iGEM 2018 — Team UMaryland

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# Team:UMaryland/Notebook

Template Title

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For Judges

- [Sitemap](https://2018.igem.org/Team%3AUMaryland/Sitemap)
- [Judging Form](https://igem.org/2018_Judging_Form?team=UMaryland)

Notebook

- [Notebook](https://2018.igem.org/Team%3AUMaryland/Notebook)
- [Protocols](https://2018.igem.org/Team%3AUMaryland/Protocols)
- [Safety](https://2018.igem.org/Team%3AUMaryland/Safety)

About Us

- [Our Team](https://2018.igem.org/Team%3AUMaryland/Team)
- [Attributions](https://2018.igem.org/Team%3AUMaryland/Attributions)
- [Sponsors](https://2018.igem.org/Team%3AUMaryland/Sponsors)

Engagement

- [Human Practices](https://2018.igem.org/Team%3AUMaryland/Human_Practices)
- [Outreach](https://2018.igem.org/Team%3AUMaryland/Public_Engagement)
- [Collaborations](https://2018.igem.org/Team%3AUMaryland/Collaborations)
- [InterLab Study](https://2018.igem.org/Team%3AUMaryland/InterLab)
- [2018 Mid-Atlantic Meetup](https://2018.igem.org/Team%3AUMaryland/meetup)

[Parts](https://2018.igem.org/Team%3AUMaryland/Parts)

[Modeling](https://2018.igem.org/Team%3AUMaryland/Model)

- [TPA vs PCA detection](https://2018.igem.org/Team%3AUMaryland/TPAvPCA)
- [Cellulose Binding](https://2018.igem.org/Team%3AUMaryland/BCmodel)

[Project](https://2018.igem.org/Team%3AUMaryland/Description)

- [iGEM PET History](https://2018.igem.org/Team%3AUMaryland/History)
- [PET NET: Improve](https://2018.igem.org/Team%3AUMaryland/Improve)
- [- Hardware](https://2018.igem.org/Team%3AUMaryland/Hardware)
- [Measurement](https://2018.igem.org/Team%3AUMaryland/Measurement)
- [- TPA Enzymes](https://2018.igem.org/Team%3AUMaryland/TPA)
- [- Demonstrate](https://2018.igem.org/Team%3AUMaryland/Demonstrate)

[Home](https://2018.igem.org/Team%3AUMaryland)

Notebook

Follow us throughout our journey

Week of May 29th

This was our first week in lab! We took a week off after school to take a break and destress. Since our team is relatively new to lab techniques and our parts haven’t come in yet, we decided to spend this first week working on basic lab techniques and the Interlab Study. We attempted to amplify the A3, C3, and K3 backbones while we waited for parts to arrive.

Week of June 4th

Our first week went well and we were happy with the way we completed the Interlab study. This week, we wanted to get started on our plastic project and assembled the HlyBD into an iGEM backbone for our PETase secretion system. Transformations and overnights for the HlyBD part were successful. We sent out our HlyBD part to be sequenced. We still had more difficulty with working on amplifying the iGEM backbones and decided to work on doing some troubleshooting to determine where our process is going wrong.

Week of June 11th

We got back our sequencing results and found out that we did correctly assemble our plasmid. We attempted to troubleshoot the amplification of iGEM backbones. Unfortunately, our G-blocks were delayed again so we weren’t able to do too much wet lab work. We started planning our Mid-Atlantic Meetup and other outreach activities for our local community.

Week of June 18th

Another week while our G-block wasn’t able to be synthesized. We really do need to get this PETase G-block so while IDT is still attempting to synthesize it for us, we are reaching out to other teams that have worked on it in the past to see if we could possible get it from them instead. We worked on cleaning the lab and ordering new reagents. We also learned theory for many different lab techniques so that we can instantly learn how to troubleshoot the problems that occur in lab. Flowcharts are our friend!

Week of June 25th

Our part still hasn’t come in, so our lab work has slowed to a crawl. In the meantime, we worked on some outreach. Another student research team on campus came to tour our lab and we talked to them about synthetic biology and our project. We also started a journal club! We look at one or two different articles every week (typically in the field of synthetic biology). One student gives a presentation on the article and afterwards we discussed the implications that this could have on our project and what we learned from it. It went really well this first week and helped promote critical thinking. Hopefully, we can do things like this every week.

Week of July 2nd

Again, we haven’t received all the parts that we need to produce our completed plasmid. In the meantime, we are working on producing bacterial cellulose for hardware for our plastic degradation. We also started brainstorming good designs for our hardware project. We started designing T-shirts and other graphics for our project to make it simpler to explain. This week, the lab members who weren’t in lab as frequently were concerned that they were losing understanding of our progress so we had a long lab meeting on the weekend to catch everyone up and update each other on all aspects of the project. The modeling team did a good job explaining what they discovered in terms of rate of plastic degradation. This encouraged us to allow at least a month for incubated PETase enzymes to degrade plastic and run experiments on it.

Week of July 9th

The door to our lab broke early this week so we were having problems getting in and out. Luckily, it was fixed quite fast. Our PETase Gblock finally came in so we can really get started on the primary plasmid for our project. Everyone was very excited for this. Also, our Mid-Atlantic Meetup was this Friday so we spent a lot of time preparing to explain our project and the modeling that we have done up to this point. It went really well and we could see a lot of different teams talking to each other and fostering connections and sharing knowledge. Holly from iGEM also came by and was an absolute delight to talk to. We learned a lot about other teams and their outreach in local communities.

Week of July 16th

This week we assembled our PETase Gblock and transformed it. We ran a gel on the PETase and sent it for sequencing. Unfortunately, it seems like the backbone annealed onto itself rather than assembling with the Gblock. We are going to run it again but attempt to further optimize the process so it hopefully doesn’t happen again. It’s starting to get to the point of summer where people are going to have to leave the housing near campus which will make it more difficult for students to come in and continue doing work.

Week of July 23rd

Once again, we attempted to assemble the PETase from the Gblock and transformed and miniprepped before sending for sequencing. This time, it ended up working for us and we can now 3A assemble it with the cellulose-binding domain and our secretion system. The transformation of PETase with the HlyBD was successful so next week we will make overnights and send it for sequencing to confirm that it was assembled correctly.

Week of July 30th

We made overnights of our plasmid and sent it for sequencing. As we are getting closer to finishing up our plasmid, we started making plans for experiments to test the efficacy of our project when it comes to degradation. We also are now planning on making a way to detect plastic degradation through the use of other enzymes and plasmids. We looked at past iGEM projects for any ideas and started contacting other labs for certain plasmids to detect the byproducts of PET degradation. We also started working on a rough draft of a Jamboree presentation to display all the data and planning that has gone into our project so far. Professor Masai and Professor Jha were contacted regarding TPH enzymes and PcaU, respectively.

Week of August 6th

At this time, a bunch of lab members went on vacation since we didn’t have campus housing during August. Also, we only have two more weeks until school starts so we decided to give people a break and only a few people were in lab at that time. TPH plasmids arrived from Masai! Transformed and verified by SDS PAGE.

Week of August 13th

pNBP, transform pGLO and PcaU attempted, but failed

Week of August 20th

Initial PNPB results obtained and positive! PcaU g block arrived. Outreach trip to MRF at Shady Grove.

Week of August 27th

PcaU g block gibsoned into pGLO. Outreach trip to Anacostia Watershed.

Week of September 3rd

PETase HLYBD sequenced and transformed into BL21 cells. Schedule for lab during classes discussed.

Week of September 10th

PcaU sequencing primers ordered, PcaU sequenced in pGLO backbone. Sequence confirmed PcaU transformed into BL21 DE3 cells. Preliminary experiments were conducted for the scale up of PETase CBD bioproduction.

Week of September 17th

Initial PcaU results for high and low range detection were carried out. PcaU detection protocol was discussed and finalized. PNPB assay performed testing the effect of cellulose binding on PETase CBD esterase activity. Another PNPB assay performed testing activity of PETase CBD HLYBD under varying induction conditions.

Week of September 24th

Completed PcaU tests and showed that PcaU could differentiate 1uM PCA concentrations. Expressed PETase HLYBD under the dual promoter system. The condition induced with both arabinose and IPTG showed signs of secretion, with a white substance accumulating on the sides of the tube. The substance was harvested for testing. All 2018 teams involved with PET degradation were contacted and we offered to test our PCA detection device on their degradation product. Makerere, Yale, and RHIT responded, however none were able to supply product at the time.

Week of October 1st

We spent the week preparing parts submissions. PcaU was assembled into C3 backbone and we removed an EcoR1 site for submission. We also ordered a g block just in case our work ran into difficulty. Fresh PETase enzyme was expressed and harvested. We began experimenting with various buffers to determine which would be best as TPH buffer, and ordered necessary reagents including ferrous ammonium sulfate and L-cysteine HCl. We tested PcaU sensitivity in DH5a vs BL21 cells and found that PcaU was functional in 5a cells but not as sensitive.

Week of October 8th

SEM images of PETase activity were obtained. It was at this point that we discovered the HlyA secretion system was not working for PETase. The modified PcaU sequence with removed EcoR1 was tested in pGLO and found to be equally sensitive as the original. Parts were shipped out. Over the weekend, TPH buffer was made and fresh cells expressing fresh TPH enzymes were sonicated.

Week of October 15th

We walked into lab on Monday to find out our freezer was broken, warm, and very smelly. Luckily our bacteria happened to be in another lab. On the weekend we performed tests using PCAU to detect TPH enzyme activity and then used PCAU to test a solution for the presence of TPA.

Contact Us
umarylandigem@gmail.com
Biology - Psychology Building
4094 Campus Dr, College Park, MD 20742

© University of Maryland 2018

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# Team:UMaryland/Experiments

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[HOME](https://2018.igem.org/Team%3AUMaryland)

TEAM

[Team Members](https://2018.igem.org/Team%3AUMaryland/Team)
[Collaborations](https://2018.igem.org/Team%3AUMaryland/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AUMaryland/Description)
[Design](https://2018.igem.org/Team%3AUMaryland/Design)
[Experiments](https://2018.igem.org/Team%3AUMaryland/Experiments)
[Notebook](https://2018.igem.org/Team%3AUMaryland/Notebook)
[InterLab](https://2018.igem.org/Team%3AUMaryland/InterLab)
[Model](https://2018.igem.org/Team%3AUMaryland/Model)
[Results](https://2018.igem.org/Team%3AUMaryland/Results)
[Demonstrate](https://2018.igem.org/Team%3AUMaryland/Demonstrate)
[Improve](https://2018.igem.org/Team%3AUMaryland/Improve)
[Attributions](https://2018.igem.org/Team%3AUMaryland/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AUMaryland/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUMaryland/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AUMaryland/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AUMaryland/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AUMaryland/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AUMaryland/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUMaryland/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AUMaryland/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AUMaryland/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AUMaryland/Hardware)
[Measurement](https://2018.igem.org/Team%3AUMaryland/Measurement)
[Model](https://2018.igem.org/Team%3AUMaryland/Model)
[Plant](https://2018.igem.org/Team%3AUMaryland/Plant)
[Software](https://2018.igem.org/Team%3AUMaryland/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=UMaryland)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:UMaryland/Results

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[![](http://placehold.it/350x150/e4dede/c4baba)](https://2018.igem.org/Team%3AUMaryland)
[HOME](https://2018.igem.org/Team%3AUMaryland)

TEAM

[Team Members](https://2018.igem.org/Team%3AUMaryland/Team)
[Collaborations](https://2018.igem.org/Team%3AUMaryland/Collaborations)

PROJECT

[Description](https://2018.igem.org/Team%3AUMaryland/Description)
[Design](https://2018.igem.org/Team%3AUMaryland/Design)
[Experiments](https://2018.igem.org/Team%3AUMaryland/Experiments)
[Notebook](https://2018.igem.org/Team%3AUMaryland/Notebook)
[InterLab](https://2018.igem.org/Team%3AUMaryland/InterLab)
[Model](https://2018.igem.org/Team%3AUMaryland/Model)
[Results](https://2018.igem.org/Team%3AUMaryland/Results)
[Demonstrate](https://2018.igem.org/Team%3AUMaryland/Demonstrate)
[Improve](https://2018.igem.org/Team%3AUMaryland/Improve)
[Attributions](https://2018.igem.org/Team%3AUMaryland/Attributions)

PARTS

[Parts Overview](https://2018.igem.org/Team%3AUMaryland/Parts)
[Basic Parts](https://2018.igem.org/Team%3AUMaryland/Basic_Part)
[Composite Parts](https://2018.igem.org/Team%3AUMaryland/Composite_Part)
[Part Collection](https://2018.igem.org/Team%3AUMaryland/Part_Collection)

[SAFETY](https://2018.igem.org/Team%3AUMaryland/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AUMaryland/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AUMaryland/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3AUMaryland/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3AUMaryland/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3AUMaryland/Hardware)
[Measurement](https://2018.igem.org/Team%3AUMaryland/Measurement)
[Model](https://2018.igem.org/Team%3AUMaryland/Model)
[Plant](https://2018.igem.org/Team%3AUMaryland/Plant)
[Software](https://2018.igem.org/Team%3AUMaryland/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=UMaryland)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)