iGEM 2022 — Team uppsala

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

## Inspirations

---

* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

{% endblock %}

[![](https://static.igem.wiki/teams/4378/wiki/img/logo.svg)](index.html)

Project

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D)
[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)
[Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)

Lab

[Overview](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27overview%27%29%20%7D%7D)
[General protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27general_protocols%27%29%20%7D%7D)
[LCC](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27LCC%27%29%20%7D%7D)
[PETase](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27PETase%27%29%20%7D%7D)
[MHETase](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27mhetase_%27%29%20%7D%7D)
[TPA transporter](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27TPA-transporter%27%29%20%7D%7D)
[TPADO](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27TPADO%27%29%20%7D%7D)
[DCDDH](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27dcddh%27%29%20%7D%7D)
[Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D)

Team

[The Team](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27team%27%29%20%7D%7D)
[Attributions](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27attributions%27%29%20%7D%7D)

Human Practices

[Overview](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D)
[Proposed Implementation](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D)
[Collaborations](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27collaborations%27%29%20%7D%7D)
[Education & Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27communication%27%29%20%7D%7D)
[Inclusivity](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27inclusivity%27%29%20%7D%7D)
[Sustainable development](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27sustainable%27%29%20%7D%7D)

[Safety](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27safety%27%29%20%7D%7D)

[Parts](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27parts%27%29%20%7D%7D)

![engineering.svg](https://static.igem.wiki/teams/4378/wiki/img/headers/engineering.svg)

[# Engineering](#main_cont_link)

1. [Construct design](#sec_1_link)
2. [Cloning and Transformation](#sec_2_link)
3. [Overexpression](#sec_3_link)
4. [Reflection](#sec_4_link)
5. [References](#sec_5_link)

# Engineering cycles

![](https://static.igem.wiki/teams/4378/wiki/img/terminator-glasses.png)Construct design

### Design

PET accounts for 12% or all solid waste materials worldwide (George, N. & Kurian, T. 2014) even though it could be theoretically integrated into a recycling or upcycling process.
Since enzymatic depolymerisation possess great potential for the utilization of PET waste it was decided to attempt the integration of all enzymes needed for PET upcycling into PCA as an easy to handle and cheap option.
After extensive research our team identified the necessary enzymes for this task and thought of a design to assemble those enzymes in a single organism as it has never been done before.
To allow characterization of all the necessary enzymes individually in sufficient amounts all were designed to have the cassette necessary for inducible protein overexpression through the T7-RNA-Polymerase system.
All the coding sequences (CDS) were carefully collected and codon-optimised for expression in *E. coli* and contained the following elements: BioBrick prefix, containing restriction sites for EcoRI and XbaI, and suffix with sites for restriction by SpeI and PstI were added on ends of the construct for easier insertion into vectors.
Our construct was also designed with RBS, T7 promoter and terminator and lac operator for optimal induction of protein expression(Du, L., Villarreal, S. & Forster, A. C. 2012) (Shepherd, T. R. *et al.* 2017) and C-terminal histidine tag for enzyme purification on immobilized metal affinity chromatography.

### Problem

Initially the CDS of PETase, MHETase and LCC were ordered from Integrated DNA Technologies (IDT) and designed to be compatible with any BioBrick vector.
After the order was shipped, it was noticed that the necessary flanking regions next to the restriction sites to allow effective restriction were missing.

### Fix

Fortunately, this mistake was noticed early enough to design primers which could introduce extra base pairs in both ends of the construct by annealing to the prefix (fwd) or suffix (rev). Since all the constructs initially ordered ended with the prefix and suffix one universal primer pair was able to fix the constructs via PCR. PCR primers used were:

Forward 5’-NNNNNGGAATTCGCGGCCGCTTCTAG,

Reverse 5’-NNNNNNCTGCAGCGGCCGCTACTA-3’.

Further orders from IDT were made containing the necessary flaking regions.

![](https://static.igem.wiki/teams/4378/wiki/img/terminator-glasses.png)Cloning and Transformation

### Design

To allow proper overexpression of the different enzymes the now fixed sequences should be ligated into a pET24a vector which was modified in 2009 to be compatible with BioBrick cloning.

### Problem

The transformation plates usually showed no or very few colonies after one night of incubation.
The few colonies which grew also showed in restriction analysis religation of the pET24 vector instead of the desired assembled construct.

### Fix

To overcome this problem NEB® 5-alpha Competent *E. coli* (High Efficiency) were used.
Those showed after transformation high amounts of colonies. Screening of those colonies showed that usually 25-50% of those contained the correct assembled construct.
This showed that the cloning protocols used showed low efficiency which could be caused by enzymes with lowered activity due to suboptimal storage and handling.
Another factor which could have had influence on our results was the before mentioned PCR to fix the construct as the PCR samples needed gel purification due to undesired side products.
These purification usually resulted in low yield and impure DNA samples. The commercial cells which had a transformation efficiency 10^4 higher than our prepared cells most likely just overcame the low efficiency of the cloning procedure by its efficiency.

![](https://static.igem.wiki/teams/4378/wiki/img/terminator-glasses.png)Overexpression

### Design

As multiple Lab groups struggled with the use of pET24a as a vector they changed to a pSB1C3 vector containing a cassette for constitutive protein expression of the chromoprotein mRFP1 as it allowed easier screening for successful transformation by color of the colony instead of restriction analysis.

### Problem

After a Lab group succeeded cloning the fixed construct into pSB1C3 they attempted protein overexpression.
Multiple tries didn’t show any sign of expression of the desired protein in SDS-PAGE analysis even though an overexpression of the T7-Polymerase in the BL21 cells was observed.
The reason for this wasn’t ultimately found.

### Fix

The same expression cassette was later cloned into a pET24a vector.
With this vector which is specifically designed for overexpression a band at the desired size was observed in SDS-PAGE which shows the importance of expression vectors over vectors designed for different purposes.

![](https://static.igem.wiki/teams/4378/wiki/img/terminator-glasses.png)Reflection

Our team went through the mentioned engineering cycles over the course of our lab period to overcome these obstacles.
In the end these efforts paid of as we managed the overexpression of two of our enzymes and one of them showed a surprisingly high over expression.
Many of these problems we encountered were new to us, but were overcame them through troubleshooting.
In some cases when we were unsure about the root cause of the problem but controls allowed us to come up with reasonable hypotheses.
Through using the scientific method and eliminating possibilities, we managed to make progress towards our set goals.
Overall we learned a lot during this summer and especially through these engineering cycles.

![](https://static.igem.wiki/teams/4378/wiki/img/terminator-glasses.png)References

Du, L., Villarreal, S. & Forster, A. C. Multigene expression in vivo: Supremacy of large versus small terminators for T7 RNA polymerase. Biotechnology and Bioengineering 109, 1043–1050 (2012).

George, N. & Kurian, T. Recent developments in the chemical recycling of postconsumer poly(ethylene terephthalate) waste. Ind. Eng. Chem. Res. 53, 14185–14198 (2014).

Shepherd, T. R. et al. De novo design and synthesis of a 30-cistron translation-factor module. Nucleic Acids Research 45, 10895–10905 (2017).

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© 2022 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International license](https://creativecommons.org/licenses/by/4.0/).
The repository used to create this website is available at [gitlab.igem.org/2022/uppsala](https://gitlab.igem.org/2022/uppsala).

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

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

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## 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 also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* 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.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}