iGEM 2020 — Team FDR-HB_Peru

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:FDR-HB Peru/Notebook

# 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://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:FDR-HB Peru/Engineering

iGEM - FDR\_HB Home

[![](https://static.igem.org/mediawiki/2020/6/64/T--FDR-HB_Peru--logo.svg)](https://2020.igem.org/Team%3AFDR-HB_Peru)

Team

[Members](https://2020.igem.org/Team%3AFDR-HB_Peru/Team)
[Attributions](https://2020.igem.org/Team%3AFDR-HB_Peru/Attributions)
[Collaborations](https://2020.igem.org/Team%3AFDR-HB_Peru/Collaborations)

Project

[Description](https://2020.igem.org/Team%3AFDR-HB_Peru/Description)
[Contribution](https://2020.igem.org/Team%3AFDR-HB_Peru/Contribution)
[Partnership](https://2020.igem.org/Team%3AFDR-HB_Peru/Partnership)
[Implementation](https://2020.igem.org/Team%3AFDR-HB_Peru/Implementation)

Engineering

[Engineering](https://2020.igem.org/Team%3AFDR-HB_Peru/Engineering)
[Parts](https://2020.igem.org/Team%3AFDR-HB_Peru/Parts)

Human Practices

[Integrated Human Practices](https://2020.igem.org/Team%3AFDR-HB_Peru/Human_Practices)
[Education](https://2020.igem.org/Team%3AFDR-HB_Peru/Education)

[Hardware](https://2020.igem.org/Team%3AFDR-HB_Peru/Hardware)

# [ Engineering ]

![](https://static.igem.org/mediawiki/2020/e/e8/T--FDR-HB_Peru--standardboat.png)
![](https://static.igem.org/mediawiki/2020/2/2e/T--FDR-HB_Peru--underwater_wave.png)

# Content

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[## Flowchart](#flowchart)

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[## Table 1](#table-1)

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[## Table 2](#table-2)

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[## Table 3](#table-3)

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[## Table 4](#table-4)

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[## Table 5](#table-5)

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[## Table 6](#table-6)

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[## Table 7](#table-7)

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[## Table 8](#table-8)

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[## Back to Top](#top)

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# Engineering

## Description

Our project centers on using a cell-free system to detect the concentration of Cadmium in fresh anchovies on fishing boats, before the production of fishmeal. 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 anchovy sample, it will express a shade of red which we can measure comparatively to gauge the concentration of Cadmium.

For this project, we designed the BBa\_K3466000 plasmid to detect the presence of Cadmium. 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.

## Flowchart

Use the interactive flowchart below by **clicking on the different sections** to go different tables.

![](https://static.igem.org/mediawiki/2020/0/0f/T--FDR-HB_Peru--3d-flowchart-v3.svg)

## 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 sponsor, TASA, cannot export fishmeal containing over 2ppm of Cd due to international regulations. We want to test how efficient our plasmids are at expressing RFP in the presence of Cd concentrations below, over, and at the 2ppm limit. | | | | | |
| 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 |

## Table 4

| Table 4: Troubleshooting Freeze-dried Cd Sensor with Pure Cd Solution | | |
| --- | --- | --- |
| Problem | Possible reason | Future steps |
| No RFP expression in freeze-dried RFP positive control | Freeze-drying obstructed the expression of RFP | Research what in the freeze-drying procedure could’ve obstructed the plasmid / try a different freeze-drying method. |
| No GFP expression in freeze-dried cell-free system positive control | Freeze-drying was not successful, interfered with function of the plasmid | Try a different freeze-drying procedure. |
| Weak / No RFP expression in freeze-dried Cd sensor | Freezedrying lowered the plasmids sensitivity to Cd | Use one of our more sensitive plasmids (2-6), Redesign the plasmid to increase Cd sensitivity, use a different device (colorimeter, app, plate reader, etc.) to read the results. |

## Table 5

| Table 5: Testing freeze-dried Cd sensor with fishmeal | | | | | |
| --- | --- | --- | --- | --- | --- |
| (For the non-freeze-dried sensor the experiment stays the same except for the column “Freeze-dried Solution” is the DNA column, and the “Dipping Solution” column becomes the Additions column) | | | | | |
| Our sponsor, TASA, gave us multiple batches of fishmeal with varying Cd concentrations. We want to test if RFP expression in the pure Cd solution varies from RFP expression in the fishmeal solution when tested with the same Cd concentration. If the RFP expression from the fishmeal solution is weaker, then we know factors from the anchovies (salinity, pH, etc.) affect the efficiency of our plasmids in the cell-free system. | | | | | |
| Filter paper # | Freeze-dried solution | Dipping solution | Test | Expected results | Evaluation of expected results |
| 1 (Negative 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 |
| 2 (Positive 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 |
| 3 | Sigma 70 Master Mix + Plasmid #1 | Fishmeal (3ppm) | Obstruction of RFP expression due to fishmeal factors (salinity, pH, etc.) | Strongest RFP expression | Factors in the fishmeal might disturb the plasmids recognition / sensitivity to Cd, making the RFP expression weaker than in the pure Cd solution, but the plasmid should still detect Cd in fishmeal. |
| 4 | Sigma 70 Master Mix + Plasmid #1 | Fishmeal (2ppm) | Obstruction of RFP expression due to fishmeal factors (salinity, pH, etc.) | Weaker RFP expression | Lower Cd concentration = Weaker RFP expression |
| 5 | Sigma 70 Master Mix + Plasmid #1 | Fishmeal (1ppm) | Obstruction of RFP expression due to fishmeal factors (salinity, pH, etc.) | Weakest RFP expression | Lowest Cd concentration = Weakest RFP expression |
| 6 | Sigma 70 Master Mix + Plasmid #7 | Fishmeal (3ppm) | Obstruction of RFP expression due to fishmeal factors (salinity, pH, etc.) | Strongest RFP expression | Higher Cd concentration = stronger RFP expression |
| 7 | Sigma 70 Master Mix + Plasmid #7 | Fishmeal (2ppm) | Obstruction of RFP expression due to fishmeal factors (salinity, pH, etc.) | Weaker RFP expression | Lower Cd concentration = Weaker RFP expression |
| 8 | Sigma 70 Master Mix + Plasmid #7 | Fishmeal (1ppm) | Obstruction of RFP expression due to fishmeal factors (salinity, pH, etc.) | Weakest RFP expression | Lowest Cd concentration = Weakest RFP expression |

## Table 6

| Table 6: Troubleshooting freeze-dried Cd sensor with Fishmeal | | |
| --- | --- | --- |
| Problem | Possible reason | Future steps |
| No RFP expression in freeze-dried RFP positive control | Freeze-drying procedure was not successful | Re-do freeze-drying procedure |
| No GFP expression in freeze-dried cell-free positive control | Freeze-drying was not successful | Re-do freeze-drying procedure |
| Weak / No RFP expression in freeze-dried Cd sensor | Factors in the fishmeal (salinity, pH, etc.) obstructed with the plasmids sensitivity to Cd | Use one of our more sensitive plasmids (2-6), Redesign the plasmid to increase Cd sensitivity, use a different device (colorimeter, app, plate reader, etc.) to read the results |

## Table 7

| Table 7: Testing freeze-dried Cd sensor with blended anchovies | | | | | |
| --- | --- | --- | --- | --- | --- |
| (For the non-freezedried sensor the experiment stays the same except for the column “Freeze-dried Solution” is the DNA column, and the “Dipping Solution” column becomes the Additions column) | | | | | |
| Our goal is to detect Cd levels in fresh anchovies on fishing boats, before the fishmeal production process. A similar experiment “Determination of toxic elements (mercury, cadmium, lead, tin and arsenic) in fish and shellfish samples. Risk assessment for the consumers” used 0.7g of anchovies in a 1:4 dilution in order to test for heavy metals. Therefore, we have decided to use 1:3, 1:4, 1:5, and 1:6 dilutions with a 0.7g anchovy sample to test for the efficiency of our plasmid. | | | | | |
| Filter paper # | Freeze-dried solution | Dipping solution | Test | Expected results | Evaluation of expected results |
| 1 (Negative 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 |
| 2 (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 |
| 3 | Sigma 70 Master Mix + Plasmid #1 | Anchovy Dilution 1:3 | What anchovy dilution allows for better RFP expression | RFP expression | Our part is designed to detect the Cd concentration in fresh anchovies through RFP expression |
| 4 | Sigma 70 Master Mix + Plasmid #1 | Anchovy Dilution 1:4 | What anchovy dilution allows for better RFP expression | RFP expression | Our part is designed to detect the Cd concentration in fresh anchovies through RFP expression |
| 5 | Sigma 70 Master Mix + Plasmid #1 | Anchovy Dilution 1:5 | What anchovy dilution allows for better RFP expression | RFP expression | Our part is designed to detect the Cd concentration in fresh anchovies through RFP expression |
| 6 | Sigma 70 Master Mix + Plasmid #7 | Anchovy Dilution 1:6 | What anchovy dilution allows for better RFP expression | RFP expression | Our part is designed to detect the Cd concentration in fresh anchovies through RFP expression |
| 7 | Sigma 70 Master Mix + Plasmid #7 | Anchovy Dilution 1:3 | What anchovy dilution allows for better RFP expression | RFP expression | Our part is designed to detect the Cd concentration in fresh anchovies through RFP expression |
| 8 | Sigma 70 Master Mix + Plasmid #7 | Anchovy Dilution 1:4 | What anchovy dilution allows for better RFP expression | RFP expression | Our part is designed to detect the Cd concentration in fresh anchovies through RFP expression |
| 9 | Sigma 70 Master Mix + Plasmid #7 | Anchovy Dilution 1:5 | What anchovy dilution allows for better RFP expression | RFP expression | Our part is designed to detect the Cd concentration in fresh anchovies through RFP expression |
| 10 | Sigma 70 Master Mix + Plasmid #7 | Anchovy Dilution 1:6 | What anchovy dilution allows for better RFP expression | RFP expression | Our part is designed to detect the Cd concentration in fresh anchovies through RFP expression |

## Table 8

| Table 8: Troubleshooting Freeze-dried Cd Sensor with Blended Anchovies | | |
| --- | --- | --- |
| Problem | Possible reason | Future steps |
| No RFP expression in freeze-dried RFP positive control | Freeze-drying procedure was not successful | Re-do freeze-drying procedure |
| No GFP expression in freeze-dried cell-free system positive control | Freeze-drying was not successful | Re-do freeze-drying procedure/ consult Arbor Biosciences |
| Weak / No RFP expression in freeze-dried Cd sensor | Factors in the anchovies (fats, salinity, etc.) and/or the consistency of the anchovy dilution obstructed with the plasmids detection of Cd. | Make the plasmid more sensitive/redesign plasmid, or use a different device to read results, Extract Cd from anchovies,or use a different freeze-drying method. |

![](https://static.igem.org/mediawiki/2020/b/b7/T--FDR-HB_Peru--corals.png)

![](https://static.igem.org/mediawiki/2020/6/69/T--FDR-HB_Peru--logo_white.svg)

# Contact Us

[Instagram](https://www.instagram.com/igem_peru/)
[Twitter](https://twitter.com/fdrhb_peru)
[Youtube](https://www.youtube.com/channel/UC7Pkjp9fELZM2J67rPm7jqQ)
igemfdr@amersol.edu.pe

# Team

[Members](https://2020.igem.org/Team%3AFDR-HB_Peru/Team)
[Attributions](https://2020.igem.org/Team%3AFDR-HB_Peru/Attributions)
[Collaborations](https://2020.igem.org/Team%3AFDR-HB_Peru/Collaborations)

# Project

[Description](https://2020.igem.org/Team%3AFDR-HB_Peru/Description)
[Contribution](https://2020.igem.org/Team%3AFDR-HB_Peru/Contribution)
[Partnership](https://2020.igem.org/Team%3AFDR-HB_Peru/Partnership)
[Implementation](https://2020.igem.org/Team%3AFDR-HB_Peru/Implementation)

# Engineering

[Engineering](https://2020.igem.org/Team%3AFDR-HB_Peru/Engineering)
[Parts](https://2020.igem.org/Team%3AFDR-HB_Peru/Parts)

# Human Practices

[Integrated Human Practices](https://2020.igem.org/Team%3AFDR-HB_Peru/Human_Practices)
[Education](https://2020.igem.org/Team%3AFDR-HB_Peru/Education)

# Other

[Hardware](https://2020.igem.org/Team%3AFDR-HB_Peru/Hardware)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:FDR-HB Peru/Experiments

# 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

* [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://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:FDR-HB Peru/Results

# 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.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)