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.
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
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.
