iGEM 2021 — Team EPFL

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# Team:EPFL/Notebook

* Project

  + [Description

    Project background, inspiration, etc.](https://2021.igem.org/Team%3AEPFL/Description)
  + [Background

    Quantification of the problem](https://2021.igem.org/Team%3AEPFL/Background)
  + [Design

    Monomer and dimer design](https://2021.igem.org/Team%3AEPFL/Design)
  + [Modelling

    Bioreactor fluid mechanics models](https://2021.igem.org/Team%3AEPFL/Model)
  + [Results

    Main results of the project](https://2021.igem.org/Team%3AEPFL/Results)
  + [Engineering Success

    Design, build, test, learn](https://2021.igem.org/Team%3AEPFL/Engineering_Success)
  + [Implementation

    Bioreactor](https://2021.igem.org/Team%3AEPFL/Implementation)
  + [Proof of Concept

    Showing yeast captures copper](https://2021.igem.org/Team%3AEPFL/Proof_Of_Concept)
  + [Contribution

    Contribution to future teams](https://2021.igem.org/Team%3AEPFL/Contribution)
  + [Safety

    Chemicals used, security measures](https://2021.igem.org/Team%3AEPFL/Safety)
  + [Protocols

    PCR, Western Blot, transformation, etc.](https://2021.igem.org/Team%3AEPFL/Protocols)
  + [Notebook

    Detailed records of all experiments](https://2021.igem.org/Team%3AEPFL/Notebook)
* People

  + [Team](https://2021.igem.org/Team%3AEPFL/Team)
  + [Attributions](https://2021.igem.org/Team%3AEPFL/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AEPFL/Collaborations)
* Parts

  + [Parts Overview](https://2021.igem.org/Team%3AEPFL/Parts_Overview)
  + [Parts Collection](https://2021.igem.org/Team%3AEPFL/Parts_Collection)
* Human Practices

  + [Human Practices](https://2021.igem.org/Team%3AEPFL/Human_Practices)
  + [Integrated Human Practices](https://2021.igem.org/Team%3AEPFL/Integrated)
  + [Education & Communication](https://2021.igem.org/Team%3AEPFL/Communication)
* [Medals](https://2021.igem.org/Team%3AEPFL/Medals_and_Awards)

![](https://static.igem.org/mediawiki/2021/f/f3/T--EPFL--background--notebook.jpg)

### Contents

* [Cloning the monomer](#heading-0)
  + [Gene PCR](#heading-1)
  + [PCR clean-up](#heading-2)
  + [Plasmid digestion](#heading-3)
  + [Gibson assembly](#heading-4)
  + [Bacteria transformation and plating](#heading-5)
  + [Colony picking and culture inoculation](#heading-6)
  + [Miniprep](#heading-7)
  + [Sequencing](#heading-8)
  + [Yeast growth](#heading-9)
  + [Yeast transformation](#heading-10)
  + [Western blot 1](#heading-11)
  + [Immunostaining 1](#immuno1)
  + [Western blot 2](#web2)
  + [Western blot 3](#heading-12)
  + [Yeast growth curves](#heading-13)
  + [Copper absorption assay](#heading-14)
* [Cloning the dimers](#heading-15)
  + [Plasmid digestion](#heading-16)
  + [Gene PCR](#heading-17)
  + [PCR and digestion clean-up](#heading-18)
  + [Gibson assembly](#heading-19)
  + [Bacteria transformation and plating](#heading-20)
  + [Colony picking and culture inoculation](#heading-21)
  + [Miniprep](#heading-22)
  + [Sequencing](#heading-23)
  + [Yeast transformation](#heading-24)
  + [Western blot 2](#heading-25)
  + [Immunostaining 2](#heading-26)
  + [Yeast growth curves](#heading-27)
  + [Copper absorption assay](#heading-28)
* [References](#heading-29)

## Cloning the monomer

### Gene PCR

2021-07-12

**Aim** Extract and amplify our genomic CUP1 sequence from yeast DNA by Polymerase Chain Reaction (PCR).

**Protocol** [Gene PCR](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-gene-pcr)

We had 3 genomic variants that we named Y1, Y2 and Y3. The samples were normalized to similar concentrations and the PCR was run with 20ng of DNA material.

We ran a 2% agarose gel electrophoresis to check our PCR product size. We prepared two sets of tubes, one destined to the gel electrophoresis and one to be cleaned up as we do not have a UV “table” in our lab.

**Results**

![](https://static.igem.org/mediawiki/2021/b/b7/T--EPFL--notebook--monomer--0.jpg)

Figure 1Genomic CUP1 PCR amplification.On the right is the low range ladder, the three lanes correspond to the three genomic variants amplified. All our amplified CUP1s have a size of around 200 base pairs (bp) which matches the theoretical size of CUP1: 183 bp.[1](#ref-0)

**Conclusion** We successfully amplified the CUP1 gene using PCR. We could move on and do a PCR clean up on our samples to purify the DNA.

### PCR clean-up

2021-07-12

**Aim** Purify DNA fragments from the PCR mix to remove excess nucleotides and primers.

**Protocol** [PCR clean-up](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-pcr-cleanup)

**Conclusion** Samples are ready for the following Gibson assembly with our to be digested plasmid backbone.

### Plasmid digestion

2021-07-13

**Aim** Linearization of the pCT-con2-V5 plasmid by restriction enzyme digestion for subsequent Gibson assembly.

**Protocol** [Plasmid digestion](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-plasmid-digestion)

**Results** To verify our digestion worked properly, we ran a 1% agarose gel electrophoresis of our plasmid digested with different combinations of our restriction enzymes: BamHI alone, NheI alone, BamHI and NheI together or no enzymes.

![](https://static.igem.org/mediawiki/2021/e/e5/T--EPFL--notebook--monomer--1.jpg)

Figure 2Gel picture of the digested vector backbone.Lane 1: GeneRuler 1 kb DNA Ladder. Lane 2: Plasmid digested with BamHI HF. Lane 3: Plasmid digested with NheI HF. Lane 4: Plasmid digested with both BamHI HF and NheI HF. Lane 5: Undigested plasmid. The bands in lane 2, 3 and 4 are slightly bigger than 6000 bp while the band in lane 5 is above the 10,000 band of the ladder. pCTcon2-V5 is 6468 bp.

The size of our plasmid, pCTcon2-V5[2](#ref-1), is 6468 bp. We should observe bands of this size when cutting the plasmid with either BamHI or NheI.

Linear DNA encounters less friction while running through the gel than circular DNA. Thus, on our gel, we expect to see the plasmid cut with either BamHI or NheI run faster than the undigested plasmid, as the plasmid would have been linearized.

The size of the insert between BamHI and NheI is 189 bp while the size of the plasmid without the insert is 6279 bp. In lane 4 where our plasmid was cut with both restriction enzymes, we thus expect a band near 6000 bp. Since the size difference is so small with the plasmid that was cut with only one restriction enzyme, we do not expect a difference on the gel. Furthermore, the insert band of 189 bp is too small to be seen on this gel thus we do not expect to see it.

**Conclusion** We were able to digest the pCTcon2V5 plasmid with BamHI and NheI and it worked properly. Thus we could continue with the next experiment.

### Gibson assembly

2021-07-13

**Aim** Gibson assembly will allow to anneal our CUP1 gene to the now linearized vector backbone. The recomposed gene-containing plasmid will be ready to be transformed into bacteria.

**Protocol** [Gibson assembly](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-gibson-assembly)

**Conclusion** We moved on to bacteria transformation and this would confirm that the Gibson Assembly worked well.

### Bacteria transformation and plating

2021-07-14

**Aim** Bacteria transformation with the newly assembled plasmid and selection on ampicillin plates. These plates allow for positive selection of the transformed bacteria as the plasmid contains an ampicillin resistance gene.

**Protocol** [Protocol bacteria transformation and plating](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-bacteria-transformation)

Plates were put in the inoculator at 37°C overnight. The next morning, we observed some colonies on our plates.

**Results**

![](https://static.igem.org/mediawiki/2021/3/3c/T--EPFL--notebook--monomer--2.jpg)![](https://static.igem.org/mediawiki/2021/6/62/T--EPFL--notebook--monomer--3.jpg)![](https://static.igem.org/mediawiki/2021/8/8b/T--EPFL--notebook--monomer--4.jpg)![](https://static.igem.org/mediawiki/2021/1/10/T--EPFL--notebook--monomer--5.jpg)

Figure 3Picture of ampicillin plates A, B, C and D.A: pCTcon2-CUP1\_Y1-V5 transformed bacteria. B: variant Y2. C: variant Y3. D: negative control, linearized pCTcon2-V5 transformed bacteria.

The colonies observed have the AmpR gene, that gives resistance to the bacteria against ampicillin.

**Conclusion** Our transformed bacteria had successfully taken up the plasmid containing the ampicillin resistance gene. Moreover, as we had asserted that the digestion was successful and as we know that bacteria can only function properly with circular DNA we could conclude that our Gibson assembly was successful.

### Colony picking and culture inoculation

2021-07-15

**Aim** pCTcon2-CUP1-V5 amplification

**Protocol** Under the flame, with a sterile pipette, scoop up a colony and release the pipette tip into a 5 ml tube. Add 4 ml of sterile LB medium and place it in the shaker at 37°C.

**Conclusion** We could move on to the next step: Miniprep.

### Miniprep

2021-07-16

**Aim** Purify the plasmid DNA from our overnight culture of bacteria transformed with the pCTcon2-CUP1-V5 plasmid.

**Protocol** [Miniprep](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-miniprep)

Once the Miniprep performed, we measured our DNA concentration as well as purity using Nanodrop.

**Results**

Table 1Quantification of digested samples

| Sample | Concentration | A260/A280 |
| --- | --- | --- |
| Sample 1 (Y1) | 446.0 ng/μl | 1.88 |
| Sample 2 (Y2) | 350.4 ng/μl | 1.88 |
| Sample 3 (Y3) | 330.0 ng/μl | 1.98 |

The last column is the absorbance ratio A260/A280 and represents the quality of the DNA. We consider a sample to be of good quality if the ratio lies between 1.8 and 2.

**Conclusion** These samples had a high concentration of DNA and appeared to be of good quality.

### Sequencing

2021-07-16

**Aim** Compare our now amplified plasmid with the theoretical sequence to observe potential mutations that may have occurred during amplification in bacteria.

**Protocol** Send samples to a DNA sequencing company (Mycrosynth in our case).

We sent 2 clones for each transformant variant: YXCX where X stands for the number of the variant and clone respectively.

**Results** The alignment of the sequencing results are on [this document](https://static.igem.org/mediawiki/2021/a/aa/T--EPFL--notebook--monomer--13.pdf), where the sequences number 1 to 12 correspond to, respectively Y1C1 forward, Y1C1 reverse and so on until Y3C2 reverse.

Y2C1 has a mutation (G to C) in both forward and reverse strands. We thus choose to ignore this one. It also seems like the forward primer of Y2C2 did not anneal, therefore we do not have a full strand. To avoid errors, we decide to only keep Y1 and Y3 variants.

**Conclusion** We decided to proceed with only Y1 and Y3 genomic variants and had two clones for each of them.

### Yeast growth

2021-07-19

**Aim** Monitor the growth of yeast culture to identify the time window by which they are competent for transformation, that is a measured OD600 of 0.8 to 1.

**Protocol** In a 15 ml falcon tube, incubate 12 μl of wildtype yeast in 4 ml of YPD in a shaker at 30°C and 220 rpm. Measure the absorbance at 600 nm (OD600) every hour using a spectrophotometer. A calibration curve may be needed before the actual measurements.

We made 3 technical replicates and 2 biological replicates, but limited by the space on the shaker we could only do one.

**Results** On average between 2 experiments, OD600 of 1 was reached within approximately 12 hours.

### Yeast transformation

2021-07-20

**Aim** Transform the yeast with our pCTcon2-CUP1-V5 plasmid and plate our transformants on a selective medium.

**Protocol** [Yeast transformation](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-yeast-transformation)

We prepared 3 samples: a negative control (no plasmid), a positive control (our plasmid backbone without the CUP1 insert) and the plasmid containing the CUP1 gene.

Our yeast strain, EBY100, is incapable of synthesizing tryptophan on its own and needs it from an external source, thus when plated onto tryptophan-less medium plates, they cannot grow. Our plasmid backbone encodes a Trp+ gene allowing the yeast to grow without tryptophan in its medium, allowing for positive selection of our yeast transformants.

We picked clones from each plate and amplified them in liquid SDCAA medium. In total we picked 6 clones from the yeast transformed with the recombinant plasmid : A, B, C, D, E and F.

**Results**

![](https://static.igem.org/mediawiki/2021/7/78/T--EPFL--notebook--monomer--14.jpg)(a)

![](https://static.igem.org/mediawiki/2021/d/d4/T--EPFL--notebook--monomer--15.jpg)(b)

![](https://static.igem.org/mediawiki/2021/d/d1/T--EPFL--notebook--monomer--16.jpg)(c)

Figure 4Plates A, B and C with transformed yeast.(a) Plate A: yeast transformed with pCTcon2-CUP1-V5 plasmid, colonies observed. (b) Plate B: positive control, yeast transformed with pCTcon2-V5 plasmid, colonies observed. (c) Plate C: negative control, wild type yeast (transformation with only water and no DNA). One colony was observed, probably a mutant. Small white dots are yeast colonies.

**Conclusion** We could conclude that the yeasts had been transformed successfully. We moved on to the next step: checking protein expression.

### Western blot 1

2021-07-30

**Aim** Verify the expression of our protein of interest in yeast.

**Protocol** [Western blot](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-western-blot)

We used two mouse anti-V5 and anti-Tubulin as primary antibodies and a goat anti-mouse as a secondary.

The first Western blot was performed on clone A and B and had for negative controls, the wild type yeast as well as the yeast transformed with the plasmid backbone cultured in SDCAA (without galactose). For positive controls, the yeast transformed with the plasmid backbone cultured in SGCAA (with galactose) as well as a fly tubulin were runned onto the gel.

![](https://static.igem.org/mediawiki/2021/0/09/T--EPFL--notebook--monomer--11.jpg)

Figure 5Western blot 1.

![](https://static.igem.org/mediawiki/2021/e/eb/T--EPFL--notebook--monomer--12.jpg)

Figure 6Analysis of western blot 1.Aga2-V5 has a molecular weight of 21.3 kDa.

We loaded the following samples onto our gel: pCTcon2-V5 in SDCAA, pCTcon2-V5 in SGCAA, EBY100 (WT) in YPD, and two pCTcon2-CUP1-V5 clones (A and B) in SGCAA and SDCAA each. We obtained a band of ~ 50 kDa in the WT and plasmids with and without galactose, showing the presence of α-tubulin (49.8 kDa[3](#ref-2)) in our samples. We also have a band of ~ 20 kDa in plasmid + galactose showing that our Aga2-V5 protein was expressed (figure 6a). Figure 6b is showing the fly control expressing tubulin as well as Relish-V5, a protein harboring the V5 tag. Figure 6c shows the ratio of V5 signal versus tubulin.

However we didn’t obtain any signal, neither for the tubulin nor the protein of interest in the clone A and B lines.

**Conclusion** The clones were probably picked prematurely, and thus didn’t show anything on the gel. That’s why a second Western Blot was performed in order to check protein expression in our 4 other clones that were picked later: C, D, E and F.

### Immunostaining 1

2021-08-09

**Aim** Check the expression of our CUP1 protein on the surface of our yeast transformants, clones C, D, E and F.

**Protocol** [Indirect immunofluorescence labeling](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-immunofluorescence)

We used a mouse anti-V5 primary antibody and a goat Alexa Fluor 488 conjugated anti-mouse as a secondary antibody.

The results were observed under a confocal microscope. For each clone, two samples were analyzed, one where the cells’ membrane were permeabilized in order to see if the protein was expressed intracellularly and one where the cells’ membrane were not permeabilized.

![](https://static.igem.org/mediawiki/2021/3/33/T--EPFL--notebook--monomer--17.jpg)

Figure 7Clone C.Nuclei are stained with DAPI in blue and the V5 tag is stained with a green fluorophore.

![](https://static.igem.org/mediawiki/2021/f/fc/T--EPFL--notebook--monomer--18.jpg)

Figure 8Clone D.

![](https://static.igem.org/mediawiki/2021/c/c3/T--EPFL--notebook--monomer--19.jpg)

Figure 9Clone E.

![](https://static.igem.org/mediawiki/2021/b/b3/T--EPFL--notebook--monomer--20.jpg)

Figure 10Clone F.

![](https://static.igem.org/mediawiki/2021/a/a2/T--EPFL--notebook--monomer--21.jpg)(a)

![](https://static.igem.org/mediawiki/2021/a/af/T--EPFL--notebook--monomer--22.jpg)(b)

![](https://static.igem.org/mediawiki/2021/6/61/T--EPFL--notebook--monomer--23.jpg)(c)

Figure 11Positive control: yeast transformed with the plasmid backbone (no insert).(a) DAPI channel (b) green fluorescence (c) merged channel.

![](https://static.igem.org/mediawiki/2021/a/a9/T--EPFL--notebook--monomer--24.jpg)(a)

![](https://static.igem.org/mediawiki/2021/5/53/T--EPFL--notebook--monomer--25.jpg)(b)

![](https://static.igem.org/mediawiki/2021/2/22/T--EPFL--notebook--monomer--26.jpg)(c)

Figure 12Negative control: wild type yeast.(a) DAPI channel (b) green fluorescence (c) merged channel.

![](https://static.igem.org/mediawiki/2021/1/1e/T--EPFL--notebook--monomer--27.jpg)(a)

![](https://static.igem.org/mediawiki/2021/a/a3/T--EPFL--notebook--monomer--28.jpg)(b)

![](https://static.igem.org/mediawiki/2021/d/d5/T--EPFL--notebook--monomer--29.jpg)(c)

Figure 13Negative control: yeast transformed with the plasmid backbone (no insert), membrane permeabilized.(a) DAPI channel (b) green fluorescence (c) merged channel.

![](https://static.igem.org/mediawiki/2021/a/a4/T--EPFL--notebook--monomer--30.jpg)(a)

![](https://static.igem.org/mediawiki/2021/5/5c/T--EPFL--notebook--monomer--31.jpg)(b)

![](https://static.igem.org/mediawiki/2021/a/a6/T--EPFL--notebook--monomer--32.jpg)(c)

Figure 14Negative control: yeast transformed with the plasmid backbone (no insert), without antibody 2.(a) DAPI channel (b) green fluorescence (c) merged channel.

![](https://static.igem.org/mediawiki/2021/2/27/T--EPFL--notebook--monomer--33.jpg)(a)

![](https://static.igem.org/mediawiki/2021/2/2c/T--EPFL--notebook--monomer--34.jpg)(b)

![](https://static.igem.org/mediawiki/2021/2/2a/T--EPFL--notebook--monomer--35.jpg)(c)

Figure 15Negative control: yeast transformed with the plasmid backbone (no insert), without antibody 1.(a) DAPI channel (b) green fluorescence (c) merged channel.

![](https://static.igem.org/mediawiki/2021/1/19/T--EPFL--notebook--monomer--31-2.jpg)

Figure 16Clone F, higher magnification.

All 4 clones showed expression of CUP1 on their surface. We assumed the expression is at the surface because: first, the antibody does not enter the cell, second, we can see a darker circle around the cell on figure 16 (higher magnification) representing the membrane and third, we did some controls with membrane permeabilized samples (figure 13). The membrane permalized samples show little signal that corresponds to the signal in negative controls WT (figure 12), backbone without antibody 1 (figure 14) and backbone without antibody 2 (figure 15), which we can assume is noise or unspecific binding of antibodies.

Unfortunately, not all cells were positive in one sample, which means that either our subsets of cells were not monoclonal and that not all the cells had the plasmid or that they all had the plasmid but weren't all expressing it.

**Conclusion** In order to see if the expression level differences were due to polyclonality of the yeast population we streaked the cells on a plate and picked a single colony in order to perform an immunostaining on a monoclonal population of cells. This resulted in no different results than before, we still had an heterogeneous population with cells displaying signal and cells with no signal. We concluded that the induction process was not 100% efficient and this was supported by the results of the paper from Chao et al.[2](#ref-1)

### Western blot 2

2021-08-11

**Aim** Check the overall expression of our protein of interest in our yeast transformant clones C, D, E and F.

**Protocol** [Western blot](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-western-blot)

![](https://static.igem.org/mediawiki/2021/b/bf/T--EPFL--notebook--monomer--37.jpg)

Figure 17Analysis of Western blot 2.

We obtained bands of approximatively 25 kDa in positive control as well as in clones C, E and F, showing that our protein of interest was present in these samples. Unfortunately we didn’t have any signal for the α-tubulin so we couldn’t normalize the signal and compare the expression level of the protein. As expected, in the negative control pCTcon2-V5 backbone plasmid uninduced, no band is visible.

We also noticed two bands on the gel, and verified the DNA sequence and saw that a second methionine, a start codon, is present downstream of the first one and in frame with the promoter, resulting in a second expressed protein, probably truncated. Unfortunately this shorter protein is the most expressed one, corresponding to the signal intensity.

**Conclusion** We’ve decided to continue the experiments with our 3 clones C, E and F, since D didn’t show any expression.

### Western blot 3

2021-10-14

**Aim** Check the overall expression of our protein of interest in our yeast transformant clone F and normalize its signal relatively to α-tubulin.

**Protocol** [Western blot](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-western-blot)

Since we did not get any signal for tubulin in Western Blot 2, we decided to re-do the western with clone F to have a nice protein quantification plot.

![](https://static.igem.org/mediawiki/2021/1/11/T--EPFL--notebook--monomer--38.jpg)(a)

![](https://static.igem.org/mediawiki/2021/a/a6/T--EPFL--notebook--monomer--39.jpg)(b)

![](https://static.igem.org/mediawiki/2021/f/f6/T--EPFL--notebook--monomer--40.jpg)(c)

Figure 18Western blot 3.(a) Image of the nitrocellulose membrane with yeast transformed with plasmid backbone pCTcon2-V5 without insert, Clone F, i.e. yeast transformed with pCTcon2-CUP1-V5, both cultured with galactose in their media and wild type yeast (cultured in YPD). (b) Protein quantification plot, the black bars represent the signal for the backbone plasmid and the grey ones are for the Clone F. On the left is represented the signal of V5 tag and on the right, the tubulin signal. (c) is the normalized V5 signal for tubulin (V5 in % of tubulin).

We obtained a tubulin signal in both plasmid backbone pCTcon2-V5 and clone F (pCTcon2-CUP1-V5) but not in wild type yeast. This was probably due to the fact that a smaller quantity of cells were loaded on the gel, since the pellet was smaller in the WT sample. We could normalize the V5 signal relatively to tubulin and the result is shown on figure 18c. We can see that clone F expresses the protein a bit more than the backbone, which is consistent with what we observed in immunostaining.

**Conclusion** We were able to show that our membrane protein fusion was expressed in our engineered yeast and could quantify its relative expression in our clone F as well as in the positive control, the backbone plasmid transformed yeast.

### Yeast growth curves

**Aim** Check for the yeast growth differences between the strains. This will allow us to see whether engineering the yeast would modify its ability to grow or survive.

**Protocol**

1. Prepare yeast cultures of known initial concentration for each of the strain to be tested (3 replicates per sample).
2. Load a 96 wells-plate with your samples (2 or 3 technical replicates for each sample).
3. Put in an overnight shaking microplate reader at 30°C, measuring the OD600 every hour.
4. If you don’t have this equipment at your disposal, place your yeast cultures in a 30°C shaker and collect samples directly from it every hour to measure the OD600 in a spectrophotometer or microplate reader.

We measured the growth of our transformed yeast (pCTcon2-CUP1-V5) in SGCAA, and we compared them to the growth curves of plasmid backbone (pCTcon2-V5) in SDCAA and in SGCAA and the wild type yeast (EBY100).

**Results**

![](https://static.igem.org/mediawiki/2021/6/6a/T--EPFL--notebook--monomer--43.jpg)![](https://static.igem.org/mediawiki/2021/a/a3/T--EPFL--notebook--monomer--44.jpg)![](https://static.igem.org/mediawiki/2021/8/84/T--EPFL--notebook--monomer--45.jpg)![](https://static.igem.org/mediawiki/2021/1/16/T--EPFL--notebook--monomer--46.jpg)![](https://static.igem.org/mediawiki/2021/5/58/T--EPFL--notebook--monomer--47.jpg)

Figure 19Yeast growth curves of different strains.

### Copper absorption assay

**Aim** Measure the yeast ability to remove copper in solution for the different strains.

**Protocol** [Cuprizone Solution Preparation and Copper Concentration Measurement protocol](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-copper)

All measurements were performed with three biological replicates and three technical replicates.

The concentration of copper in the media was set at 4 mg/l for all copper absorption assay. We tested different conditions to identify the cause of bad copper absorption with our engineered yeasts (see the results below and the [Results page](https://2021.igem.org/Team%3AEPFL/Results)).

**Results**

![](https://static.igem.org/mediawiki/2021/f/fd/T--EPFL--notebook--monomer--41.jpg)

Figure (x)Measuring copper concentration over time with varying WT (EBY100) yeast concentration in the media.The yeast density was determined as a function of OD600 (OD600 of 1 corresponds to approximately 3 x 107 cells/ml).

![](https://static.igem.org/mediawiki/2021/c/c0/T--EPFL--notebook--monomer--42.jpg)

Figure (x)Measuring copper concentration over time in the media with WT (EBY100) yeast or yeast expressing CUP1.OD600 of the yeast culture was 4.0.

**Results** Wild type yeasts absorbed copper well, and we were not able to make our engineered yeast with the CUP1 monomer work better. However, the higher wildtype yeast concentration, the faster the copper could be absorbed.

## Cloning the dimers

### Plasmid digestion

2021-08-25

**Aim** Digest the backbone plasmid with restriction enzymes in order to linearize it for cloning by Gibson assembly.

**Protocol** [Plasmid digestion](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-plasmid-digestion)

To check that digestion worked properly, we ran an agarose gel electrophoresis of our plasmid digested with different combinations of our restriction enzymes: BamHI alone, NheI alone, BamHI and NheI and none.

**Results** To verify our digestion worked properly, we ran a 1% agarose gel electrophoresis of our plasmid digested with different combinations of our restriction enzymes: BamHI alone, NheI alone, BamHI and NheI together or no enzymes.

![](https://static.igem.org/mediawiki/2021/8/8f/T--EPFL--notebook--dimers--1.jpg)

Figure 20Result of the backbone digestion on a 0.7% agarose gel.Lane 1: GeneRuler 1 kb DNA Ladder. Lane 2: Plasmid digested with BamHI HF. Lane 3: Plasmid digested with NheI HF. Lane 4: Plasmid digested with both BamHI HF and NheI HF. Lane 5: Undigested plasmid. The bands in lane 2, 3 and 4 have an approximate size of 6000 bp while the band in lane 5 is around the 8,000-10,000 bands of the ladder.

The size of our plasmid, pCTcon2-V5[2](#ref-1), is 6468 bp. We should observe bands of this size when cutting the plasmid with either BamHI or NheI.

Linear DNA encounters less friction while running through the gel than circular DNA. Thus, on our gel, we expect to see the plasmid cut with either BamHI or NheI run faster than the undigested plasmid, as the plasmid would have been linearized.

The size of the insert between BamHI and NheI is 189 bp while the size of the plasmid without the insert is 6279 bp. In lane 4 where our plasmid was cut with both restriction enzymes, we thus expect a band near 6000 bp. Since the size difference is so small with the plasmid that was cut with only one restriction enzyme, we do not expect a difference on the gel. Furthermore, the insert band of 189 bp is too small to be seen on this gel thus we do not expect to see it.

**Conclusion** pCTcon2V5 plasmid was successfully linearized by BamHI and NheI digestion.

### Gene PCR

2021-08-26

**Aim** Amplify our CUP1 dimers sequences by Polymerase Chain Reaction (PCR) and add Gibson assembly overlap sequences at both extremities.

**Protocol** [Gene PCR](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-gene-pcr)

These dimer sequences are made of two copies of CUP1 fused by a linker that we designed (see [the Design page](https://2021.igem.org/Team%3AEPFL/Design) for information). We ordered the sequences from Twist and went through a PCR to amplify and add the overlap sequences we needed for the Gibson assembly. We have 7 different dimer sequences, each discriminated by a unique linker. The samples were normalized to similar concentrations and the PCR was run with 20 ng of DNA material.

We then ran a 2% agarose gel electrophoresis to check the size of our 7 samples after PCR.

**Results**

![](https://static.igem.org/mediawiki/2021/d/d1/T--EPFL--notebook--dimers--3.jpg)

Table 3Information about the dimers and the resulting transformed yeast subclones.

As we were only interested in the first bands of 420bp that correspond to the full dimers sequence, we directly cut out the DNA from the gel using a UV transilluminator. This would then be followed by a PCR clean up.

**Conclusion** We successfully amplified our CUP1 dimers by PCR and extracted the DNA of interest from the agarose gel.

### PCR and digestion clean-up

2021-08-26

**Aim** Purify DNA fragments and digested backbone samples.

**Protocol** [PCR clean-up](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-pcr-cleanup)

**Results** Samples are ready for the following Gibson Assembly with our digested backbone.

### Gibson assembly

2021-08-26

**Aim** Gibson assembly will allow us to fuse our CUP1-dimers to the now linearized vector backbone. The recomposed gene-containing plasmid will be ready to be transformed into bacteria.

**Protocol** [Gibson assembly](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-gibson-assembly)

**Conclusion** We moved on to bacteria transformation and this would confirm that the Gibson Assembly worked well.

### Bacteria transformation and plating

2021-08-26

**Aim** Bacteria transformation with the newly assembled plasmids and selection on ampicillin plates. These plates allow for positive selection of the transformed bacteria as the plasmid encodes an ampicillin resistance gene.

**Protocol** [Protocol bacteria transformation and plating](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-bacteria-transformation)

Plates were put in the inoculator at 37°C overnight. The next morning, we observed some colonies on our plates.

**Results** We observed colonies on every plate with our dimers transformants and on the positive control provided with the NEB Gibson Kit. We did not observe any colonies on the plate with the bacteria transformed with water (negative control).

**Conclusion** We could therefore assert that our Gibson assembly and transformations were successful. Our positive selection with ampicillin allowed us to be sure that the bacteria on the plates had successfully taken up our recombinant plasmids.

### Colony picking and culture inoculation

2021-08-27

**Aim** Pick 3 colonies per plate that have survived Ampicillin treatment and grow them in liquid medium in order to amplify our plasmids DNA.

**Protocol** Under the flame, with a sterile pipette, scoop up a colony and release the pipette tip into a 5ml tube. Add 4ml of sterile LB media and place in the shaker at 30°C.

**Results** We had turbid tubes, indicating that the bacteria had grown.

**Conclusion** We have sufficient bacterial liquid culture for plasmid purification by Miniprep.

### Miniprep

2021-08-30

**Aim** Purify the plasmid DNA from our overnight culture of bacteria transformed with the pCTcon2-CUP1-dimer-V5 plasmids.

**Protocol** [Miniprep](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-miniprep)

Once the Miniprep performed, we measured our DNA concentration as well as purity using Nanodrop.

**Results**

Table 2Quantification of digested samples

| Sample | Concentration | A260/A280 |
| --- | --- | --- |
| 2A | 143.7 ng/μl | 1.83 |
| 2B | 111.7 ng/μl | 1.78 |
| 2C | 102.3 ng/μl | 1.69 |
| 2D | 127.9 ng/μl | 1.61 |
| 2E | 77.8 ng/μl | 1.86 |
| 2F | 99.8 ng/μl | 1.79 |
| 2G | 89.5 ng/μl | 1.89 |

We had three genomic variants per construct, hence we use the number 2 for dimer (version 2 of our yeast expressing CUP1 copies at their surface), then the letter A to G to identify the linker. We measure their concentration using Nanodrop and send them to be sequenced.

### Sequencing

2021-08-31

**Aim** Compare our now amplified plasmid with the theoretical sequence to observe potential mutations that may have occurred during amplification in bacteria.

**Protocol** Send samples to a DNA sequencing company (Mycrosynth in our case).

**Results** Using SnapGene we selected, for each dimer, a variant that had no mutation in the reading frame.

**Conclusion** We kept only the clones without mutations for the next steps. We could move on to transform our engineered plasmids in the yeast cells.

### Yeast transformation

2021-09-01

**Aim** Transform the yeast with our pCTcon2-dimer-V5 plasmids and plate our transformants on a selective medium.

**Protocol** [Yeast transformation](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-yeast-transformation)

According to the Yeast Growth Curve previously made during the version 1 (CUP1 monomer) experiments, we let yeast grow in YPD for approximately 10 hours in order for them to reach an appropriate OD600 = 8 concentration. We prepared 9 samples: a negative control (no DNA, just water), a positive control (our plasmid backbone without insert) and each 7 plasmids containing a dimer. After transformation, we plated our transformants on plates that do not contain tryptophan (SGCAA agar plates), for positive selection as the plasmid encodes a Trp+ gene, this allows the transformed yeast to survive on such a medium.

**Results** The negative control (water) did not show any colonies and the positive control (plasmid backbone) had many colonies. We could also observe colonies on our transformants plates.

**Conclusion** We picked clones from each transformed plate (Table 1). Each time a new clone is picked, it is amplified in liquid media (SDCAA at first but then SGCAA in order to induce the protein expression).

### Western blot 2

2021-09-14

**Aim** Verify the expression of our protein of interest in yeast.

**Protocol** [Western blot](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-western-blot)

The first Western Blot was performed on all our dimers. The V5 tag is part of the membrane display system which itself is under the Gal promoter. Thus expression (and detection) should only occur in the presence of galactose.

**Results** The dimers have a molecular weight of 28.6 kDa, whereas the α-tubulin has a molecular weight of 49.8 kDa.

![](https://static.igem.org/mediawiki/2021/1/18/T--EPFL--notebook--dimers--4.jpg)

Figure 21Image of Western Blot of gel 1.

![](https://static.igem.org/mediawiki/2021/3/39/T--EPFL--notebook--dimers--5.jpg)

Figure 22Information about the dimers and the resulting transformed yeast subclones.

![](https://static.igem.org/mediawiki/2021/5/5f/T--EPFL--notebook--dimers--6.jpg)

Figure 23Protein quantification.

![](https://static.igem.org/mediawiki/2021/a/a3/T--EPFL--notebook--dimers--7.jpg)

Figure 24Protein quantification: normalized V5.

We loaded the following bands onto our Western blot: samples from yeast subclones A1 to A6 and B1 to B3 in SGCAA. We obtained a band of ~ 30 kDa (figure 22), this size corresponds to the dimers theoretical size. Figure 23 shows the signal analysis of the bands done on the ImageJ software and figure 24 shows the ratio of V5 signal versus tubulin.

![](https://static.igem.org/mediawiki/2021/4/40/T--EPFL--notebook--dimers--8.jpg)

Figure 25Image of Western Blot of gel 2.

![](https://static.igem.org/mediawiki/2021/f/fb/T--EPFL--notebook--dimers--9.jpg)

Figure 26

![](https://static.igem.org/mediawiki/2021/4/49/T--EPFL--notebook--dimers--10.jpg)

Figure 27Protein quantification.

![](https://static.igem.org/mediawiki/2021/0/0e/T--EPFL--notebook--dimers--11.jpg)

Figure 28Protein quantification: normalized V5.

We loaded the following samples onto our Western blot: sample from yeast subclones B4 to B6 and F1 to F6 in SGCAA. We obtained a band of ~30kDa (9a) corresponding to the dimer’s theoretical size. Figure 9b shows the signal analysis of the bands done on the ImageJ software and figure 9c shows the ratio of V5 signal versus tubulin.

![](https://static.igem.org/mediawiki/2021/3/3a/T--EPFL--notebook--dimers--12.jpg)

Figure 29Image of Western Blot of gel 3.

![](https://static.igem.org/mediawiki/2021/b/b1/T--EPFL--notebook--dimers--13.jpg)

Figure 30

![](https://static.igem.org/mediawiki/2021/f/f2/T--EPFL--notebook--dimers--14.jpg)

Figure 31Protein quantification.

![](https://static.igem.org/mediawiki/2021/d/d7/T--EPFL--notebook--dimers--15.jpg)

Figure 32Protein quantification: normalized V5.

We loaded on this gel two yeast subclones for dimers 3, 4 and 5, and as controls, the plasmid backbone and the wild type.

![](https://static.igem.org/mediawiki/2021/a/aa/T--EPFL--notebook--dimers--16.jpg)

Figure 33Image of Western Blot of gel 4.

![](https://static.igem.org/mediawiki/2021/d/d1/T--EPFL--notebook--dimers--18.jpg)

Figure 34Protein quantification.

![](https://static.igem.org/mediawiki/2021/1/16/T--EPFL--notebook--dimers--19.jpg)

Figure 35Protein quantification: normalized V5.

We were able to see a band of approximately 30 kDa in each dimer sample, however other smaller bands were also visible. These are probably due to the second reading frame starting at the downstream second methionine.

**Conclusion** Our yeasts cells express two different proteins, one whose size matches the theoretical size of our dimers, and another corresponding to a second reading frame in our backbone. For the following experiments, we kept the subclones who have the best tub/V5 ratio taking into account the initial V5 quantification : A4, B1, C2, D2, E2, F3 and G2.

### Immunostaining 2

2021-09-25

**Aim** Check the extracellular membrane expression of our dimers.

**Protocol** [Indirect immunofluorescence labeling](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-immunofluorescence)

We used a mouse anti-V5 as primary antibody and a goat anti-mouse coupled with the Alexa Fluor Plus 488 as secondary. The results were observed under a confocal microscope.

**Results**

![](https://static.igem.org/mediawiki/2021/0/09/T--EPFL--notebook--dimers--20.jpg)(a)

![](https://static.igem.org/mediawiki/2021/6/61/T--EPFL--notebook--dimers--20-2.jpg)(b)

![](https://static.igem.org/mediawiki/2021/7/79/T--EPFL--notebook--dimers--21.jpg)(c)

![](https://static.igem.org/mediawiki/2021/7/77/T--EPFL--notebook--dimers--22.jpg)(d)

![](https://static.igem.org/mediawiki/2021/c/c9/T--EPFL--notebook--dimers--23.jpg)(e)

![](https://static.igem.org/mediawiki/2021/5/52/T--EPFL--notebook--dimers--24.jpg)(f)

![](https://static.igem.org/mediawiki/2021/9/98/T--EPFL--notebook--dimers--25.jpg)(g)

Figure 36Nuclei are stained with DAPI in blue and the V5 tag is stained with a green fluorophore.(a) Yeast subclone A4, dimer 1 (b) Yeast subclone B1, dimer 2 (c) Yeast subclone C2, dimer 3 (d) Yeast subclone D2, dimer 4 (e) Yeast subclone E2, dimer 5 (f) Yeast subclone F3, dimer 6 (g) Yeast subclone G2, dimer 7. All our yeast clones are expressing their corresponding CUP1 dimer at their surface, although in one sample not all the cells express it. This is due to the fact that the expression yield is not 100%. We can see that some dimers are better than others, for example dimers 6 and 7 have good expression efficiency while dimer 1 have lower expression yield. We also don’t have the same amount of cells in each sample, due to experiment manipulations and not reliable OD measurements. Fortunately we can see that our protein of interest is expressed at the surface of the cell because of the darker green circle representing the cell membrane. This can be better seen on a higher magnification image.

![](https://static.igem.org/mediawiki/2021/9/9e/T--EPFL--notebook--dimers--26.jpg)(a)

![](https://static.igem.org/mediawiki/2021/4/41/T--EPFL--notebook--dimers--27.jpg)(b)

![](https://static.igem.org/mediawiki/2021/7/7c/T--EPFL--notebook--dimers--28.jpg)(c)

Figure 37Picture of the immunostaining of wild type yeast.(a) DAPI channel (b) green fluorescence channel (c) merge channel.

![](https://static.igem.org/mediawiki/2021/f/f8/T--EPFL--notebook--dimers--29.jpg)(a)

![](https://static.igem.org/mediawiki/2021/6/63/T--EPFL--notebook--dimers--30.jpg)(b)

![](https://static.igem.org/mediawiki/2021/2/28/T--EPFL--notebook--dimers--31.jpg)(c)

Figure 38Picture of the immunostaining of plasmid backbone uninduced (- galactose) in yeast.(a) DAPI channel (b) green fluorescence channel (c) merge channel.

![](https://static.igem.org/mediawiki/2021/0/08/T--EPFL--notebook--dimers--32.jpg)(a)

![](https://static.igem.org/mediawiki/2021/b/b1/T--EPFL--notebook--dimers--33.jpg)(b)

![](https://static.igem.org/mediawiki/2021/e/ea/T--EPFL--notebook--dimers--34.jpg)(c)

Figure 39Picture of the immunostaining of plasmid backbone induced (+ galactose) in yeast, without the secondary antibody.(a) DAPI channel (b) green fluorescence channel (c) merge channel.

![](https://static.igem.org/mediawiki/2021/a/ae/T--EPFL--notebook--dimers--35.jpg)(a)

![](https://static.igem.org/mediawiki/2021/1/18/T--EPFL--notebook--dimers--36.jpg)(b)

![](https://static.igem.org/mediawiki/2021/b/b9/T--EPFL--notebook--dimers--37.jpg)(c)

Figure 40Picture of the immunostaining of plasmid backbone induced (+ galactose) in yeast, without the primary antibody.(a) DAPI channel (b) green fluorescence channel (c) merge channel.

![](https://static.igem.org/mediawiki/2021/9/93/T--EPFL--notebook--dimers--38.jpg)(a)

![](https://static.igem.org/mediawiki/2021/4/46/T--EPFL--notebook--dimers--39.jpg)(b)

![](https://static.igem.org/mediawiki/2021/6/69/T--EPFL--notebook--dimers--40.jpg)(c)

Figure 41Positive control : Picture of the immunostaining of plasmid backbone induced (+ galactose) in yeast.(a) DAPI channel (b) green fluorescence channel (c) merge channel. Our positive plasmid backbone control is expressing the yeast surface display at its membrane.

![](https://static.igem.org/mediawiki/2021/d/db/T--EPFL--notebook--dimers--41.jpg)

Figure 42Immunostaining of Clone F (CUP1 monomer - pCTcon2-CUP1-V5).40x, green channel.

**Conclusion** All of our transformed yeast expressed the dimer at their surface, although the expression efficiency is not 100%. Dimer B and D seemed to be contaminated and had a lot of noise in the GFP signal. The results of dimer A, C, E, F and G are good.

### Yeast growth curves

**Aim** Check for the yeast growth differences between the strains. This will allow us to see whether engineering the yeast would modify its ability to grow or survive.

**Protocol**

1. Prepare yeast cultures of known initial concentration for each of the strain to be tested (3 replicates per sample).
2. Load a 96 wells-plate with your samples (2 or 3 technical replicates for each sample).
3. Put in an overnight shaking microplate reader at 30°C, measuring the OD600 every hour.
4. If you don’t have this equipment at your disposal, place your yeast cultures in a 30°C shaker and collect samples directly from it every hour to measure the OD600 in a spectrophotometer or microplate reader.

We measured the growth of our 3 transformed yeast clones C, D and E as well as the plasmid backbone in SDCAA and in SGCAA. We also loaded the remaining wells with medium alone, without any yeast in it.

**Results**

![](https://static.igem.org/mediawiki/2021/b/b5/T--EPFL--notebook--dimers--43.jpg)![](https://static.igem.org/mediawiki/2021/8/8f/T--EPFL--notebook--dimers--44.jpg)![](https://static.igem.org/mediawiki/2021/7/74/T--EPFL--notebook--dimers--45.jpg)![](https://static.igem.org/mediawiki/2021/d/d7/T--EPFL--notebook--dimers--46.jpg)![](https://static.igem.org/mediawiki/2021/0/03/T--EPFL--notebook--dimers--47.jpg)![](https://static.igem.org/mediawiki/2021/9/92/T--EPFL--notebook--dimers--48.jpg)

Figure 43Yeast growth curves of different strains in SGCAA with different copper copper concentrations.

### Copper absorption assay

**Aim** Measure the yeast ability to remove copper in solution for the different strains.

**Protocol** We grew our various engineered yeast strains in liquid media containing copper and we collected 2 ml aliquots of the culture at different time points after addition of yeasts in the media (t0, 15 min, 60 min, 90 min, 120 min). The yeast cultures were prepared in 500 ml Erlenmeyer flasks, and incubated at 30°C under 200 rpm shaking. Each of the collected samples were then slowly centrifuged (200g for 10 minutes) to separate the yeast cell from the media and copper sulfate solution. Note that the centrifugation step was performed at a low speed to avoid any cell lysis, hence preventing the presence of yeast components, free or bound to copper ions, in the supernatant. This is done mainly for two reasons. First, the absorbance wavelengths of yeast and copper are in the same spectrum range. Thud, any contaminations of yeast in the supernatant could lead to alteration and artefact values in the OD measurements, resulting in data with a higher concentration than reality. Second, if any pieces of yeast bound to copper ions would end up in the supernatant, these ions would also be detected by the spectrophotometer, also leading to values with a higher concentration than the one that would have been actually removed from our yeast. Once the centrifugation step was finished, the pellet, mainly characterized by yeast cells, and the supernatant, mainly characterized by media and copper sulfate, are isolated from each other and collected for different chemical downstream processes.

**Copper concentration measurement** (see also [Cuprizone Solution Preparation and Copper Concentration Measurement protocol](https://2021.igem.org/Team%3AEPFL/Protocols#protocol-copper)) In this assay, the aforementioned supernatant is mixed with cuprizone and brought to a slightly basic (pH ~ 8) condition. Cuprizone [oxalic acid bis(cyclohexylidene hydrazide)] is a selective and sensitive copper-chelating agent, typically used in spectrophotometric determination of copper(II). Indeed, this chemical compound is highly sensitive to copper and, once it interacts with a copper solution, it forms a complex that absorbs at wavelengths of 580-600 nm. The absorbance at 580-600 nm of the solution is therefore proportional to the amount of copper. However, according to Messori et al., the complex with Cu(II) and cuprizone is unstable because of the fast decomposition of the reagent, also affecting the stability of the absorbance over time[4](#ref-3). Therefore, given the strong instability of the CuCPZ complex, we analyzed its absorbance kinetics to find a stable plateau, hence an optimal time to perform the measurements, avoiding any substantial variance in the measurements. We used a spectrophotometer UV-1600PC (#634-6001) with a filter allowing the measurement of the absorbance at 600 nm for that purpose.

All measurements were performed with three biological replicates and three technical replicates.

The concentration of copper in the media was set at 4 mg/l for all copper absorption assay. We tested different conditions to identify the cause of bad copper absorption with our engineered yeasts (see the results below and the [Results page](https://2021.igem.org/Team%3AEPFL/Results)).

**Results**

![](https://static.igem.org/mediawiki/2021/c/c7/T--EPFL--notebook--dimers--49.jpg)![](https://static.igem.org/mediawiki/2021/1/1a/T--EPFL--notebook--dimers--50.jpg)

Figure 44CUP1 surface expression in SGCAA-cultured yeast interferes with endogenous bio-accumulation The (YPD) and (SGCAA) in brackets depict the medium in which the yeast grew and are staying in during the experiment.

![](https://static.igem.org/mediawiki/2021/c/c3/T--EPFL--notebook--dimers--51.jpg)

Figure 45Interference with endogenous bio-accumulation is not related to CUP1 expression nor the surface system itself.

![](https://static.igem.org/mediawiki/2021/d/de/T--EPFL--notebook--dimers--52.jpg)

Figure 46Rich medium is needed for bio-accumulation to occur.

**Conclusion** We were not able to make our engineered yeast work better than the wild type one, but we found out that the medium in which the yeast grows is an important factor to take into account when performing this kind of bioremediation experiment.

## References

1. UniProt

   Copper metallothionein 1-1 (P0CX80)

   [Full text](https://www.uniprot.org/uniprot/P0CX80)
2. Chao, Lau, Hackel, Sazinsky, Lippow & Wittrup (2006)

   Isolating and engineering human antibodies using yeast surface display

   *Nature Protocols*, vol. 1, no. 2, pp. 755-768

   [Crossref](http://dx.doi.org/10.1038/nprot.2006.94)[Google Scholar](https://scholar.google.ch/scholar?q=10.1038%2Fnprot.2006.94)
3. UniProt

   Tubulin alpha-1 chain (P09733)

   [Full text](https://www.uniprot.org/uniprot/P09733)
4. Messori, Casini, Gabbiani, Sorace, Muniz-Miranda & Zatta (2007)

   Unravelling the chemical nature of copper cuprizone

   *Dalton Transactions*, no. 21, pp. 2112

   [Crossref](http://dx.doi.org/10.1039/B701896G)[Google Scholar](https://scholar.google.ch/scholar?q=10.1039%2FB701896G)

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# Team:EPFL/Engineering

* Project

  + [Description

    Project background, inspiration, etc.](https://2021.igem.org/Team%3AEPFL/Description)
  + [Background

    Quantification of the problem](https://2021.igem.org/Team%3AEPFL/Background)
  + [Design

    Monomer and dimer design](https://2021.igem.org/Team%3AEPFL/Design)
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    Bioreactor fluid mechanics models](https://2021.igem.org/Team%3AEPFL/Model)
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    Contribution to future teams](https://2021.igem.org/Team%3AEPFL/Engineering_Success)
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    Bioreactor](https://2021.igem.org/Team%3AEPFL/Implementation)
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    Contribution to future teams](https://2021.igem.org/Team%3AEPFL/Contribution)
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    PCR, Western Blot, transformation, etc.](https://2021.igem.org/Team%3AEPFL/Protocols)
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    Detailed records of all experiments](https://2021.igem.org/Team%3AEPFL/Notebook)
* People

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* Parts

  + [Parts Overview](https://2021.igem.org/Team%3AEPFL/Parts_Overview)
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  + [Human Practices](https://2021.igem.org/Team%3AEPFL/Human_Practices#hp-silver)
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* [Medals](https://2021.igem.org/Team%3AEPFL/Medals_and_Awards)

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### Contents

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# Team:EPFL/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

* [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)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:EPFL/Results

* Project

  + [Description

    Project background, inspiration, etc.](https://2021.igem.org/Team%3AEPFL/Description)
  + [Background

    Quantification of the problem](https://2021.igem.org/Team%3AEPFL/Background)
  + [Design

    Monomer and dimer design](https://2021.igem.org/Team%3AEPFL/Design)
  + [Modelling

    Bioreactor fluid mechanics models](https://2021.igem.org/Team%3AEPFL/Model)
  + [Results

    Main results of the project](https://2021.igem.org/Team%3AEPFL/Results)
  + [Engineering Success

    Design, build, test, learn](https://2021.igem.org/Team%3AEPFL/Engineering_Success)
  + [Implementation

    Bioreactor](https://2021.igem.org/Team%3AEPFL/Implementation)
  + [Proof of Concept

    Showing yeast captures copper](https://2021.igem.org/Team%3AEPFL/Proof_Of_Concept)
  + [Contribution

    Contribution to future teams](https://2021.igem.org/Team%3AEPFL/Contribution)
  + [Safety

    Chemicals used, security measures](https://2021.igem.org/Team%3AEPFL/Safety)
  + [Protocols

    PCR, Western Blot, transformation, etc.](https://2021.igem.org/Team%3AEPFL/Protocols)
  + [Notebook

    Detailed records of all experiments](https://2021.igem.org/Team%3AEPFL/Notebook)
* People

  + [Team](https://2021.igem.org/Team%3AEPFL/Team)
  + [Attributions](https://2021.igem.org/Team%3AEPFL/Attributions)
  + [Collaborations](https://2021.igem.org/Team%3AEPFL/Collaborations)
* Parts

  + [Parts Overview](https://2021.igem.org/Team%3AEPFL/Parts_Overview)
  + [Parts Collection](https://2021.igem.org/Team%3AEPFL/Parts_Collection)
* Human Practices

  + [Human Practices](https://2021.igem.org/Team%3AEPFL/Human_Practices)
  + [Integrated Human Practices](https://2021.igem.org/Team%3AEPFL/Integrated)
  + [Education & Communication](https://2021.igem.org/Team%3AEPFL/Communication)
* [Medals](https://2021.igem.org/Team%3AEPFL/Medals_and_Awards)

![](https://static.igem.org/mediawiki/2021/1/14/T--EPFL--background--results.jpg)

### Contents

* [Introduction](#heading-0)
* [Overview](#heading-1)
* [DNA cloning](#heading-2)
* [Protein expression characterization](#heading-3)
* [Growth assay](#heading-4)
* [Copper absorption assay](#heading-5)
* [Copper recovery](#recovery)
* [In brief](#heading-6)
* [Discussion](#discussion)

## Introduction

Once our copper retrieval system was designed, lab experiments were performed with the aim of expressing one or two copies of our protein of interest, CUP1, at the outer membrane of the yeast. First, we performed a series of experiments to check our protein expression level and localisation. Next, we tested our transformed organisms by conducting copper removal tests that consist of monitoring medium copper levels over time in presence of the engineered yeast.

## Overview

In order to solve the problem of copper pollution in the field, we came up with the idea of using a microorganism capable of absorbing copper in water, by expressing the copper intracellular yeast metallothionein CUP1[1](#ref-0) at its surface and improving the capacity of the cell to retrieve copper, a phenomenon called bio-adsorption[2](#ref-1).

To express the protein extracellularly, we cloned its coding sequence into the pCTcon2-V5 plasmid[3](#ref-2) in frame with Aga2[4](#ref-3), a protein that links by disulfide bonds to Aga1, a yeast membrane protein. In order to check its extracellular expression, we first performed a Western Blot with a primary antibody targeting the V5 tag and then performed immunocytochemistry in order to visually check the presence of CUP1 on the outer membrane of our cells.

Once CUP1 extracellular expression was validated in our transformed yeast cells, we performed tests monitoring yeast's ability to remove copper in different conditions in comparison with non-transformed yeast. We then completed the same workflow with the CUP1 dimers, namely, DNA cloning, yeast transformation, Western Blot, Immunocytochemistry assay, yeast growth curves and finally, copper absorption assays.

## DNA cloning

CUP1 coding DNA fragment was obtained by PCR-amplification of yeast genomic DNA since CUP1 genomic and coding sequence are the same. Amplicon size was checked by gel electrophoresis before proceeding to a PCR clean-up. We then digested the plasmid with two restriction enzymes, NheI and BamHI, and also checked the result by gel electrophoresis. Next, we cloned our sequence into the plasmid by Gibson Assembly, transformed E.coli with the assembled product and amplified the recombinant plasmid. The [pCTcon2-V5 plasmid](https://2021.igem.org/Team%3AEPFL/Parts_Overview#plasmid) containing an ampicillin resistance gene, transformants were selected by plating the bacteria on ampicillin LB plates and amplifying clones in liquid medium supplemented with ampicillin. Plasmid purification was performed by Miniprep. Next DNA integrity and sequence were checked by absorbance measurements and Sanger sequencing respectively. Eventually, we transformed yeast competent cells with the right plasmids and selected them on trp- plates. As our plasmid contains a trp+ gene and the transformed yeast strain EBY100 is trp-, it allowed the auxotrophic selection of transformants.

## Protein expression characterization

Aga2-CUP1-V5 protein expression quantification by [immunoblot](https://2021.igem.org/Team%3AEPFL/Notebook#web2) allowed the selection of the best clones. Protein extraction was achieved by cell lysis and expression levels quantified by Western Blot. Chemiluminescence-based protein detection was based on the binding of a mouse antibody to the C-terminal V5-epitope or a mouse monoclonal antibody to a tubulin-specific epitope, both subsequently recognized by an HRP-conjugated goat secondary antibody, binding the constant region of the primary antibodies. As expected, non-transformed yeast strains (EBY100) or transformed strains (pCTcon2-V5) cultured in medium without induction (galactose -) do not show any protein expression, while transformed strains (pCTcon2-V5/plasmid backbone, pCTcon2-CUP1-V5/CUP1 monomer and pCTcon2-CUP1-linker-CUP1/CUP1 dimer) cultured in medium with induction (galactose +) show elevated protein levels, Figure 1.

![](https://static.igem.org/mediawiki/2021/7/71/T--EPFL--results--0.jpg)

Figure 1V5 tagged CUP1 expression in *S. cerevisiae*.pCTcon2-V5: surface display backbone plasmid. pCTcon2-CUP1-V5: V5-tagged CUP1 monomer expression system. pCTcon2-CUP1-V5: V5-tagged CUP1 dimer expression system. Loaded protein lysates come from representative clones, see lab notebook for detailed expression levels of each construction.

System membrane expression was tested by immunocytochemistry. Yeasts membranes were stained with a mouse primary antibody binding the V5 tag and recognized by a second antibody harboring a fluorophore, the Alexa Fluor 488[5](#ref-4), while nuclei were marked by DAPI staining. Results validated that CUP1 surface expression, CUP1 monomer in Figure 2a and CUP1 dimer in Figure 2b, has similar expression to a CUP1-less system (plasmid backbone positive control), Figure 2d, and that is specific to the cell membranes. As these are non permeabilized cells, the antibodies bind only the extracellular proteins. In order to check whether the protein was not also present intracellularly, we permeabilized the cells before [staining them](https://2021.igem.org/Team%3AEPFL/Notebook#immuno1) and confirmed that our protein was only expressed at the membrane. Background ectopic staining was checked by non-transformed yeast cells (EBY100) staining which displayed poor signal, validating our results.

![](https://static.igem.org/mediawiki/2021/5/52/T--EPFL--results--1.jpg)(a)

![](https://static.igem.org/mediawiki/2021/4/4a/T--EPFL--results--2.jpg)(b)

Figure 2V5 tagged CUP1 extracellular membrane expression in *S. cerevisiae* (yeast transformed with pCTcon2-CUP1-V5).(a) picture taken with a 20x microscope objective, (b) with 40x (only green channel)

![](https://static.igem.org/mediawiki/2021/f/f9/T--EPFL--results--3.jpg)(a)

![](https://static.igem.org/mediawiki/2021/2/2f/T--EPFL--results--4.jpg)(b)

![](https://static.igem.org/mediawiki/2021/0/01/T--EPFL--results--5.jpg)(c)

Figure 3V5 tagged CUP1 extracellular membrane expression in *S. cerevisiae*.(a) yeast transformed with pCTcon2-CUP1-linker-CUP1-V5 (b) negative control: wild type EBY100 (c) positive control: yeast transformed with the backbone plasmid pCTcon2V5.

## Growth assay

We also wanted to check whether our expression system would affect the growth of the yeast in any way. The wild type EBY100 yeast could normally only grow in a complete yeast medium, such as YPD which is yeast extract peptone dextrose. Whereas the yeasts transformed with pCTcon2V5 vector were selected in SGCAA, which is a reduced medium containing galactose but lacking tryptophan that was used for auxotrophic selection of yeast transformants.

For that purpose, we monitored the growth over time of the transformed yeasts in both rich media (YPD) and selection media (SGCAA), as well as the growth of wild type EBY100 yeast in rich media, Figure 3 by measuring the cultures OD600 every 15 minutes. With no surprise, the transformed yeasts with the backbone vector grew faster in rich media, similarly to wild type yeasts. It took longer for the engineered yeasts (transformed with backbone vector or CUP1 construct) to grow in selection media, but the growth curves still reached a plateau. Moreover, we also performed some growth assay with the wildtype EBY100 yeast in YPD media supplemented with various concentrations of copper, Figure 4. Our yeast strain demonstrated good resistance to growing concentration of copper (up to 1000 mg/l), validating our choice to use it as a chassis for copper remediation.

![](https://static.igem.org/mediawiki/2021/a/a3/T--EPFL--results--6.jpg)

Figure 4Growth curves of wild type EBY100 yeasts and transformed EBY100 yeasts in rich (YPD) and selection media (SGCAA).

![](https://static.igem.org/mediawiki/2021/c/c4/T--EPFL--results--7.jpg)

Figure 5Growth curves of wild type EBY100 yeasts and transformed EBY100 yeasts in YPD media supplemented with various concentrations of copper.

## Copper absorption assay

The ability of the engineered microorganism to remove copper from a solution was assessed by monitoring the copper concentration of medium in contact with yeast over time. To have a complete understanding of the assay technicalities, see the Copper assay design. Different yeast cultures were prepared, each one harboring a different condition. The conditions varied between yeast strain and yeast concentration. We then collected samples from the cultures at different time points, spinned the cells down by centrifugation and measured the copper concentration in the supernatant using a colorimetry test with Cuprizone and spectrophotometer.

The first strain we tested was the non-transformed EBY100, referred to as “wild type” (WT) thereafter. We tested three different concentrations of yeast in media supplemented with 4 mg/l copper, a concentration in the same order of magnitude as the pollution found in vineyards and the results are summarized on Figure 5.

![](https://static.igem.org/mediawiki/2021/5/5b/T--EPFL--results--8.jpg)

Figure 6Measuring copper concentration over time with varying WT (EBY100) yeast concentration in the media. The yeast density was determined as a function of OD600 (OD600 of 1 corresponds to approximately 3 x 107 cells/ml).

First we could conclude that the wild type strain absorbs copper, as expected because they express endogenous intracellular CUP1. Also, the denser the yeast population, the greater the ability to absorb and remove copper, Figure 5. To illustrate, the sample with the highest yeast density absorbed three times more copper than the culture with the lowest yeast concentration after 120 minutes of incubation (p value < 0.0001).

We next tested our engineered yeasts that are: (1) one single CUP1 protein expressed at its surface and (2) CUP1 dimers. We tested 7 different dimers constructions as described in [the Design page](https://2021.igem.org/Team%3AEPFL/Design). These differences essentially rely on linker properties used for the two CUP1 proteins fusion. In a first batch of experiments, we compared CUP1 monomer surface expression and three different CUP1 dimers (dimer-1, dimer-3 and dimer-7) surface expression ability to remove copper compared with the wild type strain. The results are depicted in Figure 6.

![](https://static.igem.org/mediawiki/2021/a/ae/T--EPFL--results--9.jpg)![](https://static.igem.org/mediawiki/2021/e/ed/T--EPFL--results--10.jpg)

Figure 7CUP1 surface expression in SGCAA-cultured yeast interferes with endogenous bio-accumulation The (YPD) and (SGCAA) in brackets depict the medium in which the yeast grew and are staying in during the experiment.

Unfortunately, we observed that our newly engineered yeasts, CUP1 monomer or CUP1 dimer were not only not performing better than wild type but did not even absorb copper in solution. Disappointed by this finding but not less determined to find out why, we continued digging. We had three hypotheses to explain this results as there were three variables changing from wild type to engineered yeast, the membrane expression of CUP1, the medium and the fact that the yeast is genetically modified or not:

* The extracellular membrane expression of CUP1 interferes with the endogenous bio-accumulation property of yeast by, for example, blocking the entry of copper into the cell
* Transformation of yeast with pCTcon2V5 vector (with or without CUP1 insert) interferes with the endogenous bio-accumulation property of yeast
* The reduced medium is the problem and prevents the yeast from bio-accumulate copper.

To test if the problem was coming from the extracellular CUP1 expression we tested our transformed yeast with backbone plasmid in SGCAA medium (+ galactose, induced expression) and in SDCAA (- galactose, no expression induction) and compared the results with the wild type strain EBY100 in YPD:

![](https://static.igem.org/mediawiki/2021/e/ec/T--EPFL--results--11.jpg)

Figure 8Interference with endogenous bio-accumulation is not related to CUP1 expression nor the surface system itself.

We can clearly see on this figure that neither the backbone system in SGCAA nor in SDCAA was retrieving copper from the solution. As the system backbone in SDCAA was not expressing the surface display, we could eliminate the first hypothesis. Indeed, the only differences between the wild type and backbone system strains were in the medium and in the fact that the backbone system had been genetically modified. Therefore, we decided to check if the problem was coming from the medium by testing our engineered yeast in YPD + galactose (to induce the system) as well as the WT strain in SGCAA + tryptophan (because it would not survive without tryptophan). The results are shown below in Figure 7.

![](https://static.igem.org/mediawiki/2021/1/16/T--EPFL--results--12.jpg)

Figure 9Rich medium is needed for bio-accumulation to occur.

Seeing that wild type yeast in SGCAA supplemented with tryptophan was behaving like the engineered version in SDCAA and that engineered yeast in YPD supplemented with galactose was behaving like WT in YPD made us eventually conclude that the problem was in fact coming from the medium.

Unfortunately we ultimately found that our engineered yeast, even when cultured in YPD, was not better than the wild type strain in absorbing and removing copper, Figure 8. To conclude, our strategy based on CUP1 surface expression did not improve the endogenous bio-accumulation ability of yeast.

![](https://static.igem.org/mediawiki/2021/0/00/T--EPFL--results--13.jpg)

Figure 10Surface expression of CUP1 does not improve yeast Cu-bioremediation ability.

## Copper recovery

Since the wild type yeast could remove copper in solution and seemed to be the best option to do it compared to the engineered strains we made a last experiment to test whether we could retrieve the captured copper from the yeast in order to eventually further re-use it. Our literature research led us to consider EDTA as a candidate for copper retrieval[6](#ref-5). We thus tested this chemical on loaded (1 mg/l) wild type yeast with different times of reaction as a function of EDTA concentration. The results are shown in the following figure:

![](https://static.igem.org/mediawiki/2021/7/71/T--EPFL--results--14.jpg)

Figure 11Copper release in function of EDTA concentration.

From this, we found that the use of EDTA for copper retrieval to be effective with the best profitability using a 20 min reaction time with 20 mM. During our second meeting with Ludovic Vincent, the CEO of Biomede, we shared these results with him. It seemed that EDTA was indeed a great option for what we were trying to do. However, it is difficult to subsequently remove EDTA from the copper containing water. We must take this into account when thinking about the future uses of this copper.

Thus a rather complicated chemical question arises : we would need to find an effective chelating ligand (a compound that triggers complexe formation), which is not too expensive and not too harmful to the environment.

In our case, if we consider this copper solution to be reusable by wine growers, EDTA at a low concentration is not in itself a major problem as it is already used as a fertilizer. However, other options should be considered, such as separating EDTA from the copper-filled water to avoid further contamination of plants and soils.

## In brief

To sum, we were able to express different CUP1 configurations on the extracellular membrane of the yeast S.cerevisiae. However we did not succeed in improving the endogenous ability for bio-accumulation of the yeast with our engineered strain. Instead we found out that the medium in which the yeast is growing is a major factor to take into account when performing bioremediation experiments. Indeed, our engineered yeast were not able to retrieve copper when growing in SGCAA, a reduced medium used for transformants selection, but was acting like the wild type when put in YPD, a rich medium used to grow wild type yeast.

In conclusion, WT yeast already performs well in removing copper by bioaccumulation from a solution in a 4 mg/l range, relevant to the fungicide contaminated water copper concentration. Surface expression of CUP1 does not improve yeast ability to remove copper from a solution. Nonetheless, since autogenous bioaccumulation seems to be an effective process on its own, we suspect that increasing it further by rising endogenous intracellular CUP1 expression would make the yeast a very efficient genetically modified engineered machine for copper removal.

## Discussion

In this project we identified an underappreciated problem related to organic or ‘bio’ based horticulture in particular of wine grape vines and tea plants. Treatment with copper based fungicides, while not detrimental to the food produced (e.g. wine), is deleterious to the soil these plants are cultivated in as copper accumulates to toxic levels from rain runoff. These copper fungicide treatments have a long history, they are approved for organic use and their use is unlikely to decline in the near future given the additional environmental issues with alternate fungicides, and public sentiment against novel compounds (see [our survey](https://2021.igem.org/Team%3AEPFL/Collaborations#survey)). Copper ground contamination inhibits cultivation of new plants in the polluted soil. Given the already limited soil types and regions suitable for wine and tea production, which are becoming even further limited due to global warming, finding a solution to stop this problem becomes even more pressing.

Our solution, CuRe, aimed to employ bioremediation to remove copper from fungicide contaminated rainwater. Our strategy was to express the endogenous yeast copper-binding protein CUP1 on the surface of yeast to bind to and allow the removal of copper from water. We generated constructs which allow the expression of single and multiple copies of CUP1 on the surface of yeast separated by linker protein regions of varying flexibility. We confirmed that these proteins are produced and present on the surface of yeast using both western blotting and immunofluorescence.

![](https://static.igem.org/mediawiki/2021/f/f2/T--EPFL--design--9.jpg)

Figure 12Yeast surface display for linkers.

To couple with these novel yeast strains, we designed a bioreactor to allow yeast suspended in beads to interact with rainwater and remove pollutants such as copper. We designed, manufactured and built a device easy to use in the field by implementing a graphical user interface on an associated tablet that allows control of the bioreactor activities.

![](https://static.igem.org/mediawiki/2021/3/3f/T--EPFL--implementation--8.jpg)

Figure 13Graphical user interface used to control the bioreactor.

Evaluation of the efficiency of CUP1 expressing strains was more circumspect. We established a copper removal chemical assay and growth conditions for our yeast strains suitable for introduction to copper containing solutions. A key unexpected variable however was the potent ability of our background yeast carrier strain to bioaccumulate copper from solution. Comparison of our CUP1 surface expressing strains to our chassis strain revealed little additional removal of copper from solution compared to the control background yeast strain. From these experiments we can conclude:

1. Control yeast strains have a potent ability to absorb copper from solution.
2. CUP1 monomers or multimers could be expressed on the surface of yeast though retention of their copper binding activity could not be verified.
3. Addition of single or multiple copies of CUP1 provided no significant additional removal of copper from solution.

In sum, we discovered yeast can remove copper efficiently from solution, though our efforts to further enhance this process were not fruitful. We also provide [a strategy whereby copper can be released from yeast](https://2021.igem.org/Team%3AEPFL/Results#recovery), for return back into fungicides for example, forming a closed loop for the recycling of this metal.

Our central goal of depleting copper from solution has been achieved, albeit not enhanced by our synthetic biology approach. **Ironically, this finding could make real-world application of our technology easier to achieve.** The release of genetically modified organisms is highly restricted in Europe and other countries. The finding that wild type yeast strains allow efficient removal of copper removes an important stumbling block to exploitation of our bioremediation strategy. **As it stands, wildtype yeast strains in our bioreactor prototype could be quickly made ready for field testing, with little or no regulatory impedance.**

From our data and experiments, we can envision the next steps upon which to iterate and improve.

Firstly, we would suggest other ‘wild type’ yeast strains used in laboratory experiments or even food or beer production be tested for their ability to bioaccumulate copper. Existing strains may have an even higher ability to absorb copper from solution and bring the advantages for direct application described above.

Second, in a synthetic biology strategy, our data suggests that bioaccumulation rather than biosorption should be the cellular process upon which to aim to enhance. Yeast's natural ability to bioaccumulate copper was far more potent than our attempt to promote biosorption. One can envision strategies to further enhance copper accumulation through the modification of the copper import and detoxification machinery, such as increasing intracellular CUP1 levels or activity, increasing the copper ions influx by enhancing transporters activity or raising intracellular copper metabolism. A combination of these approaches could further enhance the existing effective ability of yeast to deplete copper in solution.

Through [our outreach activities](https://2021.igem.org/Team%3AEPFL/Communication), we have sought to encourage the next generation of bioengineers to rise to this challenge by both promoting synthetic biology in schools through [a children's book](https://2021.igem.org/Team%3AEPFL/Communication#book), by [encouraging EPFL students to get involved in iGEM](https://2021.igem.org/Team%3AEPFL/Communication#event) and by outreach the public through [surveys](https://2021.igem.org/Team%3AEPFL/Communication#survey), [podcasts](https://2021.igem.org/Team%3AEPFL/Communication#podcast) and [lay public articles](https://2021.igem.org/Team%3AEPFL/Communication#dimensions). We believe our results have laid the first foundations upon which a solution can be generated by future investigators to prevent copper pollution in wine and tea growing areas and preserve both the productivity and beauty of these regions including in [the world heritage Lavaux wine growing site](https://whc.unesco.org/en/list/1243/) immediately adjacent to our institute.

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