Team:FDR-HB Peru/Engineering

• Home
• Team
  • Members
  • Attributions
  • Collaboration
• Project
  • Description
  • Contribution
  • Notebook
  • Proposed Implementation
• Engineering
  • Engineering Success
  • Parts
• Human Practices
  • Integrated Human Practices
  • Education & Communication
• Project Modeling
• Safety

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.

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

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). 

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

Gene	Wild-Type Function	Use in our project
SMF1	Suppressor of Mitochondria import Function 1.  Gene involved in the production of a protein involved in divalent and trivalent metal transportation through the cell membrane.	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.  Produces a protein involved in vacuolar transportation of Fe2+ and Mn2+. Prevents the accumulation of metals inside the cell's cytoplasm.	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 The gene codes for a protein that accelerates heavy metal vacuolar transportation by leading Smf1p and Smf2p transporters.	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.

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.