Team:UChicago/Collaborations

Collaborations

Calgary JulyGEM

We participated in Calgary's JulyGEM, where we were able to get feedback on our project presentation, as well as give feedback to other teams. This was incredibly useful for us in terms of figuring out how to present our project in a way that was concise but also covered everything content wise. The judges were very supportive, and gave us great advice on how to improve our project presentation wise.

Darmstadt Meeting

In May, we met one on one with team TU Darmstadt, who had experience with working with laccases. We went over our project ideas and they gave us a framework for one possible direction we could go with out computational analysis.

The Laccase Meeting

In May, we met with several other teams to discuss our project proposals. All of the teams were working on laccase related projects, so we were able to get feedback on plans we had for our laccase related computational work. This is also where me met the Aboa team for the first time, which eventually would lead to our summer long collaboration.

Collaboration with Aboa

Our main collaboration was with Aboa, where we started the summer by agreeing to work our projects around each other; we would do simulations to confirm their results, and vice versa. We both ran into project difficulties throughout the summer, but we were able to depend on each other for feedback and kept each other accountable. Below is a log of our (almost) weekly meetings.

Meeting 5/27

First meeting with Aboa. Introduced ourselves to each other and went over our project outlines. We decided we could try and computationally confirm their lab results and vice versa. Decided to also make a Whatsapp group chat.

Meeting 6/17

Gave short project updates. Talked about structural differences between bacterial and fungal laccases and how that was relevant in the context of modeling and Aboa's chosen laccases. Discussed what concrete steps we could take to include the other team in our project.

Meeting 6/23

Gave short project updates as usual. Aboa is waiting for DNA so is doing their test measurements in water. They gave us the Turku water plant parameters; pH of 7-7.5 and temp of 20-25 C. Lowest temp is 8 C.

Meeting 7/01

Aboa Updates
-Received DNA distribution kit information from iGEM
-Starting in the lab next week
-Plasmid received
-Borrowing reagents from Malin’s lab
-Next week: transforming plasmid into E. Coli for cloning. Analytical digestion.
-Measure diclofenac toxicity to cyanos
-Diclofenac measurements
-Have HPLC mass spec
-Phototrophs meetup
-Human practices: inclusive website, educational module
-“There is plenty of time” - Malin

Meeting 7/07

Went over project updates. Agreed that we needed to discuss pH and temp in the role of experiments more. Worked on scheduling issues because Aboa had a conflict w the Nordic iGEM conference.

Meeting 7/14

Did project updates. We have our virtual wastewater plant tour scheduled today so we updated them about what we are expecting to have for that later today. They talked about how they have a week off from lab so we can discuss on the Whatsapp if we want to still have a meeting next week.

Meeting 7/21

Gave them a comprehensive update of the wastewater plant tour. The tour was on the longer end so we had a lot to discuss. Aboa had already visited their waste water plant, so we talked about the differences in the purification process. One component we didn't realize was different was water chlorification: this is a step that the Turku water plant does not have. We also updated them with the problems we are having with simulations and docking with the homology model of Yak, as well as gave them a time estimate for the simulations.

Meeting 7/28

We went over pH and temperatures as we agreed upon previously. After realizing we wouldn’t be able to get to all of the simulations we discussed what specific data they needed from us. We suggested that we could either run the avg temp across the year, or we could run the highest and the lowest. We also agreed to only run one of their laccases; they suggested that it might be good for us to use Yak but considering the homology model issues we decided we would look into it more before making a decision.

Meeting 8/04

We did the usual updates. Aboa told us about the Nordic iGEM Conference as well as the fact that they were making good progress in the lab. In turn, we gave them information about how our instagram page was progressing, as well as the update that we had finally started our simulations. We discussed the fact that we were going to run our work at 2 pHs (5.1 and 7.1) and the two temperatures we discussed earlier.

Meeting 8/12

A big part of todays meeting was project direction. They let us know that two of their team members needed to take time off to prioritize their health, and that they might have to skip some of the pH and temperature measurements. Aside from this we did our usual project updates; we let them know that we were having trouble with the copper parameters for the active site.

Meeting 8/19

We were slow to make progress, and updated them with the little progress we made on the copper parameters. Based on what they told us last week about their team members, we decided to come up with a plan B to figure out how we can work things out if they cant provide us with the measurements. We were brainstorming that potentially we could do something that would look at both the Turku and Chicago water plant from a technical implementation perspective, but we did not commit to anything concrete.

Meeting 8/26

We largely discussed implementation and human practices this meeting. We decided that we wanted to have a robust implementation that we could share with them, and then would later find a time that would work for both teams to go over the proposals. We also discussed how we could communicate with our waste plant about this for the purpose of getting external feedback, and Aboa agreed that they would do the same. We need to also figure out legal differences in terms of our implementation, because Finland has much stricter laws regarding this compared to the US. Laura has been doing a lot of work with human practices so we need to match her level of commitment.

Meeting 9/02

Aboa gave us some really good news about how they were preparing to do the temp and pH measurements next week. In return, we gave them the update of how our topology files are progressing. We decided to keep our ideas regarding plan B on the back burner because things are going well, but not abandon it completely just in case. We also updated them about how we started writing for the wiki, and they told us about recruiting.

Meeting 9/09

Aboa gave us some really good news about how they were preparing to do the temp and pH measurements next week. In return, we gave them the update of how our topology files are progressing. We decided to keep our ideas regarding plan B on the back burner because things are going well, but not abandon it completely just in case. We also updated them about how we started writing for the wiki, and they told us about recruiting.

9/9

Aboa gave us a large update with the pH and temp measurements. Notes from the meeting are as follows:
ABTS assay issue:
Have purified enzymes, ran gel kinda confirming proteins are okay, might be slightly bigger. For all three purified laccases looked good for CotA and CueO, Yak was missing, not sure why since saw the unpurified product. Will discard, won't try to do measurements.
Mixed buffers, made solutions of ABTS. First tried in cuvette buffer + ABTS + enzyme, measure absorbance to find amount of ABTS + buffer to use, and for how long. Unfortunately, when dissolve ABTS (yellow powder), made the entire solution yellow. The way the exp should work is go from colorless to green to indicate reaction, but since the solution is already yellow there's kinda an issue.
96 well asssay multiple pH and different buffers (phosphoric(?) acid and citric acid) - share screen need Ethan.
0-10mM substrate concentration
5 or 7 pH
buffer 1 or citric acid
Measured 30min ever 2 min, next hour every 5 min, next 6 hr every 30 mins
- absorbance values (ABTS) depended on substrate concentration, so nothing really happened
Additional test w/ hydrogen perodxide as positive control, should react really fast with ABTS
- all looked the same, nothing happened w hydrogen peroxide, suggests ABTS already oxidized
Tested buffer
- citric acid phosphate buffer (?)
- PBS test w/ ABTS mentioned on spec sheet, wanted to check if there will be the same issue. Still same issue, so ABTS is probably the issue
Next steps
- rebuy ABTS
- reduce ABTS
- another assay for laccase activity: Syringaldazine, https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/enzyme-activity-assays/enzymatic-assay-of-laccase
- dissolve ABTS first in DMSO, but not sure if it'll solve the yellow issue thing
Diclofenac tests?
- HPLC w/ company, lost contact
Next plate run:
- remove 1ul enzyme concentration for H2O2 as positive control
Collaboration for implementation
- all ask WWTP people the same questions
- one big meeting with us, Aboa, and each respective WWTP people

Meeting 9/15

We let Aboa know that we were running into problems with out simulations, specifically with the altered pH state, so for now we are planning on just running them at pH 7. Similarly, for temperature, we are just planning on doing the summer temps because we are running out of time to do simulations. We also briefly discussed the registry regarding parts, and what they would need to do for that. Lastly, we went over human practices collaboration work, and talked about next steps for collaborating with the WWTPs.

Meeting 9/30

Meeting notes:
* Engineering cycle almost done since they got the alternative substrate instead of ABTS and a control Laccase
* saw some activity with CotA but not CueO
* Control laccases were very concentrated so the data might be misleading but they are going to use what they have, however it is not enough for a partnership since they weren’t able to get out any info about pH or temperature
* Wiki:
* Partnership page: how to organize it? 
* Do the partnership page based on our own themes, but make sure content is simmilar
* Have a different zoom session just to go over what is on our wikis, also have a collaborative google doc to write the blurbs together 
* Plan B Collaboration:
* They wrote questions down, we would each ask the same questions to a waste water treatment plant in our city about our own proposed implementation and each other’s implementation. Goal is to know if the other team’s implementation would be feasible in each other’s country with regards to regulations on GMOs 
* Need to do research into what exactly we want our implementation to be (our team should have a meeting to make sure we know what our implementation would be, and update the other team about this)
* Look at the initial questions on the google doc and let them know if there’s anything else we need

Meeting 10/7

We went over plan b again and discussed what information they needed from us so we could ask their experts about our implementation. We also created a shared partnership document because these final days are likely going to be wiki crunch time.