iGEM 2022 — Team fdr-hb-peru

![](https://static.igem.wiki/teams/4338/wiki/t-fdr-hb-peru-logo-1.jpg)

[* Homepage](index%20)

* Team

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

* Project

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D%20)
 [Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)
 [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
 [Proposed Implementations](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D)
 [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

* Engineering

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

* Human Practices

[Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D%20)
 [Education & Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D%20)

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

# Notebook

## September 10th, 2022

This was our first important lab of the year. Today we performed a transformation of competent e-coli cells with the IDT plasmid constructs 1-8 our team had designed and stored since 2020. To evaluate the effectiveness of the antibiotic, we placed competent cells in plates with LB+AMP and LB as negative control and LB control, respectively.

![](https://static.igem.wiki/teams/4338/wiki/notebook/notebook-table-10-9.png)

\*Results from transformation of competent cells with constructs 1-8

There were e coli colonies in the negative control, we noted that some possible conclusions could be that the ampicilin is not working properly, or that there was some contamination to the negative control with one of the transformed cells. We noted a lot of growth on the plates with constructs, but we were expecting only a few colonies.

## September 15th, 2022

Based on the results, we decided to make a new AMP/H2O solution. We then poured LB+AMP plates to test the antibiotic with the competent cells we received this year and an LB plate with cells as a control. Similarly, we streaked the plates from the previous experiment to see if the transformation was carried out effectively.

Remaking AMP solution

Testing with Competent Cells (No transformed cells):

![](https://static.igem.wiki/teams/4338/wiki/notebook/notebook-table-15-9.png)

We believe that the 6 colonies in the LB + AMP are probably E Coli, so there wasn’t contamination in the previous experiment. However, we can’t certainly determine that the ampicillin is the problem because there is less growth on the LB than the plates with ampicillin.

Replating transformed bacteria on the new LB + AMP:

![](https://static.igem.wiki/teams/4338/wiki/notebook/c.png)

There was growth on all the plates with transformed cells and smaller colonies that are closer to the result we were expecting (single cell colonies).

## September 16th, 2022

We created a new ampicillin sodium solution diluting it in ethanol 1 gram of ampicillin with 5 ml of ethanol and 5 ml of distilled water. We tested the K1724000 tube and there was no growth in LB+AMP or LB.

![](https://static.igem.wiki/teams/4338/wiki/notebook/d.png)

We concluded that the cells from 2019 are dead, but can’t be sure the AMP is not working.

Finally, we placed some of the colonies of transformed cells from the plates into tubes with LB+AMP to store them.

## September 17th, 2022

Final Testing of AMP:

We used the AMP/Etoh solution to make LB+AMP plates with 12.5 LB and 7.5 Agar. The plates were prepared on a 500ml bottle. We have four tubes of LB+AMP to store.

For this experiment we decided to test the AMP/H2O from September 10th, the one from September 15th, and the AMP/Etoh we made on September 16th. In this way we could identify if it was a problem with the mixture or if the entire supply of ampicillin had expired. Finally we used chloramphenicol plates as negative control.

![](https://static.igem.wiki/teams/4338/wiki/notebook/e.png)

Everything grew on LB+Amp, we concluded that our supply of ampicillin was no longer working.

Testing the transformation (RFP in presence of Cadmium):

![](https://static.igem.wiki/teams/4338/wiki/notebook/f.png)

We created a new concentration of 0.001M cadmium concentration.

Cadmium concentration:

|  |  |
| --- | --- |
| Cadmium concentration: |  |
| 0.1 M |  |
| 0.01 M |  |
| 0.001 M              → | 100μL in 1 ml + 900μL distilled water |
| 0.0001  M           → | 100μL in 1 ml + 990μL distilled water |

We added 600 microliters of the bacteria with constructs 4, 7, and the single RFP stored on LB + AMP from the tubes incubated since yesterday. In total we have 16 tubes. Four negative controls (no cells), four of construct 4, four of construct 7, and four of the single RFP.

We added 600 microliters of the bacteria with constructs 4, 7, and the single RFP stored on LB + AMP from the tubes incubated since yesterday. In total we have 16 tubes. Four negative controls (no cells), four of construct 4, four of construct 7, and four of the single RFP.

Expected results:

![](https://static.igem.wiki/teams/4338/wiki/notebook/g.png)

Results:

![](https://static.igem.wiki/teams/4338/wiki/notebook/h.png)

There was no shade of red shown in the tubes. Hence, we can conclude that the transformation didn’t work or there was a problem with the plasmids for the RFP expression.

## September 22nd, 2022

We bought new ampicillin from a local provider in Centro de Lima and used it to pour new LB+AMP plates.

Testing if the transformation worked (ampicillin resistance):

To test if the cells were properly transformed or there was a problem with our RFP expression constructs we needed to see if the cells at least had acquired ampicillin resistance. Therefore, we decided to grab different colonies from the plates of supposedly transformed cells and streak them on the new LB+AMP and LB.

![](https://static.igem.wiki/teams/4338/wiki/notebook/i.png)

Since there is no growth on the antibiotic, we can conclude that the cells were not transformed correctly.

## September 23rd, 2022

Reviews from last experiment: The streaks were not transformed since they grew on lB but not LB+AMP.

Our next steps are to streak more plates with more bacteria to check if there are any transformed bacteria on the transformation plates. We will also do another bacterial transformation getting DNA in E Coli Cells and plating them on LB+AMP.

Transforming three constructs only using a negative control:

![](https://static.igem.wiki/teams/4338/wiki/notebook/j.png)

All constructs are getting 50μl of E coli competent cells.

We are following heat shock transformation in bacterial transformation protocol using a negative control instead of a positive control of iGem 2019.

![](https://static.igem.wiki/teams/4338/wiki/notebook/k.png)

## September 28th, 2022

To test the concentration of the DNA we were working with, we ran a gel electrophoresis of the 8 constructs we were working with alongside the 100bp DNA and lambda hindIII DNA that we have stored in our fridge. Fortunately, the bands show that the DNA is a supercoiled DNA, which is considered the healthiest and thus viable.

![](https://static.igem.wiki/teams/4338/wiki/notebook/l.png)![](https://static.igem.wiki/teams/4338/wiki/notebook/m.png)

## September 29th, 2022

After finding the receipt from IDT and reading the description of the product, we realized that we had confused the linear DNA constructs for a plasmid. This explains why the transformation didn’t work, along with the bad ampicillin we used in the process: the linear DNA needs to be cloned before transformation. However, we were able to find the real plasmids at the back of the freezer.

We started a new transformation with the competent cells we had left and the constructs 1, 2, 3, 6, and the single RFP. Then, the negative control was just competent cells.

![](https://static.igem.wiki/teams/4338/wiki/notebook/n.png)![](https://static.igem.wiki/teams/4338/wiki/notebook/o.png)

Since there wasn’t growth on the plates with ampicillin, the transformation didn’t work. The possible reasons for this could be because:

The ampicillin from the local provider was too strong because it was probably pharmaceutical grade.
The cells were no longer competent.
The plasmid no longer works.
There was an error in our protocol

![](https://static.igem.wiki/teams/4338/wiki/t-fdr-hb-peru-logo-1.jpg)

[* Homepage](index%20)

* Team

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

* Project

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D%20)
 [Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)
 [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
 [Proposed Implementations](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D)
 [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

* Engineering

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

* Human Practices

[Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D%20)
 [Education & Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D%20)

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

# Engineering Success

## Design

This year we decided to change our approach, not so much focusing on the detection and bioremediation of cocoa liquor, but rather on the land where the plant grows. That way we can prevent cadmium from reaching the plant and solve the problem from the ground up.

### Design: Part A- Creating the Cd biosensor

Our team was finally able to return to the lab this year, but there were many restrictions and setbacks including limited access to essential materials in Peru due to cumbersome and expensive local custom processes. However our team tried our best to research and execute our preliminary protocols and experiments to check whether our competent cells and plasmids were functioning with the pharmaceutical grade antibiotics we could obtain, as lab grade ampicillin took 60 days to ship. Due to the obstacles faced from the limited supplies we had, we weren’t able to create successful transformed cells with our proposed plasmids by the Jamboree date. Hence, our first step in our next season would be to create viable transformed cells.

After creating transformed cells that contain the correct plasmid with cadmium dependent RFP, our plan is to test the transformed bacteria in different cadmium concentrations and measure its level of red fluorescence with a spectrophotometer. From the data collected a mathematical model can be created to provide a key of the level of red fluorescence and the corresponding cadmium concentration. Then we can test our soil samples from cacao plantations, make a solution from the soil by adding dH2O, and examine the solution’s cadmium levels using our cadmium biosensor and the key that we produced.

![](https://static.igem.wiki/teams/4338/wiki/arrowchart.jpg)

### Design: Part B- Cell free system

![](https://static.igem.wiki/teams/4338/wiki/flowchart1.jpg)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **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 construct 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: 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: Testing freeze-dried Cd sensor with Cd solution** | | | | | |
| **We want to test how efficient our plasmids are in expressing RFP at different concentrations of cadmium in the soil.** | | | | | |
| **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: 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. |

### Design: Part C- Detection and Bioremediation

### Application of Biosensor

In order to make our biosensor our team designed 7 possible plasmids in 2019, which will make E Coli cells express RFP in the presence of cadmium. Bacteria modified with our construct will then become a cell-free system to detect this heavy metal and indicate the concentration through the intensity of the red color it displays.

To make the distribution easier and simplify the use of our solution, we decided that the best presentation for our biosensor would be a dipstick. And as we mentioned last year, we want to take into account the environmental impact, so a possible material for the dipstick could be paper. Finally, due to the fact that in Peru cocoa is grown in more than three provinces, we believe that the sample data such as place, date, time, results, and test number should be recorded. That way, a bioremediation or conservation plan can be implemented depending on the level of cadmium found in the area.

One of the experiments regarding the usage of the dipstick would be to test a prototype with soil samples that contain different concentrations of cadmium, in this way we analyze the effectiveness of our solution and the accuracy of the results. Likewise, we have to test whether the dipstick can be used on land or the soil sample has to be dissolved first. Through the results of these experiments, we will be able to improve the prototype, provide clear instructions for the users, and have the best possible product for cocoa farmers.

![](https://static.igem.wiki/teams/4338/wiki/synbio/design/cd1.png)

### Bioremediation

Once we create a biosensor of cadmium using cell free systems and dipsticks, our next step is to remediate cadmium through genetically modified bacteria. Some genes promote bioremediation of cadmium in naturally occurring bacteria. Our goal is to find these genes and aim to overexpress them so that the bacteria specializes in accumulating cadmium. We also have to keep in mind that the bacteria we are using should also be resistant to heavy concentrations of cadmium to ensure its survival. To do this, we can also aim to overexpress cadmium resistant genes like “gene zntA in the genome of E. coli” and capb gene (“Improving Cadmium Resistance in Escherichia Coli Through Continuous Genome Evolution”). There are also survival mechanisms like efflux systems in bacteria that we have to consider when considering the approximate amount of cadmium we want each bacteria to bioaccumulate.

![](https://static.igem.wiki/teams/4338/wiki/synbio/design/cd2.png)

Our final goal of this project would be to hopefully apply these bioengineered bacteria to act as rhizobacteria to not only remove cadmium from the soil, but would also further enhance the growth of hyperaccumulating plants that also remediate cadmium from the soil by reducing cadmium concentration in soil which may be otherwise too high for the plants to grow.

### Progress

This year we started with the transformation of E Coli cells to test our constructs from 2019-2020. However, we encountered several obstacles. During the first transformation we realized that our ampicillin had expired, so the cells we had used had not been well selected by the antibiotic. In the second transformation we did not obtain growth in the new ampicillin and after performing gel electrophoresis and finding the IDT receipts, we realized that there had been a mix-up of plasmids with linear DNA. For that reason, we tried to carry out a third transformation with the real plasmids, but it did not work. Therefore, we did not obtain a successful bacterial transformation. However, we did learn a lot about lab protocols and perseverance. Also, we have done research that will contribute a lot in the future of our project.

### Flowchart of the whole troubleshooting:![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-flowchart.png ) Plating the results from transformation #1:![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-testing-amp-resistance-table.png) Remaking Lb Amp and testing:![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-final-testing-of-amp-table.png) After the transformation we made new LB+AMP and plated competent cells to test it. We expected to not have any growth as the cells weren’t resistant to ampicillin. However, competent cells were able to grow on LB+AMP, which made us conclude that the ampicillin wasn’t working. We knew that the cells were alive because there was growth in the plate with just LB. Re-testing Amp with transformed cells:![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-testing-amp-with-old-cells-table.png) The results indicated that the transformed cells we had from 2019 were dead, as there wasn’t even growth on the plate that only had LB. No clear conclusions can be drawn about the ampicillin. Final testing of Amp:![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-final-testing-of-amp-table.png) The results indicated that the ampicillin (either diluted in water or ethanol) did not work as competent cells without ampicillin resistant genes grew on LB+AMP plates. This means that we have to purchase new ampicillin for our future experiments for viability. Testing the transformation (RFP expression):![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-testing-rfp-expression-table.png) To test the transformation, we put transformed E. Coli in LB+AMP with different concentrations of cadmium. If the transformation were successful, the liquid media with the bacteria would glow with hints of red, signifying that the bacteria is alive with the ampicillin resistant gene and was thus producing RFP that creates the red glow. However, we could not find any red glows, meaning that the bacteria did not transform and died from the ampicillin. Testing the transformation (Amp resistance):![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-testing-amp-resistance-table.png) For this experiment, we purchased new ampicillin from a local supplier and poured plates. These are the plates that have ¼ with different colonies of the supposedly transformed bacteria. But as we can see, they did not grow on the ampicillin, so we concluded that they had not been transformed well. We already expected that the bacteria would not grow because there was no expression of RFP in the previous experiment. Testing transformation #2:![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-transformation-2-table.png) After buying new ampicillin and finding out that our transformation didn’t work, we decided to make a new transformation with Constructs 1, 2, and 7. However, the transformed cells did not grow on the plates with LB+AMP, which indicated that the transformation was not successful. Testing transformation #3:![](https://static.igem.wiki/teams/4338/wiki/engineering/engineering-transformation-3-table.png) Finally, after realizing that the constructs we had been using for the last two transformations were linear DNA, we looked for the plasmids in our freezer and decided to start a third transformation. Nevertheless, the transformation didn’t work and there wasn’t any growth on the LB+AMP plates. Possible reasons behind the failed transformation: 1. The pharmaceutical ampicillin we bought wasn’t suitable for our experiment and was so strong that it killed off all transformed and non-transformed bacteria 2. Because we incubated the cells in ice for only 25 minutes instead of 30 minutes. 3. The cells were no longer competent. In this case, our only source of competent cells was Universidad Cayetano Heredia, meaning that we had limited supply. Similar to ampicillin, importing competent cells would take about 60-90 days, and thus getting new competent cells for this season wasn’t possible.

{% 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 %}

![](https://static.igem.wiki/teams/4338/wiki/t-fdr-hb-peru-logo-1.jpg)

[* Homepage](index%20)

* Team

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

* Project

[Description](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27description%27%29%20%7D%7D%20)
 [Contribution](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27contribution%27%29%20%7D%7D)
 [Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)
 [Proposed Implementations](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27implementation%27%29%20%7D%7D)
 [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

* Engineering

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

* Human Practices

[Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D%20)
 [Education & Communication](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27education%27%29%20%7D%7D%20)

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

# Results

As already mentioned, our three attempts to transform the e coli cells with our 2019-2020 constructs did not work. In the first attempt, the main problem we had was that the ampicillin was expired, so the cells were not selected properly. After buying new ampicillin from a local provider, we made a second bacterial transformation. However, the bacteria did not grow in the LB+AMP plates. For this reason, we made a gel electrophoresis to find out what the problem was and looked for the IDT receipts, realizing that we had confused linear DNA with plasmid. Finally, with the last competent cells we had, we made a third transformation with the plasmids of constructs 1, 2, 3 and 6. However, there was still no growth in the antibiotic.

Despite not having obtained satisfactory results for the progress of our project, we were able to learn a lot about laboratory protocols and the necessary perseverance to work in a scientific environment. Likewise, we were able to work on the research so we have more knowledge about the next steps to make our solution a reality.

## Future Plans:

Once we confirm the transformation of cells with the plasmid constructs, we are planning to test if the DNA displays RFP by doing what we did back in September 17th 2022: adding transformed bacteria in different concentrations of cadmium to see any different shades of red produced by different concentrations of produced RFP.

### Cell free system (with small bits of soil)

Once confirmed that the transformation has been completed successfully and there are no issues regarding the expression of RFP, our group will proceed to the next phase of our investigation. This would consist of obtaining data concerning how well our plasmid and cell-free system design works within its intended objective. For this we plan to dry freeze the cell free system with our plasmids into dipstick-like devices, similar to those found in water PH testing kits. For which we would later evaluate our design's success or failure rate with genuine soil. We expect to accumulate diverse data when exposing our plasmids to contaminated and non-contaminated soil. From this, we will conclude from the previously mentioned data to either modify or analyze specific features of our methodology or our tested variables. This section would also include testing the sensitivity of the plasmids, ensuring that they give both accurate and precise readings of the data they are meant to report.

### Biorremediation: Hyperaccumulation & Rhizoremediation

Rhizoremediation uses “mutual interaction of plant roots and suitable microbial species” (Verma et al.) in the rhizosphere and is considered the most effective, ecological method of biodegration and bioremediation of contaminants (like Cadmium). Our project strives to reach this point where we can suggest the usage our bioengineered bacteria for Cadmium remediation in cacao plantations.

However, through further research, we also found that the plant-microbe relationship may also further promote the efficiency of phytoremediation. Phytoremediation is a method used to remediate contaminants using accumulator/hyperaccumulator plants, which are plants that can “accumulate metals at high concentrations” at parts that can be harvested and later incinerated. (Verma et al.) However, even hyperaccumulator plants may not function optimally beyond certain levels of toxicity. Hence, plant growth promoting rhizobacteria (PGPR), which are mircroorganisms that live “in and around the roots” (Verma et al.) of plants, are implemented to reduce stress and support the plant through numerous mechanisms, including accumulation, transformation, sequestration, and degradation of heavy metals.

![](https://static.igem.wiki/teams/4338/wiki/results/verma-et-al.png)

![](https://static.igem.wiki/teams/4338/wiki/results/phytoremediation.png)

## Considerations for replicating the experiments.

### Difficulty in obtaining certain substances:

DNA.
Due to strict customs regulations in Peru, it is expected that there will be difficulties in importing redesigned DNA constructs from IDT and Twist.

Antibiotics (laboratorial). In Peru, we only have access to pharmaceutical antibiotics which may not be suitable for our experiment because it may be too strong or may not be pure anitbiotics. Hence, we also have to import laboratory antibiotics from the US which takes 60-90 days.

Competent cells.
In Peru we only have access to limited supply of competent cells from a local university. It would take about 60-90 days to import competent cells from abroad.

Hence, mostly concerning time, it is optimal that we create as few errors in our experiments as possible for these essential ingredients are difficult to obtain.

### Before transformation, recheck if what we are transforming are plasmids or linearized DNA with the receipt:

We discovered that we were using linearized DNA (Which has to be cloned before transformation) at the very end, meaning that all of our attempts of transformation undoubtingly failed and thus our ‘transformed’ products did not show any signs of antibiotic resistance nor RFP.