iGEM 2021 — Team Thrace

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

# Team:Thrace/Notebook

[![](https://static.igem.org/mediawiki/2021/b/bb/T--Thrace--salica_logo_short.png)](https://2021.igem.org/Team%3AThrace)

* [Home](https://2021.igem.org/Team%3AThrace)
* Team

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

  [Project Description](https://2021.igem.org/Team%3AThrace/Description)
  [Contribution](https://2021.igem.org/Team%3AThrace/Contribution)
  [Engineering](https://2021.igem.org/Team%3AThrace/Engineering)
  [Results](https://2021.igem.org/Team%3AThrace/Results)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3AThrace/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3AThrace/Implementation)
* Wet Lab

  [Project Design](https://2021.igem.org/Team%3AThrace/Project_Design)
  [Notebook](https://2021.igem.org/Team%3AThrace/Notebook)
* Dry Lab

  [Model](https://2021.igem.org/Team%3AThrace/Model)
  [Hardware](https://2021.igem.org/Team%3AThrace/Hardware)
  [Software](https://2021.igem.org/Team%3AThrace/Software)
* [Safety](https://2021.igem.org/Team%3AThrace/Safety)
* Public Engagement & Entrepreneurship

  [Communication](https://2021.igem.org/Team%3AThrace/Communication)
  [Education & Public Engagement](https://2021.igem.org/Team%3AThrace/Education)
  [Entrepreneurship](https://2021.igem.org/Team%3AThrace/Entrepreneurship)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3899)
![](https://static.igem.org/mediawiki/2021/7/74/T--Thrace--notebook.png)

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SPONSORS

[![](https://static.igem.org/mediawiki/2021/8/80/T--Thrace--bodossakis-foundation.png)](https://www.bodossaki.gr/en/)
[![](https://static.igem.org/mediawiki/2021/a/aa/T--Thrace--BFRAA.png)](http://www.bioacademy.gr/?lang=en)
[![](https://static.igem.org/mediawiki/2021/f/fe/T--Thrace--nueria.png)](https://www.linkedin.com/company/nueriaproperties/about/)
[![](https://static.igem.org/mediawiki/2021/6/6d/T--Thrace--etaireia-diaxeirisis.png)](https://duth.gr/en/University/Property-Development-and-Management-Company)

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

# Team:Thrace/Engineering

[![](https://static.igem.org/mediawiki/2021/b/bb/T--Thrace--salica_logo_short.png)](https://2021.igem.org/Team%3AThrace)

* [Home](https://2021.igem.org/Team%3AThrace)
* Team

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

  [Project Description](https://2021.igem.org/Team%3AThrace/Description)
  [Contribution](https://2021.igem.org/Team%3AThrace/Contribution)
  [Engineering](https://2021.igem.org/Team%3AThrace/Engineering)
  [Results](https://2021.igem.org/Team%3AThrace/Results)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3AThrace/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3AThrace/Implementation)
* Wet Lab

  [Project Design](https://2021.igem.org/Team%3AThrace/Project_Design)
  [Notebook](https://2021.igem.org/Team%3AThrace/Notebook)
* Dry Lab

  [Model](https://2021.igem.org/Team%3AThrace/Model)
  [Hardware](https://2021.igem.org/Team%3AThrace/Hardware)
  [Software](https://2021.igem.org/Team%3AThrace/Software)
* [Safety](https://2021.igem.org/Team%3AThrace/Safety)
* Public Engagement & Entrepreneurship

  [Communication](https://2021.igem.org/Team%3AThrace/Communication)
  [Education & Public Engagement](https://2021.igem.org/Team%3AThrace/Education)
  [Entrepreneurship](https://2021.igem.org/Team%3AThrace/Entrepreneurship)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3899)

![](https://static.igem.org/mediawiki/2021/c/cc/T--Thrace--Engineering-title.png)

![](https://static.igem.org/mediawiki/2021/2/22/T--Thrace--Engineering-blue-border.png)

In cooperation with our Dry Lab, our first option was to implement an electrochemical potentiostat,
as in theory it achieves the best accuracy to cost ratio. The initial process of designing and electronically
simulating a potentiostat model took many months, as we extensively studied the literature, past
electrochemical potentiostat models such as SimpleStat etc. Due to Covid restrictions and global
semiconductor shortages however, we had to adapt to a colorimetric model, despite all the research
and simulation time already invested on a potentiostat model. Our team is highly skilled and even
faced with the last minute seemingly inescapable odds we managed to proceed with a viable, even
more economically viable alternative.

![](https://static.igem.org/mediawiki/2021/1/18/T--Thrace--Engineering-1.png)

Using a photo standardization 3D printable cube that allows the use consumer photo sensors,
cutting the cost for the final product considerable, and developing a color normalization
algorithm of the photo taken to quantize the fluorescence of the test strip, enabling the use
of an AI system in the future to classify the test cases, complementing the work of the medical
personnel that is already hard at work at these difficult times

![](https://static.igem.org/mediawiki/2021/5/58/T--Thrace--Engineering-2.png)

We started by discovering the physical restrictions of the colorimetric model,
especially the non standard environmental lighting of each consumer, so the first iteration of the required
solution was a folding dark paper mockup, that through testing we shaped accordingly to make the photo
acquisition easy and as environment agnostic as possible. After finalizing the shape through several paper
mockups and dozens of tests in a variety of lighting conditions, we arrived at the conclusion that a LED of known
color temperature is needed as well as a mirror to prevent the light from the LED from blinding the camera sensor.

![](https://static.igem.org/mediawiki/2021/4/4a/T--Thrace--Engineering-3.png)

![](https://static.igem.org/mediawiki/2021/9/9f/T--Thrace--Engineering-4.png)

At the same time, the Dry
lab was hard at work, modeling the optimal temperature used for target
multiplication, the enzyme kinetics used by the Wet Lab, and the fluorescence to enzyme ratio of
the final solution, using the Michael-Menten equations, while linearizing the results using the
Lineweaver–Burk equation for easier representation. Using mathematical series and approximations
we showed that fluorescence intensity is directly proportional to concentration, thus aiding th
e further interpretation of the collected data.

![](https://static.igem.org/mediawiki/2021/7/77/T--Thrace--Engineering-5.png)

The modeling process started by
exploring all the popular mathematical options and implementations in enzyme kinetic modeling
found in literature, testing the validity, runtime and consistency of each method, we developed
an implementation on Matlab mathematical modeling software that is consistent, has high accuracy
(>95%) and runs almost instantaneously. Stepping on the above milestones, we are ready to integrate
a consumer device capable of detecting colon cancer with high accuracy while collecting anonymous
data valuable to medical researchers and data analysts, further expanding the knowledge in the field,
long after our device hits the market.

## Wet Lab

We followed the engineering cycle regarding the primer (stem loop) that is used to reverse transcribe miR-766-8p. Firstly we created a modified stem loop primer from protocols [1,2] that had a melting temperature between 30 - 40 C in order for it to stay in its hairpin structure while the reverse transcription reaction takes place and to be linear while the recombinase polymerase amplification was in action. Unfortunately, after many tries, during our experiments, the reverse transcription and recombinase polymerase amplification of the cDNA did not work. Also the stem loop primer binds to only the last 6 bases at the 3’ end of the microRNA that we are trying to detect, thus there is room for improving the specificity of the design. So we tried to make a new stem loop primer that has a higher melting temperature (>50 C) than the first one in order to take advantage of a higher temperature reverse transcription reaction and increase the specificity for our intended microRNA target by binding to its 5’ and 3’ ends. The new stem loop primer has to have a hairpin structure with higher GC content and two tails at its 5’ and 3’ prime ends where the microRNA can bind. Actually, there is a design [1] that already exists but is made to work with PCR. Taking inspiration from that article and combining our knowledge about designing the previous stem loop primer that is compatible with RPA we designed the new primer and tested it using UNAfold DNA Folding Form [2] and this is the result:

![](https://static.igem.org/mediawiki/2021/b/b5/T--Thrace--Engineering-wet.png)

### References:

1. Androvic, P., Valihrach, L., Elling, J., Sjoback, R., & Kubista, M. (2017). Two-tailed RT-qPCR: a novel method for highly accurate miRNA quantification. Nucleic Acids Research, 45(15), e144-e144. <https://doi.org/10.1093/nar/gkx588>

2. DNA Folding Form. Unafold.org. (2021). Retrieved 21 October 2021, from <http://www.unafold.org/mfold/applications/dna-folding-form.php>.

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SPONSORS

[![](https://static.igem.org/mediawiki/2021/8/80/T--Thrace--bodossakis-foundation.png)](https://www.bodossaki.gr/en/)
[![](https://static.igem.org/mediawiki/2021/a/aa/T--Thrace--BFRAA.png)](http://www.bioacademy.gr/?lang=en)
[![](https://static.igem.org/mediawiki/2021/f/fe/T--Thrace--nueria.png)](https://www.linkedin.com/company/nueriaproperties/about/)
[![](https://static.igem.org/mediawiki/2021/6/6d/T--Thrace--etaireia-diaxeirisis.png)](https://duth.gr/en/University/Property-Development-and-Management-Company)

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# Team:Thrace/Experiments

[![](https://static.igem.org/mediawiki/2021/b/bb/T--Thrace--salica_logo_short.png)](https://2021.igem.org/Team%3AThrace)

* [Home](https://2021.igem.org/Team%3AThrace)
* Team

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

  [Project Description](https://2021.igem.org/Team%3AThrace/Description)
  [Contribution](https://2021.igem.org/Team%3AThrace/Contribution)
  [Engineering](https://2021.igem.org/Team%3AThrace/Engineering)
  [Results](https://2021.igem.org/Team%3AThrace/Results)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3AThrace/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3AThrace/Implementation)
* Wet Lab

  [Project Design](https://2021.igem.org/Team%3AThrace/Project_Design)
  [Notebook](https://2021.igem.org/Team%3AThrace/Notebook)
* Dry Lab

  [Model](https://2021.igem.org/Team%3AThrace/Model)
  [Hardware](https://2021.igem.org/Team%3AThrace/Hardware)
  [Software](https://2021.igem.org/Team%3AThrace/Software)
* [Safety](https://2021.igem.org/Team%3AThrace/Safety)
* Public Engagement & Entrepreneurship

  [Communication](https://2021.igem.org/Team%3AThrace/Communication)
  [Education & Public Engagement](https://2021.igem.org/Team%3AThrace/Education)
  [Entrepreneurship](https://2021.igem.org/Team%3AThrace/Entrepreneurship)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3899)

# 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.

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).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [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)

![](https://static.igem.org/mediawiki/2021/2/28/T--Thrace--iGEM_Thrace_Logo_Black.png)
![](https://static.igem.org/mediawiki/2021/9/96/T--Thrace--SaliCa.png)

## Follow us:

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SPONSORS

[![](https://static.igem.org/mediawiki/2021/8/80/T--Thrace--bodossakis-foundation.png)](https://www.bodossaki.gr/en/)
[![](https://static.igem.org/mediawiki/2021/a/aa/T--Thrace--BFRAA.png)](http://www.bioacademy.gr/?lang=en)
[![](https://static.igem.org/mediawiki/2021/f/fe/T--Thrace--nueria.png)](https://www.linkedin.com/company/nueriaproperties/about/)
[![](https://static.igem.org/mediawiki/2021/6/6d/T--Thrace--etaireia-diaxeirisis.png)](https://duth.gr/en/University/Property-Development-and-Management-Company)

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

# Team:Thrace/Results

[![](https://static.igem.org/mediawiki/2021/b/bb/T--Thrace--salica_logo_short.png)](https://2021.igem.org/Team%3AThrace)

* [Home](https://2021.igem.org/Team%3AThrace)
* Team

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

  [Project Description](https://2021.igem.org/Team%3AThrace/Description)
  [Contribution](https://2021.igem.org/Team%3AThrace/Contribution)
  [Engineering](https://2021.igem.org/Team%3AThrace/Engineering)
  [Results](https://2021.igem.org/Team%3AThrace/Results)
* Human Practices

  [Integrated Human Practices](https://2021.igem.org/Team%3AThrace/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3AThrace/Implementation)
* Wet Lab

  [Project Design](https://2021.igem.org/Team%3AThrace/Project_Design)
  [Notebook](https://2021.igem.org/Team%3AThrace/Notebook)
* Dry Lab

  [Model](https://2021.igem.org/Team%3AThrace/Model)
  [Hardware](https://2021.igem.org/Team%3AThrace/Hardware)
  [Software](https://2021.igem.org/Team%3AThrace/Software)
* [Safety](https://2021.igem.org/Team%3AThrace/Safety)
* Public Engagement & Entrepreneurship

  [Communication](https://2021.igem.org/Team%3AThrace/Communication)
  [Education & Public Engagement](https://2021.igem.org/Team%3AThrace/Education)
  [Entrepreneurship](https://2021.igem.org/Team%3AThrace/Entrepreneurship)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3899)
![](https://static.igem.org/mediawiki/2021/7/71/T--Thrace--results.png)

References

1. Kramer, M. (2011). Stem‐Loop RT‐qPCR for miRNAs. Current Protocols In Molecular Biology, 95(1).
   <https://doi.org/10.1002/0471142727.mb1510s95>
2. Chen, C. (2005). Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Research, 33(20),
   e179-e179.
   <https://doi.org/10.1093/nar/gni178>

![](https://static.igem.org/mediawiki/2021/2/28/T--Thrace--iGEM_Thrace_Logo_Black.png)
![](https://static.igem.org/mediawiki/2021/9/96/T--Thrace--SaliCa.png)

## Follow us:

[![Facebook](https://static.igem.org/mediawiki/2021/6/69/T--Thrace--Facebook.png)](https://www.facebook.com/igemthrace/)
[![Twitter](https://static.igem.org/mediawiki/2021/a/af/T--Thrace--Twitter.png)](https://twitter.com/igemthrace)
[![Instagram]( https://static.igem.org/mediawiki/2021/f/f6/T--Thrace--Instagram.png)](https://www.instagram.com/igemthrace/)
[![LinkedIn](https://static.igem.org/mediawiki/2021/5/52/T--Thrace--LinkedIn.png)](https://www.linkedin.com/company/igem-thrace/)

SPONSORS

[![](https://static.igem.org/mediawiki/2021/8/80/T--Thrace--bodossakis-foundation.png)](https://www.bodossaki.gr/en/)
[![](https://static.igem.org/mediawiki/2021/a/aa/T--Thrace--BFRAA.png)](http://www.bioacademy.gr/?lang=en)
[![](https://static.igem.org/mediawiki/2021/f/fe/T--Thrace--nueria.png)](https://www.linkedin.com/company/nueriaproperties/about/)
[![](https://static.igem.org/mediawiki/2021/6/6d/T--Thrace--etaireia-diaxeirisis.png)](https://duth.gr/en/University/Property-Development-and-Management-Company)