firsthand experience. We had a couple more questions regarding this email. Firstly, we were wondering about how you planned to implement the under-sink bioreactors ethically, and while given permission from the home-owners, because as we see it, many people would be scared about putting a bioreactor within their house? We also wondered if you still had the papers you referenced, because we do have some papers that we have read, those ones might be useful for us to look into. If you don't still have copies, no worries, but thank you again! I'm sure we will continue to come up with questions for you, as it is much appreciated that you would be willing to collaborate with us and share your knowledge! Thanks, Our Lady of the Snows Catholic Academy iGEM Team

Hi, I'll have to talk to some of my team about the bioreactor thing. If I remember rightly we weren't looking to make them compulsory, but would have put an incentive scheme in place with water companies for people that used them. We definitely interviewed some people about whether they'd be comfortable having one in their home, and most people weren't as worried as we'd assumed they would be. I'll have a look for the papers tonight, I don't have access to my google drive from work. If I don't get back to you with them, just send me a reminder email. Thanks, Lara

University of Chicago

Our second collaboration was also with another team who previously attempted this project, the University of Chicago. The University of Chicago 2013 iGEM team designed a construct to degrade feathers in the poultry industry but did not complete their construct. They also planned to use the byproducts in the reaction to make useful products “By developing an efficient process for the digestion of keratin, we would not only drastically reduce the cost of disposal but also allow the keratin to be used in products like animal feed, fertilizers and biodegradable plastics. Our final product would be a one-step continuous process in which feather waste could be continually added to B. subtilis cultures.” -University of Chicago. From the collaboration with the University of Chicago we hoped to get advice from them concerning our project and the issue we are tackling, as well as advice for implementation.

University of Chicago Correspondence

To whom it may concern: My name is Freya Morgan, and I am a member of the Our Lady Of the Snows Catholic Academy iGem team. We are a small high school based based out of Canmore, Alberta, Canada. We are contacting you because we have chosen to tackle build ups of keratin in wastewater treatment, and well as the poultry industry as this year’s project, and your team’s 2013 project involved Keratinases too. When talking about wastewater treatment, these build ups of hair can lead to clogs causing flooding and the breakage of water pumps and pipes, something very costly to repair. The only current way to deal with this issue is to have a worker manually remove the blockage, which is slow, unpleasant for the worker and inefficient. Upon further research our team discovered that the reason hair is such a huge issue for waste treatment facilities is because of the protein keratin, which as you probably know is a very hard protein to break down due the tightly wrapped helixes and hydrogen bonds. Once we discovered this pressing issue we looked into keratin more and realized it is also a component of the basic construct of feathers and they to build up and are hard to break down. With more searching on the iGEM parts registry we discovered your project, as well as one other project, both of whom attempted to solve the keratin issue. With this research and the exploration of previous iGEM projects, we decided to create a construct that would express a Keratinase enzyme to break down the keratin in the hair and feathers, thus solving the issue. Because iGEm requires collaboration between teams, we decided to try contact you due to your decision to tackle this project in 2013. As we understand it, you reached the planning stages of the project and then dropped it? As much of our project is based on building on, and optimizing previous constructs, we are looking for some more in-depth information concerning the cleaving process, and some issues you would've tackled there, and how you would've dealt with them. We know that Keratinases are difficult to express in E. Coli, due to the double membrane, and because when expressed, the export tag does not always get cleaved properly, so if you could give us any insight upon this, that would be appreciated. We would also like to get some of your current opinions on the issues, if you have any advice for us that could make us more successful at the competition and if you had devised a way in which you would have deployed your construct outside of the lab, if you had completed the project. We also were wondering what your plans were for implementation of the biobrick into the real-world, and safety precautions to be taken outside the lab through implementation. Looking forward to hearing back from you soon and we understand if you can't answer some of our questions. Thank You, Sincerely, Our Lady Of The Snows Catholic Academy 2015 iGem Team.

Hello Our Lady Of The Snows Catholic Academy iGEM Team, Nice to receive your email. I'm Kevin, and I'm the president of our student organization on campus here at UChicago. Our entire team during the school year is larger, but our summer team consists of me, as well as three other undergraduates (CC'd here on this email, in case they have anything to add that I forgot). So obviously this is not the project we are currently pursuing, but I and others did quite a bit of background research into what we could do with this project before deciding against it. I can answer some of your questions hopefully, as well as describe issues that made us stop pursuing this project (that you may be able to overcome). As you know, our 2013 team pursued the Keratinase expression idea, primarily with a focus on digesting feather waste. We didn't get very far, but that year we aimed to express KerA in B. subtilis. Obviously, ideally you would want to express in E. Coli if you're doing it on an industrial scale due to growth rate, but as you may have read on our website, the issue is with KerA getting trapped in the periplasm since it's not endogenously expressed. On the other hand, B. subtilis has the export machinery required to So here is the format for a couple of theoretical functional systems you could think about that we were deciding between when considering this project, as well as a brief evaluation of each approach: 1. Express in B. Subtilis: Advantage: You don't have the problem of it getting stuck in the periplasm like E. coli since B. subtilis actually recognizes KerA. The Disadvantage: B. Subtilis growth rate is majorly terrible compared to E. coli and people have reported difficulty getting it to grow on an industrial scale, which is why it would be extremely great to get it in E. coli. Also, when we tried it with B. subtilis, the reason we didn't get very far with the 2013 team is because we never got the transformation into B. Subtilis to work after several repeats (but that's possibly because of issues with our protocol/technique, it's not totally clear)- in either case, obviously working with E. coli would be much nicer/easier. 2. Express in E. Coli, part I: You could potentially get around the issue of KerA getting trapped in the periplasm by inserting the Tat and Sec pathways in E. coli (which we are actually using in our project right now in a completely different context). You would remove the endogenous KerA signal peptide and add an alternative signal peptide, which could be recognized and cleaved by one of these two secretion systems (I forget which- one system cleaves the tag and the other one doesn't, sorry definitely another person on our team working on these systems know them better than me). You may also want to look at other signal peptides/systems- though I think Tat/Sec are the most famous and most efficient ones. Advantage: Gets around the issue of the stuff getting stuck in the periplasm. Disadvantage: This could be a little difficult to get to work, as getting recombinant proteins to secrete in E. coli is kind of a flip of a coin- sometimes it works and sometimes it doesn't. It also wouldn't be very novel, as it's already been done before: http://pubs.acs.org/doi/abs/10.1021/jf100803g- maybe you could improve on their efficiency though, not sure if that's good. 3. Express in E. coli, part II: Advantage: You could just not secrete the protein and instead lyse E. coli and purify the KerA later. This means you don't have to think about secretion systems. Disadvantage: The problem with this approach is that misfolding and the formation of big clumps of the protein called inclusion bodies within the acidic environment of the cytoplasm is common with overexpression of recombinant proteins in E. coli (http://www.nature.com/nbt/journal/v22/n11/full/nbt1029.html). So with this approach after lysis, you'd have to figure out the conditions for separating out the inclusion bodies and refolding the Keratinase properly. This type of an approach where you lyse then refold is well-documented with something long-known like insulin (I think this is actually how it's industrially produced), but refolding would probably be pretty tedious to work out for something like Keratinase where it hasn't been done before. Possibly doable though, just might take a while to figure out. 4. Express in other organisms: there's expression in a yeast that's been reported (we didn't look very much into it- I forget which one off the top of my head), but I think it might have a similar problem as B. subtilis in terms of growth rate. 5. Other approaches we haven't thought of. That's a possibility too. It's probably important to understand mechanistically how the enzyme works. Not much is known (after much searching by us), but we found that most Keratinases are simply just serine proteases. They can't actually cleave the disulfide bonds of feathers which make feathers so mechanically strong- it requires the presence of a strongly reducing environment or (speculated) reductases to get the job done. There's also an incredible number of Keratinases that have been identified that differ in mechanism (while most are serine proteases, some are metalloproteases I think), and optimal functional environment- pH, temperature, etc. For us, if we were to do the project, we would have chosen a Keratinase that functions at a high temperature due to the high temperatures at which industrial processes like these usually function at and possibly at a lower pH (instead of KerA). I probably have a reference paper somewhere with a big list of known Keratinases. It might be that it doesn't matter which one you choose and you might just choose a well known one like KerA or KerBL, but this is something that should definitely be considered. The main reasons we didn't pursue this project is because it didn't seem like there was anything novel we could do with it when looking at the literature/what had been done with the past, as well as the fact we got discouraged by the various disadvantages we'd listed above and didn't think it was possible to accomplish the goal we'd want to aim for of production at industrially relevant levels. In terms of other novel things we considered, we thought of potentially using directed evolution to make it work better, but we couldn't find any specific characteristic that we could do directed evolution on- finding a high temperature functioning mutant is a possibility, but many Keratinases have already been identified from hot-spring bacteria (I think, might be wrong on this) that naturally function at high temperatures; simply finding a mutant that "has greater activity" doesn't seem particularly fruitful since the main problem isn't low activity intrinsic to the enzyme but instead its recombinant overexpression at industrially useful levels. We also tried looking at was possible applications of using the byproduct of the reaction besides chicken feed (the degraded feather waste is sometimes reused as feed for chickens), maybe to make a useful biomaterial or something- but had no luck after a decent amount of searching. Of course, it would be cool if we could get an actually functional Biobrick of any given Keratinase in the registry, so that by itself would be a great potential goal for you- we just wanted to go beyond that. Obviously recombinant Keratinases have been successfully expressed in many other contexts/papers by multiple different approaches, so it is definitely possible. Also, if you find anything else that we missed, that would be cool too- for instance we hadn't thought of the wastewater plant application. Let me know if we can help you with anything else, as we would definitely be interested in collaborating/helping to mentor your team. We'd definitely be very interested to find out what approach your team ends up going with this project. I'm sure you've read some papers on Keratinases already, but I've attached the reading that I've found to be most helpful in terms of overall orientation in the field (starts Chapter 33, p. 881). Sorry if I went too detailed into things- I had a bit of time between experiments so decided to elaborate a bit. Hope this was helpful. Best, Kevin Yang UChicago iGEM Team

Consort iGEM Team

Our third collaboration has taken place with our friends, the Consort, Alberta iGEM team from Consort Alberta. We have also been able to present in front of them and talk to them one on one about our projects meaning that we both have a lot of information regarding each other’s projects. For this reason consort contacted us and asked us if we would right a short report talking about their project, concerns that we have with their project, ways to address these concerns, general advice and more.

Here are our two collaborative pieces:

Canmore’s Write Up for Consort: This year the iGem team from Consort is continuing with their ECOS project, bacteria designed to detect the presence of aromatic hydrocarbons in order to prevent any harm to the environment. Consort’s ongoing project is a great concept for many reasons. Firstly it is something that is a local problem in their community and especially hits home for some of the team members, which means that they devote themselves fully to the project. Secondly because the outcome of this project could be very helpful for surrounding communities and possibly could hold some wealth in it. Finally because this project could help save countless amounts of crops and livestock from what is a major threat to them.

The science behind the project is very intriguing as well. The indicator protein is atoned to xylene, which is a very volatile liquid hydrocarbon, which in itself is a good thing to find and remove but it is tied to other dangerous compounds. When these compounds are detected the proteins emit a color indication that would alert the user to the presence of said dangerous compounds. Whilst this all seems simple the biobrick is not, and they have spent years developing this to perfection to start with their prototypes.

The prototype and models of prototypes look to be a very effective method of using ECOS. I was able to see one of the prototypes and it looked good even though I was unable to see it in action, this shows great promise for the team in their upcoming endeavors. I can see much success in the future if this team especially if they have a working prototype at the giant Jamboree, I personally wish them the best (-Joshua Lamb)

Consort’s Collaboration Write Up for Canmore: Canmore’s project deals primarily with the issue of hair and feather build up in the water treatment systems. This is clearly a major issue and can use extensive amounts of money and time to remedy. There is an extreme amount of inconvenience to unclog the water treatment systems once they get to this point. Creating a BioBrick to produce keratinases could be an effective and cheap solution to the problem and also in preventing the problem. Our concerns for Canmore lie in the prototype and safety. The specific engineering and prototyping of a bioreactor is extremely important. The conditions required to keep the bacteria alive, the amount of keratinases that would need to be produced and what kind of a time span are all aspects to look into when designing the bioreactor. Their plan to use the membrane bioreactor is a very good and easily applicable idea but the research as to optimum conditions concerning the production of the gene are incredibly important. Having a viable analysis of the cost, time and resources required to make this something we can implement is society is also an important consideration. The time frame of how long it takes to break down the keratin is also something to look into. The comparison of the time requirements to break down other pollutants in the bioreactor chamber compared with the time to break down keratin. Knowing this will allow a design maximizing potential. Other thoughts for them to consider include: what types of bacteria are already being used in the bioreactor? What are they breaking down? Is there a way to genetically modify bacteria already in use to contain the kera or karus gene? This would eliminate the need to introduce another species and readjust conditions while allowing maximum productivity. Overall, I believe this project could introduce a valid and much needed solution to the issue of hair build up and could be quite effective, quick and safe. As the bioreactor is not connected to the main water line and is completely contained, contamination of water with E. coli would not be an issue. The proposed prototype also utilizes the current system and technologies used making it as easy as possible for implementation. Canmore has done an excellent job in looking at the practical portions of their project and with more research, their project could do a lot of good in our world. (-Sam Davies)