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.
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.
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Table 1: Testing Cd Dependent RFP Expression in Cell-Free Mix |
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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. |
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Tube # |
DNA |
Additions |
Test |
Expected results |
Evaluation of expected results |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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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 |
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Table 2: Troubleshooting Cd Dependent RFP Expression in Cell-Free Mix |
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Problem |
Possible reason |
Future steps |
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DNA contamination of cell-free negative control |
There is contamination |
Try again in a more controlled environment |
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RFP expression in RFP negative control |
The plasmid design is not specific to Cd |
Check the design of the plasmid and alter as needed |
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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) |
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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 |
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The cell-free system was not kept properly |
Use a different batch of cell-free tubes |
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Weak / no RFP expression |
Not sensitive enough |
Test other parts, change part design to be more sensitive to Cd. |
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Wrong plasmid |
Test with live bacteria ,use a restriction digest on the plasmid DNA to test for the right length |
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Plasmid not compatible with cell-free system |
Test the plasmid with live bacteria, then change design of the plasmid if necessary |
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Table 3: Testing freeze-dried Cd sensor with Cd solution |
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We want to test how efficient our plasmids are in expressing RFP at different concentrations of cadmium in the soil. |
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Filter paper # |
Freeze-dried solution |
Dipping solution |
Test |
Expected results |
Evaluation of expected results |
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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 |
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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 |
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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 |
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4 |
Sigma 70 Master Mix + Plasmid #1 |
Cd (3ppm) |
RFP expression over Cd concentration limit |
Strong RFP expression |
Higher Cd concentration = stronger RFP expression |
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5 |
Sigma 70 Master Mix + Plasmid #1 |
Cd (2ppm) |
RFP expression at Cd concentration limit |
Weaker RFP expression |
Lower Cd concentration = Weaker RFP expression |
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6 |
Sigma 70 Master Mix + Plasmid #1 |
Cd (1ppm) |
RFP expression under Cd concentration limit |
Weakest RFP expression |
Lowest Cd concentration = Weakest RFP expression |
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7 |
Sigma 70 Master Mix + Plasmid #7 |
Cd (3ppm) |
RFP expression over Cd concentration limit |
Strong RFP expression |
Higher Cd concentration = stronger RFP expression |
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8 |
Sigma 70 Master Mix + Plasmid #7 |
Cd (2ppm) |
RFP expression at Cd concentration limit |
Weaker RFP expression |
Lower Cd concentration = Weaker RFP expression |
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9 |
Sigma 70 Master Mix + Plasmid #7 |
Cd (1ppm) |
RFP expression under Cd concentration limit |
Weakest RFP expression |
Lowest Cd concentration = Weakest RFP expression |
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Table 4: Troubleshooting Freeze-dried Cd Sensor with Pure Cd Solution |
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Problem |
Possible reason |
Future steps |
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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. |
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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. |
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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. |
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.