iGEM 2019 — Team SIS_Korea

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# Team:SIS Korea/Notebook

DePet

* [Home](https://2019.igem.org/Team%3ASIS_Korea)
* Team
  + [Members](https://2019.igem.org/Team%3ASIS_Korea/Team)
  + [Collaboration](https://2019.igem.org/Team%3ASIS_Korea/Collaborations)
  + [Attribution](https://2019.igem.org/Team%3ASIS_Korea/Attribution)
* Project
  + [Description/Design](https://2019.igem.org/Team%3ASIS_Korea/Description-Design)
  + [Protocol](https://2019.igem.org/Team%3ASIS_Korea/Protocol)
  + [Results](https://2019.igem.org/Team%3ASIS_Korea/Results)
  + [Parts](https://2019.igem.org/Team%3ASIS_Korea/Parts)
* Device
  + [Software](https://2019.igem.org/Team%3ASIS_Korea/Software)
  + [Hardware](https://2019.igem.org/Team%3ASIS_Korea/Hardware)
* Human Practices
  + [Education](https://2019.igem.org/Team%3ASIS_Korea/Education)
  + [Campaign](https://2019.igem.org/Team%3ASIS_Korea/Campaign)
  + [Interviews](https://2019.igem.org/Team%3ASIS_Korea/Interviews)
* [Awards/Judging Form](https://2019.igem.org/Team%3ASIS_Korea/Awards-Judging)
* [Safety](https://2019.igem.org/Team%3ASIS_Korea/Safety)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SIS Korea/Experiments

DePet

* [Home](https://2019.igem.org/Team%3ASIS_Korea)
* Team
  + [Members](https://2019.igem.org/Team%3ASIS_Korea/Team)
  + [Collaboration](https://2019.igem.org/Team%3ASIS_Korea/Collaborations)
  + [Attribution](https://2019.igem.org/Team%3ASIS_Korea/Attribution)
* Project
  + [Description/Design](https://2019.igem.org/Team%3ASIS_Korea/Description-Design)
  + [Protocol](https://2019.igem.org/Team%3ASIS_Korea/Protocol)
  + [Results](https://2019.igem.org/Team%3ASIS_Korea/Results)
  + [Parts](https://2019.igem.org/Team%3ASIS_Korea/Parts)
* Device
  + [Software](https://2019.igem.org/Team%3ASIS_Korea/Software)
  + [Hardware](https://2019.igem.org/Team%3ASIS_Korea/Hardware)
* Human Practices
  + [Education](https://2019.igem.org/Team%3ASIS_Korea/Education)
  + [Campaign](https://2019.igem.org/Team%3ASIS_Korea/Campaign)
  + [Interviews](https://2019.igem.org/Team%3ASIS_Korea/Interviews)
* [Awards/Judging Form](https://2019.igem.org/Team%3ASIS_Korea/Awards-Judging)
* [Safety](https://2019.igem.org/Team%3ASIS_Korea/Safety)

# 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://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:SIS Korea/Results

DePet

[![](https://static.igem.org/mediawiki/2019/9/9c/T--SIS_Korea--logodesign.jpg)](https://2019.igem.org/Team%3ASIS_Korea)* [Home](https://2019.igem.org/Team%3ASIS_Korea)
* Team
  + [Members](https://2019.igem.org/Team%3ASIS_Korea/Team)
  + [Collaboration](https://2019.igem.org/Team%3ASIS_Korea/Collaborations)
  + [Attribution](https://2019.igem.org/Team%3ASIS_Korea/Attribution)
* Project
  + [Description/Inspiration](https://2019.igem.org/Team%3ASIS_Korea/Description-Design)
  + [Design](https://2019.igem.org/Team%3ASIS_Korea/Design)
  + [Labnotes](https://2019.igem.org/Team%3ASIS_Korea/Experiment)
  + [Protocol](https://2019.igem.org/Team%3ASIS_Korea/Protocol)
  + [Results](https://2019.igem.org/Team%3ASIS_Korea/Results)
  + [Parts](https://2019.igem.org/Team%3ASIS_Korea/Parts)
* Device
  + [Software](https://2019.igem.org/Team%3ASIS_Korea/Software)
  + [Hardware](https://2019.igem.org/Team%3ASIS_Korea/Hardware)
* Human Practices
  + [Introduction](https://2019.igem.org/Team%3ASIS_Korea/Introduction)
  + [Education](https://2019.igem.org/Team%3ASIS_Korea/Education)
  + [Campaign](https://2019.igem.org/Team%3ASIS_Korea/Campaign)
  + [Interviews](https://2019.igem.org/Team%3ASIS_Korea/Interviews)
  + [Survey Analysis](https://2019.igem.org/Team%3ASIS_Korea/SurveyAnalysis)
  + [Integrated
    Human Practices](https://2019.igem.org/Team%3ASIS_Korea/IntegratedHumanPractices)
* [Awards/Judging Form](https://2019.igem.org/Team%3ASIS_Korea/Awards-Judging)
* [Safety](https://2019.igem.org/Team%3ASIS_Korea/Safety)

# Results

## 1. Cloning of Recombinant DNA for expressing PETase and MHETase *E. coli*

![](https://static.igem.org/mediawiki/2019/4/46/T--SIS_Korea--r1.png)

Figure 1. Part of agarose gel image used for DNA cloning in this project

Our basic parts were synthesized from Twist Bioscience and IDTDNA. When needed, the 5’-nucleotide of synthetic gene fragments were phosphorylated by T4 polynucleotide kinase. After that, they were inserted into the blunted plasmid. After selecting the positive clones with the use of restriction enzymes, these plasmids were finally sequenced. The genes used in this project were cut with NdeI and Xhol and were subcloned into the expression vectors.

## 2. Reporter gene Assay for light inducible promoter in *E. coli*

![](https://static.igem.org/mediawiki/2019/4/4e/T--SIS_Korea--r2.png)

Figure 2. Light inducible expression of mScarlet in *E. coli*.

To overexpress the PET degrading enzymes in *E. coli*, we used two different expression systems. The
first is the light inducible expression system, which was used by many iGem teams. To check whether the
regulatory part was functioning properly under our testing conditions, we incubated *E. coli*
BL21 with pDawn-mScarlet(BBa\_K3288054) under white light in 37 degrees in a shaking incubator. This expression
system successfully functioned without much issues in not only the Normal LB broth(pH6.6), but also the
maximum activation pH of the PET degrading enzymes (8.5).

## 3. Reporter gene Assay for selection of constitutive promoter in *E. coli*

![](https://static.igem.org/mediawiki/2019/7/77/T--SIS_Korea--r11.jpg)

Figure 3. Constitutive expression of mSCarlet in *E. coli*.

Next, in order to express constitutively, we combined the following to check expression: J23119, constitutive
strong promoter, and reporter gene. However, for an unknown reason, regulatory part did not function in our
experiment condition (Figure 3B). Therefore, we fused 4 strong constitutive promoters that were chosen based
on research papers and experiments - CP25[(BBa\_K1509003)](http://parts.igem.org/Part%3ABBa_K1509003),
CP44[(BBa\_K864511)](http://parts.igem.org/Part%3ABBa_K864511) , J23119[(BBa\_J23119)](http://parts.igem.org/Part%3ABBa_J23119) - with 3 strong ribosome binding sites - RBS,
Cdog(ref), NO29(ref), Dawn(from pDawn)- and constructed 12 constitutive expression units (CEU)-mScarlet[(BBa\_K3288032,](http://parts.igem.org/part%3ABBa_K3288032) [BBa\_K3288033](http://parts.igem.org/part%3ABBa_K3288033)  [BBa\_K3288034,](http://parts.igem.org/part%3ABBa_K3288034) [BBa\_K3288035,](http://parts.igem.org/part%3ABBa_K3288035) [BBa\_K3288036,](http://parts.igem.org/part%3ABBa_K3288036) [BBa\_K3288037,](http://parts.igem.org/part%3ABBa_K3288037) [BBa\_K3288038,](http://parts.igem.org/part%3ABBa_K3288038) [BBa\_K3288039,](http://parts.igem.org/part%3ABBa_K3288039) [BBa\_K3288040,](http://parts.igem.org/part%3ABBa_K3288040) [BBa\_K3288041,](http://parts.igem.org/part%3ABBa_K3288041) [BBa\_K3288042,](http://parts.igem.org/part%3ABBa_K3288042) [BBa\_K3288043,)](http://parts.igem.org/part%3ABBa_K3288043) to create to create a new regulatory part.
As it is shown in Figure 3A, CEU(pCEU03([BBa\_K3288034](http://parts.igem.org/part%3ABBa_K3288034)),
pCEU06([BBa\_K3288037](http://parts.igem.org/part%3ABBa_K3288037)), pCEU09(<BBa_K3288040>), pCEU12([BBa\_K3288043](http://parts.igem.org/part%3ABBa_K3288043) that include RBS
(Dawn;tttgtttaactttaagaaggaga), which originated from pDawn, had the highest expression rate regardless of the
promoter. Furthermore, fluorescence was observed in pCEU04([BBa\_K3288035](http://parts.igem.org/part%3ABBa_K3288035)), pCEU07([BBa\_K3288038](http://parts.igem.org/part%3ABBa_K3288038)), pCEU11([BBa\_K3288042](http://parts.igem.org/part%3ABBa_K3288042)). In order to quantify 12 pCEU activation,
we sonicated overnight cultured *E.coli* and obtained whole lysate. We used Spectrophotometer to measure
A280(total protein) of whole lysate and A569(excitation wavelength of mScarlet). Results showed that pCEU09
and pCEU12 have the high expression rates.

## 4. The finding optimal condition for pCEU12 promoter in *E. coli*

![](https://static.igem.org/mediawiki/2019/d/d1/T--SIS_Korea--r4.png)

Figure 4. Expression of pCEU12-mScarlet in *E. coli*.

Among the 4 strong constitutive expression units that were checked in Figure 3, we used pCEU12 that did not
disturb the growth of *E.coli* (data not shown) to check mScarlet expression in various conditions.
Results showed that at 37℃, mScarlet expression rate decreased in LB(pH8.5), and after 12 hours of incubation,
no mScarlet were observed at 25 and 30 degrees celsius, but after 24 hours or more incubation, we were able to
observe mScarlet.

## 5. The expression of PETase and MHETase by two different promoters at low temperature and high pH conditions.

![](https://static.igem.org/mediawiki/2019/4/4f/T--SIS_Korea--r5.png)

Figure 5. Production of PET degrading enzymes in low temp. and high pH condition.

Based on original research, when PET degrading enzymes are overexpressed at 37 degrees Celsius, they create
insoluble aggregates(inclusion body) that cannot show enzyme activation. Therefore, they have to be expressed
slowly in low temperature. We checked PET degrading enzyme expressions in low temperature conditions In two
kinds of expression systems. In Figure 5A, our constitutive system could not produce PETase in low temperature
condition. On the other hand, light inducible expression system was able to induce both PETase and MHEtase
expressions. Therefore, we used light inducible expression system for every protein expression after that.

## 6. Prediction of secretion signal peptide fused PETase by SignalP.

![](https://static.igem.org/mediawiki/2019/a/af/T--SIS_Korea--r6.png)

Figure 6. Prediction of secretion signal fused PETase by SignalP.

According to previous research (Hogyun Seo et al. (2019), Ideonella sakaiensis’s PETase was not properly
secreted into extracellular matrix of *E.coli*. In order to increase PET degradation enzyme secretion
rate in *E.coli*, we researched and chose 2 secretion signal peptides(SPLamB, NSP4). In the research
paper (Hogyun Seo et al. (2019), LamB’s signal peptide most effectively secreted PETase than other signal
peptides(PelB, MalE, etc). The PETase used in the aforementioned research had removed 5 other amino acids
(Q28TNPY32) in addition to predicted signal peptide(M1NFPRASRLMQAAVLGGLMAVSAAATA27). We included the 5 amino
acids and again computed. Results showed that there is a higher probability of cleavage site and signal
peptide likelihood than SPLamB-PETase(33-290). Novel signal peptide NSP4 that was reported in Soojin Han el
al. (2017)’s research paper was attached to PETase(28-290), and when predicted, NSP4-PETase(28-290) was
estimated to have a higher probability of secretion than SPLamB-PETase(28-290).

## 7. Prediction of secretion signal peptide fused PETase by SignalP.

![](https://static.igem.org/mediawiki/2019/0/04/T--SIS_Korea--r7.png)

Figure 7. Prediction of secretion signal fused PETase by SignalP.

In order to confirm the predicted data, we induced PETase that has each signal peptide attached with light at
18 °C for 24 hours. We centrifuges and removed *E.coli*, finally concentrating the media. We purified
concentrated LB media with Nickel column and the purified proteins were analyzed with SDS-PAGE and immunoblot
assay. As it is shown in Figure 7, SPLamB-PETase(28-290) was secreted 20% more, NSP4-PETase(28-290) was
secreted 50% more than SPLamB-PETase(33-290).

## 8. Degradation of PET disc by extracellular PETase and MHETase.

![](https://static.igem.org/mediawiki/2019/e/e8/T--SIS_Korea--r8.png)

Figure 8. Degradation of PET disc by extracellular PET degrading enzymes.

After PETase and MHETase was analyzed based on protein structures, some of them were reported to be point
mutated with increased enzyme activation. Our team checked PETase wild type, W159H/S238F, and
S121E/D186H/R280A’s PET degradation activities to choose enzymes for PET degrading device. These enzymes were
produced at 18 °C in LB (buffered 50mM BTP (pH8.5) for 24 hours. After centrifuged, we put PET disc into
cleared media with no *E.coli* and reacted it for 24 hours at 40 degrees Celsius. Everyday, we replaced
media with ones that has new PET degrading enzyme secreted. After 5 days, we washed PET disc and dried it up
to measure the change in weight. Results showed that NSP4-PETase (S121E/D186H/R280A) degraded the most PET.

Based on previous data, we constructed PET degrading device(DePET). DePET has two parts. For the first part,
the temperature is kept at 18 degrees Celsius and LB media (50 mM BTP, pH 6.6) is used to produce PET
degrading enzyme stably. For the second part, the temperature is kept at 40 °C and pH is kept at 8.5 to
effectively degrade PET. We used NSP4-PETase(S121E/D186H/R280A) and NSP4-MHETase(W397A) as PET degrading
enzyme.

## 9. Growth curve of engineered *E. coli at 18 ℃*.

![](https://static.igem.org/mediawiki/2019/e/ec/T--SIS_Korea--r9.png)

Figure 9. *E. coli* growth curve at 18 ℃

Also, due to the fact that we needed to decide when to replace the media (for the automation of DePET), we
had to first determine the growth curve of the *E. coli* under our expression conditions. As it can be
seen in Figure 9, *E. coli* BL21 reaches a stationary phase at 18 °C after 24 hours. Also, PET degrading
enzyme loses half of its activation after 24 hours at 40 degrees Celsius. Therefore, we set it up so enzyme
production and PET degradation would circulate every 24 hours. After, we checked if PET disc is effectively
degraded at DePET in this condition.

## 10. Degradation of PET by DePET (Prototype)

![](https://static.igem.org/mediawiki/2019/3/3d/T--SIS_Korea--r10.png)

Figure 10. Degradation of PET by DePET.

First, PET disc were treated for 2 days at DePET. In order to find out about the change in PET disc’s
surface, we used atomic force microscopy and obtained an image. As it is shown in Figure 10A, PET disc that
was treated for 2 days in DePET had more hollow parts than negative control. In order to check whether DePET
production and degradation circulation work properly, we drove the DePET for 20 days. In DePET, the weight of
PET gradually decreased over time. These results indicate that the PET degradation function of DePET is
operating normally (Figure 10B).

## References

Han, S., Machhi, S., Berge, M., Xi, G., Linke, T., & Schoner, R. (2017). Novel signal peptides improve the
secretion of recombinant Staphylococcus aureus Alpha toxin H35L in Escherichia coli. AMB Express, 7(1), 93.

Eckdahl, A. J., Neal, R., Campbell, A. M., & Eckdahl, T. T. (2017). rClone: a synthetic biology tool that
enables the research of bacterial translation. Journal of Young Investigators, 32(3).
Chen, J., Zhu, X., Tan, Z., Xu, H., Tang, J., Xiao, D., & Zhang, X. (2014). Activating C 4-dicarboxylate
transporters DcuB and DcuC for improving succinate production. Applied microbiology and biotechnology, 98(5),
2197-2205.

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Email: igemsiskorea@gmail.com

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