iGEM 2021 — Team UIUC_Illinois

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# Team:UIUC Illinois/Notebook

Notebook | UIUCiGEM

* Team

  [Team](https://2021.igem.org/Team%3AUIUC_Illinois/Team)[Attributions](https://2021.igem.org/Team%3AUIUC_Illinois/Attributions)[Collaborations](https://2021.igem.org/Team%3AUIUC_Illinois/Collaborations)
* Project

  [Contribution](https://2021.igem.org/Team%3AUIUC_Illinois/Contribution)[Description](https://2021.igem.org/Team%3AUIUC_Illinois/Description)[Engineering Success](https://2021.igem.org/Team%3AUIUC_Illinois/Engineering)[Excellence in another area](https://2021.igem.org/Team%3AUIUC_Illinois/Package)[Experiments](https://2021.igem.org/Team%3AUIUC_Illinois/Experiments)[Model](https://2021.igem.org/Team%3AUIUC_Illinois/Model)[Notebook](https://2021.igem.org/Team%3AUIUC_Illinois/Notebook)[Partnership](https://2021.igem.org/Team%3AUIUC_Illinois/Partnership)[Proposed Implementation](https://2021.igem.org/Team%3AUIUC_Illinois/Implementation)[Results](https://2021.igem.org/Team%3AUIUC_Illinois/Results)
* [Parts](https://2021.igem.org/Team%3AUIUC_Illinois/Parts)
* [Safety](https://2021.igem.org/Team%3AUIUC_Illinois/Safety)
* [Human Practices](https://2021.igem.org/Team%3AUIUC_Illinois/Human_Practices)

[ ]

# Notebook

Documenting our progress and timeline of our project

---

##### Contents

# March

##### [Week 1](https://docs.google.com/presentation/d/1QsMs5O9dXddDGm6dOUSNo-jtgfl_qNP77RfKf1uZuoE/edit?usp=sharing)

* First team meeting, brainstormed ideas for the project

##### [Week 2](https://docs.google.com/presentation/d/1ct75cn6P3tdHqn7d1uOC8eB6tiNB4VVTNMSjk2wf2tU/edit#slide=id.p)

* Continuation of Week 1

##### [Week 3](https://docs.google.com/presentation/d/1ewm9bSkwOJqjLO6MO2zrWWgQ-CpccWu5hi2LQyTdNsA/edit#slide=id.p)

* Narrowed down our project ideas into top two contenders

# April

##### [Week 1](https://docs.google.com/presentation/d/16mGN80M4SeKDFyraB4mOoL7qHQRSVn2fe-bXS2BXFjs/edit#slide=id.p)

* Decided our project topic: Plastic Degradation

##### [Week 2](https://docs.google.com/presentation/d/1mL1wPnVBBH3IET4NTy8y2t0uNuXKPg2HBiFtm1poLEU/edit#slide=id.p)

* Planned for our project to be composed of a drylab component and a wetlab component

##### [Week 3](https://docs.google.com/presentation/d/1mL1wPnVBBH3IET4NTy8y2t0uNuXKPg2HBiFtm1poLEU/edit#slide=id.p)

* Brainstormed end goals and tasks for each component of the project

##### [Week 4](https://docs.google.com/presentation/d/1bHYOngWQqoKQGqgpKpaxqRcXzaGb0AudkvwBUNOSSho/edit#slide=id.p)

* Continuation of Week 3

# May

##### [Week 1](https://docs.google.com/presentation/d/11sG14wPYblFdip2gozcdJ6hmnL3QZ9BFtYuw5GUWD2E/edit#slide=id.p)

* Began literature review for PETase and PET degradation studies

##### Week 2

* Off week for finals

##### [Week 3](https://docs.google.com/presentation/d/1wH2a9YTCVhDeQHsOzf00dOrrgOYNyrUolY52JRQeexI/edit#slide=id.p)

* Wetlab: Developed initial protocols
* Drylab: Evaluated different methods of creating a model for new protein sequences

##### [Week 4](https://docs.google.com/presentation/d/1CP6EWY59IrhPJcV3P623Et0aHCNZG_a-Mx6REjDn2Ag/edit#slide=id.p)

* Continuation of Week 3

# June

##### [Week 1](https://docs.google.com/presentation/d/1-hA1KMC0a3G69YG1lB8Ooe7FXrh8YUs1s99g8RfalbI/edit#slide=id.p)

* Wetlab: Reagents prepared for initial testing of plasmids
* Drylab: Evaluated HMMER and RNN models and their efficacy for sequence analysis and generation

##### [Week 2](https://docs.google.com/presentation/d/1JkmoeHbtuNdLG3UUHCLSfVrMtDNvYVqo0z9oK29NCRY/edit#slide=id.p)

* Wetlab: Began practice for plasmid transformation and purification
* Drylab: Decided on using an RNN pipeline, researched prior iGEM teams’ computational work with PETase

##### [Week 3](https://docs.google.com/presentation/d/1BklqAkUwnTEajw7hmNL0NIjF3P0AcCJZv8g6I_7HJas/edit#slide=id.ge2014a9313_0_124)

* Wetlab: Received plasmids, continue work with competent cells
* Drylab: Continued research into previous iGEM work, specifically 2019 iGEM Toronto. Analyzed their pipeline and researched use-cases of UniRep + Docker

##### [Week 4](https://docs.google.com/presentation/d/17E7ev24W4Sl4oF6TSZ0p3ztoG5MlSmeUQrQeONJHdsA/edit#slide=id.p)

* Wetlab: Performed plasmid purification and Nanodrop analysis, reviewing errors for coming weeks
* Drylab: Compiled initial set of training sequences from literature review. Also performed sequence alignment of Toronto iGEM’s sequences using BLAST

# July

##### [Week 1](https://docs.google.com/presentation/d/1bnAyHE6kmmF9FN4Hl38QYyU8d1GjwFQdQ5NUy1l_vPk/edit#slide=id.p)

* Wetlab: Troubleshooting restriction digest, sequenced extracted DNA
* Drylab: Continued sequence gathering for model training, and set up Docker for pipeline testing. Emailed iGEM Toronto for questions and possible

##### [Week 2](https://docs.google.com/presentation/d/1e_fsa3oVsSB6Vjj1LR-ediGt81HpuG82O8tiv4Ml5Ys/edit#slide=id.p)

* Wetlab: Performed SDS-PAGE and troubleshooting
* Drylab: Continuation of Week 1, UniRep tutorial started

##### [Week 3](https://docs.google.com/presentation/d/1732h4edFuFtsL7opWxOv2zII7vPEOd4SOkVL6_YdC_E/edit#slide=id.p)

* Wetlab: Ran more SDS-PAGEs, began preparation for protein purification
* Drylab: Began pipeline work, continued literature review for enzyme characteristics like thermostability and catalytic activity

##### [Week 4](https://docs.google.com/presentation/d/1W9yBFgRXfXQg_uo0MR4lhWxRc_zov9LZnZNO2CN_kr0/edit#slide=id.p)

* Wetlab: Additional troubleshooting for previous protocols, began collaboration with UT Austin’s wetlab group for protein purification troubleshooting
* Drylab: Tested iGEM Toronto 2019’s initial pipeline for any output, continue studying code architecture for incorporation of our training set
* Attended JulyGEM meetup with other iGEM teams to receive feedback on our progress so far

# August

##### [Week 1](https://docs.google.com/presentation/d/17_5iAI87U401O_xrUQ04Xyg3rb6hqcKarI4hcfO6AdU/edit#slide=id.p)

* Wetlab: Literature review for protocols, especially alternative lysis
* Drylab: Pipeline tested, several technical issues and platform issues with Docker and Colab debugged. Working to create CRUD table for a PETase web database

##### [Week 2](https://docs.google.com/presentation/d/1C-FhNVVKdUvdJexONQ4QcEqYbhHCq2UOJdfQp825t_A/edit#slide=id.p)

* Wetlab: Worked on protocols for coming weeks (competent cells)
* Drylab: Completed basic pipeline debugs, able to generate sequence output. Began preparation of training set for the pipeline

##### [Week 3](https://docs.google.com/presentation/d/1LLjLETke3wk_n3dQZuqzZxWiW8mHVzdNdIu2xISO3ic/edit#slide=id.p)

* Wetlab: No updates, continued work from Week 2
* Drylab: Completed further modifications to pipeline and ran model with our training data, output sequences generated flawlessly. Continued work on web PETase database in collaboration with iGEM Toronto

##### [Week 4](https://docs.google.com/presentation/d/1TggnsJ_xugIN7gk8kPTjVbrc9-qeXwC2fiJHrbi5uxg/edit#slide=id.p)

* Wetlab: Began plasmid purification and protein purification prep, update protocols
* Drylab: No updates, aside from further pipeline testing and database work
* Interviewed Dr. Ting Lu in Bioengineering

# September

##### [Week 1](https://docs.google.com/presentation/d/1GkzuJP3rYdNdVgg0MVCc-qhCz09jTsw2LfuX5AzwOhA/edit#slide=id.p)

* Wetlab: Protein and plasmid purification, ordered mutants
* Drylab: Sent three mutant candidates to wetlab for validation. Began package development

##### [Week 2](https://docs.google.com/presentation/d/15HUNq47EgKIL7vwKNleM1yt11M4WhEJ320mbNDacIJs/edit#slide=id.gf07a4d5387_0_0)

* Wetlab: Continuation of Week 1, continued contact with UT Austin for troubleshooting
* Drylab: Drafting master document for wiki and compiling all information. Continued collaboration with iGEM Toronto for PETase sequence database. Package development in progress.

##### [Week 3](https://docs.google.com/presentation/d/15HUNq47EgKIL7vwKNleM1yt11M4WhEJ320mbNDacIJs/edit#slide=id.gf07a4d5387_0_0)

* Wetlab: Obtained PET band on SDS-PAGE gel, began prep for FPLC
* Drylab: Continued work on master wiki document

##### Week 4

* Off-week for midterms and other commitments

# October

##### [Week 1](https://docs.google.com/presentation/d/1j1odutGd0F6B3LYI3fbc72fkV40GRizPUPmH3CzStjw/edit#slide=id.gef963dee75_0_27)

* Wetlab: FPLC preparation and training
* Drylab: Drafting for wiki complete, started illustrations and design work. Initial version of package published on test index

##### [Week 2](https://docs.google.com/presentation/d/1bmM6EnKCqU50CG7Y4gG0vCpFoXZDeVKAUl2GpxDPTeo/edit#slide=id.p)

* Wetlab: Finalized additional protocols for wiki, continued FPLC prep
* Drylab: Package made available on GitHub. Met with iGEM Toronto for mutant analysis and thermostability predictions. Troubleshooted the wiki pipeline and began incorporating content to the main wiki page

##### [Week 3](https://docs.google.com/presentation/u/2/d/1BlX2LE6Emv2fiA03aXMV2PaMOVdpxqPK5JgzOYjIqrc/edit)

* Wetlab: Performed FPLC and began work with mutants
* Drylab: Continued incorporation of content into the wiki before deadline
* Wiki freeze on Oct 21

##### Week 4

* Worked on the final presentation video

iGEM UIUC\_Illinois

Email: illinoisigem@gmail.com

Headquarter: [Carl R. Woese Institute for Genomic Biology](https://www.igb.illinois.edu/)

1206 W Gregory Dr. Urbana IL 61801

Sample template built using the iGEM Wiki Starter Pack by BITS Goa.

2021 UIUC\_Illinois[Judging Form](https://igem.org/2021\_Judging\_Form?id=3713)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UIUC Illinois/Engineering

Engineering Success | UIUCiGEM

* Team

  [Team](https://2021.igem.org/Team%3AUIUC_Illinois/Team)[Attributions](https://2021.igem.org/Team%3AUIUC_Illinois/Attributions)[Collaborations](https://2021.igem.org/Team%3AUIUC_Illinois/Collaborations)
* Project

  [Contribution](https://2021.igem.org/Team%3AUIUC_Illinois/Contribution)[Description](https://2021.igem.org/Team%3AUIUC_Illinois/Description)[Engineering Success](https://2021.igem.org/Team%3AUIUC_Illinois/Engineering)[Excellence in another area](https://2021.igem.org/Team%3AUIUC_Illinois/Package)[Experiments](https://2021.igem.org/Team%3AUIUC_Illinois/Experiments)[Model](https://2021.igem.org/Team%3AUIUC_Illinois/Model)[Notebook](https://2021.igem.org/Team%3AUIUC_Illinois/Notebook)[Partnership](https://2021.igem.org/Team%3AUIUC_Illinois/Partnership)[Proposed Implementation](https://2021.igem.org/Team%3AUIUC_Illinois/Implementation)[Results](https://2021.igem.org/Team%3AUIUC_Illinois/Results)
* [Parts](https://2021.igem.org/Team%3AUIUC_Illinois/Parts)
* [Safety](https://2021.igem.org/Team%3AUIUC_Illinois/Safety)
* [Human Practices](https://2021.igem.org/Team%3AUIUC_Illinois/Human_Practices)

[ ]

# Engineering Success

---

##### Contents

![Wet Lab and Dry Lab Design](https://static.igem.org/mediawiki/2021/d/dc/T--UIUC_Illinois--img--Engineering--diagram.png)

# Drylab

Our focus with drylab this year was to discover a solution to design and optimize new PETase sequences such that they have an increased rate of PET degradation. To do this, we aimed to design a machine learning algorithm that utilized a recurrent neural network (RNN) with specific filters, such that the output will display a selected number of PETase sequences, each of which are potential candidates for increased enzymatic activity.

We began the process with a literature review and research with PETase completed in the past. We saw that the iGEM Toronto team attempted this two years ago in 2019, however we did find some potential for improvement upon their pipeline. Following our research, we gathered several sequences found in literature and in other teams’ tests to include in our training dataset. We tested and reworked our pipeline with various training datasets to improve and clean up its output based on the filters created within the pipeline. Once we were able to achieve this, we generated our sequences and sent three of our candidates over to the wetlab team for validation, where they will test the mutants for enzymatic activity.

# Wetlab

Our focus with wetlab this year was to carry out wildtype PETase protein purification and ultimately purify and gather data for the mutant PETase sequences that drylab made. To do this, we began early in the summer by doing literature review on the PET degradation process itself, from looking into key enzymes involved as well as researchers in this field that we ask questions to.

Following our research, we began to slowly design protocols from scratch, as seen in our Notebook section. We’ve made countless revisions each time to our protocols based on what we found in research papers and what past iGEM teams did. During our “build” stage, we began working with e.coli and our PET sequences where we kept track of colony growth, color changes in culture media as well as beginning to run tests and assays such as induction and SDS-PAGE.

During our “build” and “test” stages, we did a lot of troubleshooting as the results were not what we expected. For a few weeks in the summer, we went through the cycle of design, build, and test which allowed us to learn a lot about why our experimental setup was not working and what we can do differently.

Along the way, we shared what we learned with UT Austin and they did the same. Both of our teams ran into similar issues but were able to troubleshoot together. At this time point, we successfully purified our wild-type PEtase protein and have begun running a nanodrop test to look at PET film degradation. We are currently in the process of purifying our mutant PETase sequences and gathering data for it.

iGEM UIUC\_Illinois

Email: illinoisigem@gmail.com

Headquarter: [Carl R. Woese Institute for Genomic Biology](https://www.igb.illinois.edu/)

1206 W Gregory Dr. Urbana IL 61801

Sample template built using the iGEM Wiki Starter Pack by BITS Goa.

2021 UIUC\_Illinois[Judging Form](https://igem.org/2021\_Judging\_Form?id=3713)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UIUC Illinois/Experiments

Experiments | UIUCiGEM

* Team

  [Team](https://2021.igem.org/Team%3AUIUC_Illinois/Team)[Attributions](https://2021.igem.org/Team%3AUIUC_Illinois/Attributions)[Collaborations](https://2021.igem.org/Team%3AUIUC_Illinois/Collaborations)
* Project

  [Contribution](https://2021.igem.org/Team%3AUIUC_Illinois/Contribution)[Description](https://2021.igem.org/Team%3AUIUC_Illinois/Description)[Engineering Success](https://2021.igem.org/Team%3AUIUC_Illinois/Engineering)[Excellence in another area](https://2021.igem.org/Team%3AUIUC_Illinois/Package)[Experiments](https://2021.igem.org/Team%3AUIUC_Illinois/Experiments)[Model](https://2021.igem.org/Team%3AUIUC_Illinois/Model)[Notebook](https://2021.igem.org/Team%3AUIUC_Illinois/Notebook)[Partnership](https://2021.igem.org/Team%3AUIUC_Illinois/Partnership)[Proposed Implementation](https://2021.igem.org/Team%3AUIUC_Illinois/Implementation)[Results](https://2021.igem.org/Team%3AUIUC_Illinois/Results)
* [Parts](https://2021.igem.org/Team%3AUIUC_Illinois/Parts)
* [Safety](https://2021.igem.org/Team%3AUIUC_Illinois/Safety)
* [Human Practices](https://2021.igem.org/Team%3AUIUC_Illinois/Human_Practices)

[ ]

# Experiments

---

##### Contents

# Protocols

[Bacterial Transformation](https://static.igem.org/mediawiki/2021/1/11/T--UIUC_Illinois--Bacterial_Transformation.pdf)

[C41 Competent Cells](https://www.lucigen.com/docs/manuals/MA031-OverExpress-Chemicallycompetent-Cells.pdf)

[Competent Cell Prep](https://static.igem.org/mediawiki/2021/6/66/T--UIUC_Illinois--Competent_Cell_Prep.pdf)

[FPLC](https://static.igem.org/mediawiki/2021/c/c5/T--UIUC_Illinois--FPLC.pdf)

[Induction](https://static.igem.org/mediawiki/2021/a/a1/T--UIUC_Illinois--Induction.docx.pdf)

[LB Media Plate Preparation](https://static.igem.org/mediawiki/2021/c/c9/T--UIUC_Illinois--LB_Media_Plate_Preparation.pdf)

[NanoDrop Kinetic Assay](https://static.igem.org/mediawiki/2021/b/b3/T--UIUC_Illinois--NanoDrop_Kinetic_Assay.pdf)

[PCR](https://static.igem.org/mediawiki/2021/f/fa/T--UIUC_Illinois--PCR.pdf)

[SDS-PAGE](https://static.igem.org/mediawiki/2021/4/4b/T--UIUC_Illinois--SDS-PAGE.docx.pdf)

[Zymo Zippy Plasmid Purification](https://static.igem.org/mediawiki/2021/8/8a/T--UIUC_Illinois--Zymo_Zippy_Plasmid_Purification.pdf)

# Wet Lab Notebook

[Master Wet Lab Notebook](https://static.igem.org/mediawiki/parts/2/23/T--UIUC_Illinois--Master-Wet-Lab-Notebook.docx.pdf)

iGEM UIUC\_Illinois

Email: illinoisigem@gmail.com

Headquarter: [Carl R. Woese Institute for Genomic Biology](https://www.igb.illinois.edu/)

1206 W Gregory Dr. Urbana IL 61801

Sample template built using the iGEM Wiki Starter Pack by BITS Goa.

2021 UIUC\_Illinois[Judging Form](https://igem.org/2021\_Judging\_Form?id=3713)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:UIUC Illinois/Results

Results | UIUCiGEM

* Team

  [Team](https://2021.igem.org/Team%3AUIUC_Illinois/Team)[Attributions](https://2021.igem.org/Team%3AUIUC_Illinois/Attributions)[Collaborations](https://2021.igem.org/Team%3AUIUC_Illinois/Collaborations)
* Project

  [Contribution](https://2021.igem.org/Team%3AUIUC_Illinois/Contribution)[Description](https://2021.igem.org/Team%3AUIUC_Illinois/Description)[Engineering Success](https://2021.igem.org/Team%3AUIUC_Illinois/Engineering)[Excellence in another area](https://2021.igem.org/Team%3AUIUC_Illinois/Package)[Experiments](https://2021.igem.org/Team%3AUIUC_Illinois/Experiments)[Model](https://2021.igem.org/Team%3AUIUC_Illinois/Model)[Notebook](https://2021.igem.org/Team%3AUIUC_Illinois/Notebook)[Partnership](https://2021.igem.org/Team%3AUIUC_Illinois/Partnership)[Proposed Implementation](https://2021.igem.org/Team%3AUIUC_Illinois/Implementation)[Results](https://2021.igem.org/Team%3AUIUC_Illinois/Results)
* [Parts](https://2021.igem.org/Team%3AUIUC_Illinois/Parts)
* [Safety](https://2021.igem.org/Team%3AUIUC_Illinois/Safety)
* [Human Practices](https://2021.igem.org/Team%3AUIUC_Illinois/Human_Practices)

[ ]

# Results

Summary of our achievements

---

##### Contents

# Drylab Results

The following pictures are snapshots of sections of our pipeline output. The RNN model is able to run based off of our training dataset “sequences\_final.txt”, and is able to put out the specified number of sequences requested in the code. In this instance, we are telling the pipeline that we want three potential PETase mutant candidates, and the pipeline is able to return the three mutants.

To view our full pipeline, training dataset, and sequence outputs, [here](https://github.com/UIUCiGEM/apPETite) is our Github.

![Running the RNN model and training it with our dataset.](https://static.igem.org/mediawiki/2021/f/f3/T--UIUC_Illinois--img--Results--DrylabOutput1.png)

![Sequences generated and printed for output.](https://static.igem.org/mediawiki/2021/b/b1/T--UIUC_Illinois--img--Results--DrylabOutput2.png)

Figure 2: Sequences generated and printed for output.

# NanoDrop Kinetic Assay

Due to our complications in our purification of wild-type PETase, we decided to begin our testing on our crude unpurified soluble sample. From our literature review, we decided to use a NanoDrop method for evaluating the enzymatic degradation kinetics of PET films as described by Zhong-Johnson et al [1]. Specifically, we utilized a 'bulk absorbance method' in which all degradation products contribute to the absorbance.

We initially took an assay of our crude soluble sample as we were still in the process of conducting FPLC and purifying the wild type PETase. We used a Thermomixer set to 200 rpm and incubated our samples at a temperature of 40 °C, which we chose based on a publication reviewing the current state of PET degrading enzymes [2]. From this assay, we were able to determine the ideal quantities of protein to use for testing the enzymatic degradation kinetics of wild-type PETase. Additionally, after leaving these samples at room temperature overnight, we found that the samples with higher concentrations of protein (≥750 μl) had noticeably higher degradation within the tube (see Crude PET Degradation Visual Assay).

Figures 3 and 4 show reactions consisting of sample, reaction buffer, and PET plastic film. All measurements were performed on a NanoDrop One. Reaction buffer plus PET plastic film was used as a blank and Dialysis buffer plus PET plastic film acted as a negative control. (Note that the uninduced sample in Figure 3 was noticeably higher in absorbance due to the high concentration of cells.)

![Plot of absorbance taken at 260 nm for crude soluble wild-type PETase sample in terms of A260 vs. enzyme sample quantity.](https://static.igem.org/mediawiki/2021/9/9e/T--UIUC_Illinois--img--Results--fig1.png)

Figure 3: Plot of absorbance taken at 260 nm for crude soluble wild-type PETase sample in terms of A260 vs. enzyme sample quantity.

![Plot of absorbance taken at 260 nm for purified wild-type PETase sample in terms of A260 vs. enzyme sample quantity.](https://static.igem.org/mediawiki/2021/6/6d/T--UIUC_Illinois--img--Results--fig2.png)

Figure 4: Plot of absorbance taken at 260 nm for purified wild-type PETase sample in terms of A260 vs. enzyme sample quantity.

Despite some inconsistencies in our results due to readings taken by the NanoDrop, we were able to observe an overall upward trend in absorbance at 260 nm for our protein sample using the “bulk absorbance method” as depicted by Figures 1 and 2, which suggests that an increase in PET degradation products is occurring over time. However, as shown in Figure 2, the 900 μl protein sample displayed an inconsistent trend in absorbance at 260 nm, which is likely due to the small quantity of reaction buffer (100 μl) in the tube. Moreover, the purified wild-type PETase produced a more consistent trend with the results found by Zhong-Johnson et al., in which the absorbance at 260 nm fell within the range of 0.0 to 1.0.

While we have only included our wild-type PETase results above, we are currently in the process of testing our mutated PETase sequences and intend to test them via the NanoDrop Assay in the near future.

# SDS-PAGE

![7/15/21. First SDS-PAGE ran on E. coli BL21 expression of PETase. We used Coomassie Blue dye to stain and Destain. There was no clear data from the gel.](https://static.igem.org/mediawiki/2021/5/5b/T--UIUC_Illinois--img--Results--SDS715.png)

Figure 5: 7/15/21. First SDS-PAGE ran on E. coli BL21 expression of PETase. We used Coomassie Blue dye to stain and Destain. There was no clear data from the gel.

![7/17/21. For this Gel, we switched the staining method to ThermoFisher SimplyBlueTM SafeStain and destained it with water and 20% NaCl Solution. This provided us with a clearer staging of the gel.](https://static.igem.org/mediawiki/2021/f/f0/T--UIUC_Illinois--img--Results--SDS717.png)

Figure 6: 7/17/21. For this Gel, we switched the staining method to ThermoFisher SimplyBlueTM SafeStain and destained it with water and 20% NaCl Solution. This provided us with a clearer staging of the gel.

![7/24/21. Staining Method: ThermoFisher SimplyBlueTM SafeStain and destained with water and 20% NaCl Solution. Lanes as follows -- Lane 15: Bio-Rad Kaleidoscope Ladder, Lane 14: Insoluble Fraction, Lane 12: Insoluble Fraction, Lane 11: Insoluble Fraction, Lane 10: Insoluble Fraction, Lane 9: Soluble Fraction, Lane 8: Soluble Fraction, Lane 6: Soluble Fraction, Lane 4: Positive Control Soluble, Lane 2: Positive Control Insoluble, Lane 1: Bio-Rad All Blue Ladder ](https://static.igem.org/mediawiki/2021/f/f0/T--UIUC_Illinois--img--Results--SDS717.png)

Figure 7: 7/24/21. Staining Method: ThermoFisher SimplyBlueTM SafeStain and destained with water and 20% NaCl Solution. Lanes as follows -- Lane 15: Bio-Rad Kaleidoscope Ladder, Lane 14: Insoluble Fraction, Lane 12: Insoluble Fraction, Lane 11: Insoluble Fraction, Lane 10: Insoluble Fraction, Lane 9: Soluble Fraction, Lane 8: Soluble Fraction, Lane 6: Soluble Fraction, Lane 4: Positive Control Soluble, Lane 2: Positive Control Insoluble, Lane 1: Bio-Rad All Blue Ladder

![7/28/21. Lanes as follows --  C: control, I: Insoluble Fraction, S: Soluble Fraction. Staining Method: ThermoFisher SimplyBlueTM SafeStain and destained with water and 20% NaCl Solution. We used Used 20 ul well 12% Gel. ](https://static.igem.org/mediawiki/2021/8/88/T--UIUC_Illinois--img--Results--SDS728.jpg)

Figure 8: 7/28/21. Lanes as follows -- C: control, I: Insoluble Fraction, S: Soluble Fraction. Staining Method: ThermoFisher SimplyBlueTM SafeStain and destained with water and 20% NaCl Solution. We used Used 20 ul well 12% Gel.

![9/25/21. Successful Induction and expression of PETase in E. coli C41. ](https://static.igem.org/mediawiki/2021/8/88/T--UIUC_Illinois--img--Results--SDS728.jpg)

Figure 9: 9/25/21. Successful Induction and expression of PETase in E. coli C41.

![10/9/21. SDS-PAGE Post FPLC Troubleshooting performed. Lanes as follows -- 1: Kaleidoscope Ladder, 4: Centrifuged Pellet Before FPC, 7 & 8: Supernatant from FPLC, 12-15: FPLC Samples. Issues encountered included high centrifugation speed which resulted in lost soluble supernatant. FPLC Method settings were also off, wells were overflowing and fractions mixed.](https://static.igem.org/mediawiki/2021/6/65/T--UIUC_Illinois--img--Results--SDS1009.jpg)

Figure 10: 10/9/21. SDS-PAGE Post FPLC Troubleshooting performed. Lanes as follows -- 1: Kaleidoscope Ladder, 4: Centrifuged Pellet Before FPC, 7 & 8: Supernatant from FPLC, 12-15: FPLC Samples. Issues encountered included high centrifugation speed which resulted in lost soluble supernatant. FPLC Method settings were also off, wells were overflowing and fractions mixed.

![10/18/21. Successful FPLC purification and dialysis of PETase. Lanes as follows --  Lane 1: Kaleidoscope Ladder, Lane 2: Induced Total (Crude soluble and insoluble), Lane 3: Uninduced (Crude Sample to IPTG), Lanes 9-13: FPLC samples ](https://static.igem.org/mediawiki/2021/7/76/T--UIUC_Illinois--img--Results--SDS1018.JPG)

Figure 11: 10/18/21. Successful FPLC purification and dialysis of PETase. Lanes as follows -- Lane 1: Kaleidoscope Ladder, Lane 2: Induced Total (Crude soluble and insoluble), Lane 3: Uninduced (Crude Sample to IPTG), Lanes 9-13: FPLC samples

# Wet Lab Data Induction Troubleshooting Assay

In this assay we had two tubes of BL21 E. coli cells with our plasmid of interest in them. Both tubes were grown in LB overnight; however, the tube to the left was induced with IPTG beforehand and the tube to the right was not. The uninduced tube showed a higher visual concentration of cells and led us to conclude that that induction of the PETase protein hindered the growth of the BL21.

![Results of Induction Troubleshooting Assay](https://static.igem.org/mediawiki/2021/9/92/T--UIUC_Illinois--img--Results--InductionAssay.png)

Figure 12: Results of Induction Troubleshooting Assay

# Wet Lab Data Crude PET Degradation Visual Assay

In this assay we set up microcentrifuge tubes with various concentrations of crude C41 cell extract with PETase, ¼ hole punch of pet film, and reaction buffer. We let the tubes sit overnight at room temperature and observed the results the following day.

![Crude Degradation Assay 500 uL](https://static.igem.org/mediawiki/2021/9/96/T--UIUC_Illinois--img--Results--Crude500.png)

Figure 13: Crude Degradation Assay 500 uL

![Crude Degredation Assay 750 uL](https://static.igem.org/mediawiki/2021/f/f4/T--UIUC_Illinois--img--Results--Crude750.png)

Figure 14: Crude Degredation Assay 750 uL

![Crude C Degredation Assay 500 uL](https://static.igem.org/mediawiki/2021/a/a6/T--UIUC_Illinois--img--Results--CrudeC500.png)

Figure 15: Crude C Degredation Assay 500 uL

![Crude C Degredation Assay 750 uL](https://static.igem.org/mediawiki/2021/a/a7/T--UIUC_Illinois--img--Results--CrudeC750.png)

Figure 16: Crude C Degredation Assay 750 uL

![Crude C Degredation Assay 100 and 250 uL](https://static.igem.org/mediawiki/2021/0/02/T--UIUC_Illinois--img--Results--CrudeC100250.png)

Figure 17: Crude C Degredation Assay 100 and 250 uL

# References

1. Zhong-Johnson, E.Z.L., Voigt, C.A. & Sinskey, A.J. (2021).

   An Absorbance Method for Analysis of Enzymatic Degradation Kinetics of Poly(Ethylene Terephthalate) Films.

   Scientific Reports vol. 11, no. 1

   [CrossRef](https://doi.org/10.1038/s41598-020-79031-5)[Google Scholar](https://scholar.google.com/scholar?q=An Absorbance Method for Analysis of Enzymatic Degradation Kinetics of Poly(Ethylene Terephthalate) Films.)[Back to text](#intext1)
2. Kawai, Fusako. (2021).

   The Current State of Research on Pet Hydrolyzing Enzymes Available for Biorecycling.

   Catalysts vol. 11, no. 2, p. 206.

   [CrossRef](https://doi.org/10.3390/catal11020206)[Google Scholar](https://scholar.google.com/scholar?q=The Current State of Research on Pet Hydrolyzing Enzymes Available for Biorecycling.)[Back to text](#intext2)

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