iGEM 2021 — Team FDR-HB_Peru

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

# Team:FDR-HB Peru/Notebook

![Logo](https://static.igem.org/mediawiki/2021/6/64/T--FDR-HB_Peru--logo.jpg)

**# Notebook**
 [Dates for organization](https://docs.google.com/spreadsheets/d/1cHqMcKvijWFjN9lWV_D6xlBcT-TqRnPc98BA17k9S4I/edit#gid=0)

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| **April 21, 2021**  **3:40PM** | | | |
| **19 Student Participants**  **Overall Goals or Objectives of next meeting:** | | | |

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| **April 28, 2021**  **3:40PM** | | | |
| **15 Student Participants**  **Overall Goals or Objectives of next meeting:** | | | |

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| **May 5, 2021**  **3:40PM** | | | |
| **15 Student Participants**  **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * Figure out new direction and if contact is interested in our help, and doable * Bio acomulators- make bacteria accumulate something you program it to do | | | |

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| **May 12, 2021**  **3:40PM** | | | |
| **13 Student Participants**  **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**     + Making a decision on the new direction  * **Discarding Anchovies as an option.** | | | |

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| **May 19, 2021**  **3:40PM** | | | |
| **13 Student Participants**  **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * Discussion on Opening Weekend Part 2 * [Discussion on pros and cons](https://docs.google.com/spreadsheets/d/1wxvtzAlb4K7HC59hxNZvBMVDKkw2WvkNvY3ZLUj_Yes/edit#gid=0): Asparagus/ Cacao Project * Final decision on new direction: [Google Forms](https://forms.gle/iagma8RRyjorDadi6) | | | |

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| **June 2nd, 2021**  **3:40PM** | | | |
| **18 Student Participants**  **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * Reminder on filling out the [personal forms information](https://docs.google.com/forms/d/e/1FAIpQLSeUjdIsupZzYfGjUTe3V7KFmKjuyOMBPnQ5lQxJ-jQCNP3Lfw/viewform) for for the wiki * Stay tuned on [how we can  apply for a $2,500 grant](https://2021.igem.org/Teams/Grants/Impact) through iGEM. * Need to begin work on [safety form](https://2021.igem.org/Safety/Final_Safety_Form) which is due June 25th.  Here are [answers from last year’s safety form](https://docs.google.com/document/d/1CLETJw0qsYIap-AoBuZqOHDSEGFGAv_H2MkZ6UjexnA/edit?usp=sharing) for reference. * New members for the production team! * Fill out forms of the different logo options | | | |

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| **June 9th, 2021**  **3:40PM** | | | |
| **17 Student Participants**  **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * Summary of meeting with Alianza Cacao * FDR is going back to the lab! → Friday, June 18th 2:00 - 4:00 and Wednesday, June 23rd 2:00 - 4:00. * Fill [form](https://docs.google.com/forms/d/e/1FAIpQLSccOqnpkzQ1zG-MbMrd-rLpWKmIUffd04BIN6MEB8EO0pc6mw/viewform?pli=1) to go to campus: * New Frontiers group | | | |

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| **June 16th, 2021**  **3:40PM** | | | |
| **17 Student Participants**  **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **TEDx-Tukuy:  Sign up ends today.  Virtual. July 3rd.  Email Dr. Munoz** * [**iGEM Impact Grant**](https://2021.igem.org/Teams/Grants/Impact?mtm_campaign=newsletter&mtm_kwd=reminder616)**.  Ms. Salter’s Grant Writing workshop (June 24th).** * [**Return to the Laboratory**](https://docs.google.com/presentation/d/1Ax36uJD-ZK-18WBpvMiATqX_rALtQaWp1A_UySpZvCE/edit?usp=sharing)**: June 18th and 23rd.  Live results/reporting to iGEM meeting on 23rd of bacteria plates/electrophoresis.** * **Announcements regarding collaboration opportunities** * **New Frontiers-Monday, June 21st 3:40PM** | | | |

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| **June 23rd, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **Promotional Video due July 16th** * **iGEM Impact Grant.** * [**Safety Form**](https://docs.google.com/document/d/1li8dW-kx9s1yefFQ-f-L7v-ZIRXwUFoYDf5awWBrmwU/edit#heading=h.29lwepxq43ba) | | | |

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| **June 30th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **Promotional Video due July 16th** * **iGEM Impact Grant is Done** * **Bootcamp Days??? July 13th AND 14th** | | | |

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| **August 4th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **Welcome back!  How was the break?** * **iGEM Impact Grant Update: (We didn’t win one...next time!)** * **Two months until competition deadlines.  Setting priorities for each group.** | | | |

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| **August 11th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **Icebreaker** * **Meeting with Washington** * **Goals for the day** | | | |

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| **August 18th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **Welcome new members!** * **Icebreaker** * **Judging Feedback from 2020** * **Registration for new members** * **iGEM 2020 wiki--look for design phase only language for 2021 discount** | | | |

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| **September 1st, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**    * **Icebreaker** * **Breakout room: Registration for new members (20 min)** * **IGEM LATAM FEST \* Thu Sep 23rd 16:00 - Sat 25th 20:00 (CST)** * **Ana Lu reminder: wiki deadlines** | | | |

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| **September 15th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **Icebreaker Secretaries** * **Secretary Tip to put some music when working independently, have fun :)** * **Latam Fest coming soon!!  Thu Sep 23rd 16:00 - Sat 25th 20:00 (CST) (registration needed)** * **Ana Lu wiki! (Did Human Practices meet the deadline?)** * **Human production Russian collaboration** * **Secretary registration last people** | | | |

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| **September 22nd, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * [**Icebreaker (5 min)**](https://www.canva.com/design/DAEqHGGUboU/share/preview?token=sPfKMJrqOwT7ahUqxZ2fcg&role=EDITOR&utm_content=DAEqHGGUboU&utm_campaign=designshare&utm_medium=link&utm_source=sharebutton) * **Secretary news: Safety Forms,** [**Judging Forms**](https://docs.google.com/document/d/1nsYolfR2do08YuCIChpKFeIdEHVyiWoFgt1OjUHbeWY/edit?usp=sharing) **and Abstract/Project Title due October 1st./ Latam Fest coming soon!! Thursday, Friday and Weekend 17:00 - (**[**registration needed**](https://www.surveymonkey.com/r/iGEMLATAMFest2021)**) (5 min)** * **Check in with leaders (5min)** * **Boot Camp! (1 hour)** | | | |

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| **September 29th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **SECRETARY REMINDER: Attendance, judging form, safety form, abstract project title.** | | | |

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| **October 6th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * [**Icebreaker (5 min)**](https://www.canva.com/design/DAEqHGGUboU/share/preview?token=sPfKMJrqOwT7ahUqxZ2fcg&role=EDITOR&utm_content=DAEqHGGUboU&utm_campaign=designshare&utm_medium=link&utm_source=sharebutton) * **Secretary news: Close deadlines** * **Ricardo will help coding for the wiki :D** * **Let’s all work in the wiki!** | | | |

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| **October 13th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * [**Icebreaker (5 min)**](https://www.canva.com/design/DAEqHGGUboU/share/preview?token=sPfKMJrqOwT7ahUqxZ2fcg&role=EDITOR&utm_content=DAEqHGGUboU&utm_campaign=designshare&utm_medium=link&utm_source=sharebutton) * **Close deadlines: Final Judging Form Freeze, Registry Pages Freeze, Wiki Freeze: October 21st. Team presentation video: October 28th.** * **Team photo!** | | | |

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| **October 18th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:**   * **WIKI WORK!!!!** | | | |

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| **October 20th, 2021**  **3:40PM** | | | |
| **Overall Goals or Objectives of next meeting:**   **Summary of main room discussion and reminders:** | | | |

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

# Team:FDR-HB Peru/Engineering

![Logo](https://static.igem.org/mediawiki/2021/6/64/T--FDR-HB_Peru--logo.jpg)

**# Engineering Success**
**## Description**

**Design: Part A  - Cd sensor**

Due to the challenges and changes that the pandemic brought to our lives, returning to the laboratories and accessing some materials was still not possible this year. Despite this difficulty, our team was resilient and continued to research and plan to be ready to make and test our previously designed Cadmium sensor, this time applying it to determine Cd-levels in chocolate liquor, cocoa powder, and soil (liquified) from cacao plantations.

Our project centers on using a cell-free system to detect the concentration of cadmium in cacao liquor and powder, before being shipped out for further chocolate production. We plan to freeze-dry the Sigma 70 Master Mix, from our sponsor Arbor Biosciences, onto paper strips to eventually create a dip-stick-like detection system. When this dip-stick is placed in a blended cacao liquor and powder sample, it will express a shade of red which we can measure comparatively to gauge the concentration of cadmium.

For this project, last year we designed the BBa\_K3466000 plasmid to detect the presence of cadmium in fishmeal. This engineering is being planned to be applied in cacao liquor and powder. In the plasmid, the MerR protein represses the PcadA promoter. When cadmium is present, the MerR protein is released and RFP is expressed. Additionally, we created 6 alternative versions of the part to test if different characteristics changed the efficiency of the plasmid.

![](https://static.igem.org/mediawiki/2021/thumb/b/b3/T--FDR-HB_Peru--synbioflowchart.png/1600px-T--FDR-HB_Peru--synbioflowchart.png)

## Table 1

| Table 1: Testing Cd Dependent RFP Expression in Cell-Free Mix | | | | | |
| --- | --- | --- | --- | --- | --- |
| Testing if our plasmids express RFP in the presence of Cd, how efficiently they express RFP, and which promotor yields better expression in the cell-free system. | | | | | |
| Tube # | DNA | Additions | Test | Expected results | Evaluation of expected results |
| 1 (Negative Control) | No DNA | Sigma 70 Master Mix | Contamination of the cell-free system | No color change | There is no DNA being added to the cell-free mix |
| 1 (Positive Control) | GFP plasmid | Sigma 70 Master Mix | Functioning cell-free system | GFP expression | The cell-free system comes with a GFP plasmid to check for proper protein synthesis |
| 3 (Cd Control) | GFP plasmid | Sigma 70 Master Mix + Cd | Cd obstruction of FP expression | GFP expression | The cell-free mix has been used successfully as a heavy metal detector by other iGEM teams |
| 4 (RFP Negative Control) | RFP plasmid | Sigma 70 Master Mix | Leakage of RFP | No growth | Plasmid is designed to only express RFP when there is Cd |
| 5 (RFP Positive Control) | RFP plasmid | Sigma 70 Master Mix + Cd | RFP expression in the presence of Cd | RFP expression | Plasmid is designed to express RFP in the presence of Cd |
| 6 | Plasmid #1 | Sigma 70 Master Mix + Cd | RFP expression in the presence of Cd | RFP expression to be quantified with colorimetry | Plasmid is designed to express RFP in the presence of Cd |
| 7 | Plasmid #7 | Sigma 70 Master Mix + Cd | RFP expression in the presence of Cd | RFP expression to be quantified with colorimetry | Plasmid is designed to express RFP in the presence of Cd |

## Table 2

| Table 2: Troubleshooting Cd Dependent RFP Expression in Cell-Free Mix | | |
| --- | --- | --- |
| Problem | Possible reason | Future steps |
| DNA contamination of cell-free negative control | There is contamination | Try again in a more controlled environment |
| RFP expression in RFP negative control | The plasmid design is not specific to Cd | Check the design of the plasmid and alter as needed |
| No GFP expression in Cd positive control | The Cd obstructed the cell-free system | Try a different route that isn’t cell-free / with this specific mix, (go back to in-vivo) |
| No GFP expression in cell-free positive control | The cell-free system doesn’t work | Go a different route that is not cell-free/consult Arbor Biosciences |
| The cell-free system was not kept properly | Use a different batch of cell-free tubes |
| Weak / no RFP expression | Not sensitive enough | Test other parts, change part design to be more sensitive to Cd. |
| Wrong plasmid | Test with live bacteria,use a restriction digest on the plasmid DNA to test for the right length |
| Plasmid not compatible with cell-free system | Test the plasmid with live bacteria, then change design of the plasmid if necessary |

## Table 3

| Table 3: Testing freeze-dried Cd sensor with Cd solution | | | | | |
| --- | --- | --- | --- | --- | --- |
| Our partner Alianza Cacao, cannot export cacao liquor over 0.8 ppm due to international regulations. We want to test how efficient our plasmids are ay expressing RFP in the present of Cd concentrations below and over 0.8 ppm | | | | | |
| Filter paper # | Freeze-dried solution | Dipping solution | Test | Expected results | Evaluation of expected results |
| 1 (Negative Control) | Sigma 70 Master Mix | No solution | Contamination of the cell-free system | No color change | There is no DNA being added to the cell-free mix |
| 2 (Positive Control) | Sigma 70 Master Mix + GFP plasmid | No solution | Function of cell-free system when freeze-dried | GFP expression | Teams have successfully freeze-dried this cell-free mix |
| 3 (RFP Control) | Sigma 70 Master Mix + RFP plasmid | Cd | RFP expression in the presence of Cd | RFP expression | Plasmid is designed to express RFP in the presence of Cd |
| 4 | Sigma 70 Master Mix + Plasmid #1 | Cd (3ppm) | RFP expression **over** Cd concentration limit | Strong RFP expression | Higher Cd concentration = stronger RFP expression |
| 5 | Sigma 70 Master Mix + Plasmid #1 | Cd (2ppm) | RFP expression **at** Cd concentration limit | Weaker RFP expression | Lower Cd concentration = Weaker RFP expression |
| 6 | Sigma 70 Master Mix + Plasmid #1 | Cd (1ppm) | RFP expression **under** Cd concentration limit | Weakest RFP expression | Lowest Cd concentration = Weakest RFP expression |
| 7 | Sigma 70 Master Mix + Plasmid #7 | Cd (3ppm) | RFP expression **over** Cd concentration limit | Strong RFP expression | Higher Cd concentration = stronger RFP expression |
| 8 | Sigma 70 Master Mix + Plasmid #7 | Cd (2ppm) | RFP expression at Cd concentration limit | Weaker RFP expression | Lower Cd concentration = Weaker RFP expression |
| 9 | Sigma 70 Master Mix + Plasmid #7 | Cd (1ppm) | RFP expression under Cd concentration limit | Weakest RFP expression | Lowest Cd concentration = Weakest RFP expression |

**Design: Part B - Cd Bioacummulation**

Having discussed our project in detail with our industry stakeholders, Alianza Cacao, we learned that their main interest is in cadmium removal from their final product, not just Cd detection, so we have also been planning to design and test a transgenic yeast strain that will bioaccumulate Cadmium from contaminated cacao liquor/powder as soon as we return to the lab.

In our project, we will focus on using baker's yeast (*S. cerevisiae)* to perform bioremediation on cadmium contaminated cacao liquor/powder. We plan to achieve this by modifying genes (CCC1, TaPCS1, BSD2, and SMF1) that produce proteins involved in membrane transport and assimilation of metals (see details in table below).

|  |  |  |
| --- | --- | --- |
| **Gene** | **Wild-Type Function** | **Use in our project** |
| **SMF1** | Suppressor of Mitochondria import Function 1. | Mutations of SMF1’s ubiquitination site K33,34 were altered to arginine. This makes this transportation specific to the cadmium along with manganese. |
| **CCC1** | Cross-Complements Ca(2+) phenotype of csg1. | Transports the cadmium from the cytoplasm to the vacuoles within the cell, protecting the cell from the cadmium and allowing for more efficient uptake. |
| **BSD2** | Bypass Sod1p Defects | Speeds up the transportation of the cadmium, allowing for faster uptake and increased resistance by reducing the time it spends outside of chelated complex |
| **TaPCS1** | This gene creates a phytochelatin synthase, which contributes to the heavy metal tolerance and metalloid homeostasis by producing phytochelatins. | Sequesters the cadmium in the yeast and increases resistance |

​​

A flowchart of the experiments we plan to work on once we return to the lab in order to create the yeast that we plan to use for the bioremediation and test it's capabilities for final use.

![](https://static.igem.org/mediawiki/2021/b/b6/T--FDR-HB_Peru--CdBioaccumulation.png)

**Results:  Future plans for Bioaccumulation**

The modified yeast will then be applied to the cocoa liquor and will be able to hyperaccumulate cadmium as a form of bioremediation. One of the tests that we have to carry out is regarding the technique for separating the cadmium contaminated yeast from the cacao liquor/cacao powder. That way we will ensure that the product is safe for consumers and does not alter its flavor or consistency.

During our planning, we have found multiple options:

Centrifugation: A process of separation that uses the application of centrifugal force to isolate particles from solutions, based on their density, size, or shape.  One potential problem with this method when using cocoa liquor is that its density (approx. 1100 kg / m3) could be an impediment to separating it from cadmium-contaminated yeast. That is why we will also try our modified yeast in cocoa powder diluted in water. That way it would be easier to separate, but an extra step would be added for the dehydration of the cocoa powder and water mixture.

Another method we have researched for the removal of yeast is induced magnetization of the yeast cells. This method would be ideal for our project because it gives us the possibility to simplify and accelerate the process of separating the contaminated cadmium yeast from the cocoa liquor, thus making it more accessible and convenient for companies that manufacture cocoa-based products. We plan on achieving this through the cultivation of yeast in liquid ferrin citrate supplemented media. However, we still have to test different cultivation times and amounts of liquid ferrin citrate to achieve the best results possible. If after experimentation the desired level of magnetism is not obtained, the option of using a ferritin expressor could be considered (Nishida K, Silver PA). When the yeast is exposed to these conditions, the yeast protein Tco89p (involved with growth related to environmental availability) will cause oxidation and create iron particles inside the cell, so that the yeast can be attracted by a magnet and separated from the cocoa liquor/cocoa powder.

The future plans for the project would be to scale up the process of separating yeast with cadmium from cocoa powder/cocoa liquor. The solution would be directed towards the companies that collect the cacao beans from the producers to make the different products because it has to be applied during the process of refinement. And although the methods could be replicated in a laboratory on a small scale, cocoa production has to be more accelerated. For that reason, as a team we would have to connect with the machinery they have available or even modify some to better suit the needs of our audience.

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

# Team:FDR-HB Peru/Experiments

![Logo](https://static.igem.org/mediawiki/2021/6/64/T--FDR-HB_Peru--logo.jpg)

# 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/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:FDR-HB Peru/Results

![Logo](https://static.igem.org/mediawiki/2021/6/64/T--FDR-HB_Peru--logo.jpg)

# Results

You can describe the results of your project and your future plans here.

### 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.

### Inspiration

See how other teams presented their results.

* [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)