iGEM 2020 — Team EPFL

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# Team:EPFL/Notebook

[iGEM EPFL 2020](https://2020.igem.org/Team%3AEPFL)

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* [Project Description](https://2020.igem.org/Team%3AEPFL/Description)
* Results

  [Proposed implementation](https://2020.igem.org/Team%3AEPFL/Implementation)
  [Engineering Success](https://2020.igem.org/Team%3AEPFL/Engineering)
  [Proof of Concept](https://2020.igem.org/Team%3AEPFL/Proof_Of_Concept)
  [Contribution](https://2020.igem.org/Team%3AEPFL/Contribution)
* [Integrated Human Practices](https://2020.igem.org/Team%3AEPFL/Human_Practices)
* Synthetic Biology

  [Overview](https://2020.igem.org/Team%3AEPFL/Overview)
  [Deletion Strains](https://2020.igem.org/Team%3AEPFL/Deletion_Strains)
  [Reporter Strains](https://2020.igem.org/Team%3AEPFL/Reporter_Strains)
  [Protocols](https://2020.igem.org/Team%3AEPFL/Experiments)
  [Notebook](https://2020.igem.org/Team%3AEPFL/Notebook)
  [Parts](https://2020.igem.org/Team%3AEPFL/Parts)
* [Hardware](https://2020.igem.org/Team%3AEPFL/Hardware)
* People

  [Team](https://2020.igem.org/Team%3AEPFL/Team)
  [Attributions](https://2020.igem.org/Team%3AEPFL/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AEPFL/Collaborations)
* [Judging Form](https://igem.org/2020_Judging_Form?team=EPFL)
* [Medals&Awards](https://2020.igem.org/Team%3AEPFL/Awards)
* [Safety](https://2020.igem.org/Team%3AEPFL/Safety)
* [Survey](https://2020.igem.org/Team%3AEPFL/Survey)

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## Espress'EAU - Notebook

[Download Notebook](https://static.igem.org/mediawiki/2020/5/53/T--EPFL--Notebook-final.pdf)

Special thanks to our sponsors!

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@EPFL iGEM 2020

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# Team:EPFL/Engineering

[iGEM EPFL 2020](https://2020.igem.org/Team%3AEPFL)

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![](https://static.igem.org/mediawiki/2020/d/d9/T--EPFL--navbar-toggler.png)

* [Project Description](https://2020.igem.org/Team%3AEPFL/Description)
* Results

  [Proposed implementation](https://2020.igem.org/Team%3AEPFL/Implementation)
  [Engineering Success](https://2020.igem.org/Team%3AEPFL/Engineering)
  [Proof of Concept](https://2020.igem.org/Team%3AEPFL/Proof_Of_Concept)
  [Contribution](https://2020.igem.org/Team%3AEPFL/Contribution)
* [Integrated Human Practices](https://2020.igem.org/Team%3AEPFL/Human_Practices)
* Synthetic Biology

  [Overview](https://2020.igem.org/Team%3AEPFL/Overview)
  [Deletion Strains](https://2020.igem.org/Team%3AEPFL/Deletion_Strains)
  [Reporter Strains](https://2020.igem.org/Team%3AEPFL/Reporter_Strains)
  [Protocols](https://2020.igem.org/Team%3AEPFL/Experiments)
  [Notebook](https://2020.igem.org/Team%3AEPFL/Notebook)
  [Parts](https://2020.igem.org/Team%3AEPFL/Parts)
* [Hardware](https://2020.igem.org/Team%3AEPFL/Hardware)
* People

  [Team](https://2020.igem.org/Team%3AEPFL/Team)
  [Attributions](https://2020.igem.org/Team%3AEPFL/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AEPFL/Collaborations)
* [Judging Form](https://igem.org/2020_Judging_Form?team=EPFL)
* [Medals&Awards](https://2020.igem.org/Team%3AEPFL/Awards)
* [Safety](https://2020.igem.org/Team%3AEPFL/Safety)
* [Survey](https://2020.igem.org/Team%3AEPFL/Survey)

![](https://static.igem.org/mediawiki/2020/7/75/T--EPFL--capsule--gif.gif)

* [Synthetic Biology](#synbio)
* [Reporter Strains](#reporter)
* [Cassette assembly](#assembly)
* [Results ans Discussion](#results)
* [Hardware](#hardware)

## Espress'EAU - Engineering Success

### Synthetic Biology

#### Reporter strains

Our aim is to build a yeast strain that generates a signal in the presence of pesticides in water. In order to accomplish this, we leverage the stress response pathway of yeast. We hypothesize that the latter should be activated by the pesticides, as yeast has previously been shown to be sensitive towards pesticides[1,2,3](#ihpref1). By combining the promoter region of stress induced genes with a reporter gene, we expect the reporter to be activated, when the yeast is grown in contaminated water, and thus, generate a signal. Here, we will describe the strategy we used to successfully synthesize these reporter strains. For more results obtained with these strains, please visit [Reporter Strains](https://2020.igem.org/Team%3AEPFL/Reporter_Strains).

After studying the stress response of yeast, we selected five genes, which are known to be activated when the yeast cell is exposed to stress: HSP12[4](#ihpref4), TRX2[5](#ihpref5), GLR1[6](#ihpref6), YCF1[7](#ihpref7), GSH1[8](#ihpref8). We therefore decided to engineer five yeast reporter strains, one for each stress response gene.

This engineering process comprises two main steps:

1. Assembling the cassette that we want to integrate into the yeast genome
2. Cassette integration (yeast transformation)

#### Cassette assembly

As mentioned above, we want to integrate a promoter and a reporter gene into the yeast genome. However, other parts are also required in order to optimize the integration and expression of the reporter. Multiple parts can be assembled together simultaneously using the Golden Gate assembly method. But, as the number of parts that are being assembled increases, the probability of them assembling in the correct order and directionality decreases. Our initial cassette design consisted of 8 separate parts that are based on the yeast toolkit described by Lee et al. 2015[9](#ihpref9).

![Figure 1](https://static.igem.org/mediawiki/2020/5/58/T--EPFL--reporter.png)

Figure 1: The eight part types of our cassette design.

After running the assembly experiment several times and not obtaining the desired results, we decided to modify our experimental design. Instead of trying to assemble the 8 parts at once, we decided to start by assembling 6 parts and then add the remaining 2 parts in a second assembly step. By decomposing this process into two assembly steps, we effectively reduce the number of parts concurrently coming together, and thus increase the probability of a successful assembly. By following this alternative method, we obtained our desired cassettes.

#### Cloning strategy

The next step is to integrate the cassettes into the yeast genome. An efficient way to achieve this is by using CRISPR/Cas9[10](#ihpref10). By transforming the Cas9 backbone along with the sgRNA and the linearized integration cassette into the yeast cells, the Cas9-sgRNA complex will be synthesized and create a double strand break at the desired locus. The yeast’s homology repair (HR) mechanism then repairs this break while introducing the given cassette at the break site.

![Figure 2](https://static.igem.org/mediawiki/2020/2/20/T--EPFL--ReporterStrains-yeast_integration2.png)

Figure 2: The linearized sgRNA has 500bp exact homology arms that are used to gap-repair with the Cas9 backbone in yeast, thereby forming a stable expression vector. Cas9 will then induce double strand breaks in the yeast genome.

#### Results and Discussion

To verify our cassette assembly, we transform the plasmids into E.coli to amplify them and perform a colony PCR. When running the PCR products on a gel (Figure 3), we observe bands at the expected sizes indicating that our five integration cassettes were assembled successfully. The cassettes were then sequenced for further validation.

![Figure 3](https://static.igem.org/mediawiki/2020/7/73/T--EPFL--ReporterStrains-Results-cPCR-EColi.png)

Figure 3: Colony PCR of E.coli colonies containing our cassettes after the first assembly step (6-part assembly). The correct cassette should yield a band with a size of 2470bp while the backbone should yield a band with a length of 1612bp. The screening process yielded several colonies that produced bands of the correct size (highlighted in green) for each of the five cassettes that we constructed.

To confirm the successful integration of the cassette, we do a selection step against the selection marker (URA3) which is found in the Cas9 backbone, and perform a colony PCR on the transformed yeast colonies. In Figure 4 we observe bands at the expected sizes for four of our strains indicating that the cassettes successfully integrated the yeast genome. The data for the GSH1 cassette integration is missing.

![Figure 4](
https://static.igem.org/mediawiki/2020/f/f1/T--EPFL--ReporterStrains-Results-cPCR-yeast.png)

Figure 4: Colony PCR of four yeast strains transformed with the final integration cassette. The primers used for this were located in the Lys2 homology arm region that was added during the second assembly. Bands of the correct sizes (highlighted in green) can be observed for each strain.

These strains were then sequenced for further validation. The following sequencing results showcase the successful integration of the cassettes into the yeast genome.

![](https://static.igem.org/mediawiki/2020/0/0f/T--EPFL--hsp.png)

![](https://static.igem.org/mediawiki/2020/8/82/T--EPFL--trx.png)

![](https://static.igem.org/mediawiki/2020/7/7b/T--EPFL--glr.png)

![](https://static.igem.org/mediawiki/2020/8/84/T--EPFL--ycf.png)

Figure 5: Sequencing alignments for the four transformed yeast strains. The forward and reverse sequencing primers are aligned with the yeast genome. We can see that the promoters along with the reporter genes and the terminators have been integrated into the yeast genome.

### Hardware

When imagining our project, we passed through several different hardware ideas. First, we planned to have a continuous monitoring device for water quality based on a bioreactor. Then, after different discussions with our stakeholders which challenged our ideas, we thought that it would be more useful to have a punctual measurement and adapted the project to the need of non-trained users. For this, a small device was conceived to enable the users to measure yeast growth along with a fluorescent signal in case of a stressing environment.

To test our components, first a calibration curve was also done using our optical density detector (based on a light-to-frequency converter) and demonstrated a linear relation between the frequency that was recorded by our detector and on a commercial spectrophotometer. This shows that the device that our Hardware Team built was successful and is functional to record yeast growth using the capsules that we designed.

![Figure 2](https://static.igem.org/mediawiki/2020/3/3f/T--EPFL--linear.png)

Figure 6: Linear relation between the OD600 measured with 11 standard samples in a spectrophotometer (x axis) and the transmitted light measured by our device (y axis).

The fluorescence sensor was also tested. As we aimed to detect a red fluorescent signal (expressed by mScarlet-I protein), the fluorescence at different wavelengths (green, red and blue) was measured. An inducible strain that can be activated by -estradiol was used as a positive control as it gives a red fluorescent signal when activated. The graph below shows a remarkable increase in the detected signal when measuring this inducible strain compared to the negative control (water) and compared to the blue and green wavelength.

![Figure 2](https://static.igem.org/mediawiki/2020/3/3d/T--EPFL--fluo.png)

Figure 7: Fluorescence measurements with inducible strain for 3 different wavelengths.

Even though none of the components are very complex, we consider the design and implementation of the device an engineering success: the prototype built runs smoothly for multiple hours (16+) with no issues, it is very compact, and every component works well in parallel. The [user feedback](https://2020.igem.org/Team%3AEPFL/Human_Practices#timeline) that we got the 23rd and 27th of October was as well very positive showing that the device is simple to use. In addition to being functional, it is close to be usable by the interested users.

### References

1. Braconi D, Bernardini G, Santucci A. Saccharomyces cerevisiae as a model in ecotoxicological studies: A post-genomics perspective. Journal of proteomics. 2016 Mar 30;137:19-34.
2. Lal S, Lal R. Bioaccumulation, metabolism, and effects of DDT, fenitrothion, and chlorpyrifos onSaccharomyces cerevisiae. Archives of environmental contamination and toxicology. 1987 Nov 1;16(6):753-7.
3. Esteve K, Poupot C, Dabert P, Mietton-Peuchot M, Milisic V. A Saccharomyces cerevisiae-based bioassay for assessing pesticide toxicity. Journal of industrial microbiology & biotechnology. 2009 Dec 1;36(12):1529-34.
4. Varela, J. C., Praekelt, U. M., Meacock, P. A., Planta, R. J. & Mager, W. H. The Saccharomyces cerevisiae HSP12 gene is activated by the high-osmolarity glycerol pathway and negatively regulated by protein kinase A. Molecular and Cellular Biology 15, 6232–6245 (1995).
5. Kuge, S. & Jones, N. YAP1 dependent activation of TRX2 is essential for the response of Saccharomyces cerevisiae to oxidative stress by hydroperoxides. The EMBO Journal 13, 655–664 (1994).
6. Grant, C. M., Collinson, L. P., Roe, J. H. & Dawes, I. W. Yeast glutathione reductase is required for protection against oxidative stress and is a target gene for yAP-1 transcriptional regulation. Mol. Microbiol. 21, 171–179 (1996).
7. Bhuiyan, M. S. U. et al. Overexpression of a yeast cadmium factor 1 (YCF1) enhances heavy metal tolerance and accumulation in Brassica juncea. Plant Cell Tiss Organ Cult 105, 85–91 (2011).
8. Wu, A. L. & Moye-Rowley, W. S. GSH1, which encodes gamma-glutamylcysteine synthetase, is a target gene for yAP-1 transcriptional regulation. Molecular and Cellular Biology 14, 5832–5839 (1994).
9. Lee ME, DeLoache WC, Cervantes B, Dueber JE. A highly characterized yeast toolkit for modular, multipart assembly. ACS synthetic biology. 2015 Sep 18;4(9):975-86.
10. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research. 2013 Apr 1;41(7):4336-43.

Special thanks to our sponsors!

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@EPFL iGEM 2020

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# Team:EPFL/Experiments

[iGEM EPFL 2020](https://2020.igem.org/Team%3AEPFL)

[![](https://static.igem.org/mediawiki/2020/1/17/T--EPFL--Project_Logo_small.png)](https://2020.igem.org/Team%3AEPFL)

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* [Project Description](https://2020.igem.org/Team%3AEPFL/Description)
* Results

  [Proposed implementation](https://2020.igem.org/Team%3AEPFL/Implementation)
  [Engineering Success](https://2020.igem.org/Team%3AEPFL/Engineering)
  [Proof of Concept](https://2020.igem.org/Team%3AEPFL/Proof_Of_Concept)
  [Contribution](https://2020.igem.org/Team%3AEPFL/Contribution)
* [Integrated Human Practices](https://2020.igem.org/Team%3AEPFL/Human_Practices)
* Synthetic Biology

  [Overview](https://2020.igem.org/Team%3AEPFL/Overview)
  [Deletion Strains](https://2020.igem.org/Team%3AEPFL/Deletion_Strains)
  [Reporter Strains](https://2020.igem.org/Team%3AEPFL/Reporter_Strains)
  [Protocols](https://2020.igem.org/Team%3AEPFL/Experiments)
  [Notebook](https://2020.igem.org/Team%3AEPFL/Notebook)
  [Parts](https://2020.igem.org/Team%3AEPFL/Parts)
* [Hardware](https://2020.igem.org/Team%3AEPFL/Hardware)
* People

  [Team](https://2020.igem.org/Team%3AEPFL/Team)
  [Attributions](https://2020.igem.org/Team%3AEPFL/Attributions)
  [Collaborations](https://2020.igem.org/Team%3AEPFL/Collaborations)
* [Judging Form](https://igem.org/2020_Judging_Form?team=EPFL)
* [Medals&Awards](https://2020.igem.org/Team%3AEPFL/Awards)
* [Safety](https://2020.igem.org/Team%3AEPFL/Safety)
* [Survey](https://2020.igem.org/Team%3AEPFL/Survey)

![](https://static.igem.org/mediawiki/2020/7/75/T--EPFL--capsule--gif.gif)

* [Protocols - General](#general)
* [Protocols - Yeast](#yeast)
* [Protocols - Bacteria](#bacteria)

## Espress'EAU - Experiments

# Protocols

### General

##### Agarose Gel Preparation

open

##### Materials

* 50ml TAE buffer
* 0.5g Agarose
* 3μl Gel Stain

##### Procedure

1. mix TAE and agarose
2. microwave for 1.5 minutes
3. add gel stain
4. pour into gel tray and insert combs

##### cPCR validation

open

#### cPCR validation

##### Materials

* Phire Plant Direct PCR Master Mix (ThermoFisher)
* Forward/Reverse primer (100µM)
* H2O
* Transformed Colonies

##### Procedure

1. Select and mark 10 colonies on the plate of interest
2. Prepare master mix according to the table below, and distribute into labeled PCR-tubes

Master mix for one PCR reaction

|  | **Volume (µl)** |
| H2O | 12.25 |
| Phire Plant Direct PCR Master Mix | 12.5 |
| Forward primer | 0.125 |
| Reverse primer | 0.125 |
| Total | 25 |

3. Touch colonies and inoculate in PCR-tubes
4. Run PCR (33 cycles)

cPCR reaction cycle parameters. Must be run 33 times.

| **Temperature (ºC)** | **Time (minutes)** |
| 98 | 5:00 |
| 98 | 0:05 |
| 62 | 0:05 |
| 72 | 1:30 |
| 72 | 1:00 |
| 4 | ∞ |

5. Prepare a 1% agarose gel
6. Load PCR reactions on gel and run the gel for 25 minutes at 120 Volts
7. Take a UV image of the gel

##### Six parts assembly

open

##### Materials

**Master mix for BsaI assembly of six parts:**

| **Reagent** | **Concentration (ng/µl)** | **Volume(µl)** |
| --- | --- | --- |
| DNA Part 1 | 83 | 0.7 |
| DNA Part 2 | 100 | 1.5 |
| DNA Part 3 | 70.8 | 1.41 |
| DNA Part 4 | 67.5 | 1.48 |
| DNA Part 5 | 84.5 | 1.18 |
| DNA Part 6 | 110 | 0.91 |
| 10x T4 ligase buffer |  | 2 |
| T4 ligase (NEB) |  | 2 |
| BsaI |  | 1 |
| dH2O |  | 7.81 |
| **TOTAL:** |  | 20 |

- DNA part 1: assembly connector [ConLS]
- DNA part 2: 5 promoters [HSP12pr], [TRX2pr], [YCF1pr], [GSH1pr], [GLR1pr]
- DNA Part 3: coding sequence [yomScarlet-i]
- DNA Part 4: Terminator [tADH1]
- DNA Part 5: Assembly connector [ConRE]
- DNA Part 6: E.coli marker and origin [AmpR-ColE1]

##### Procedure

**Mix:**

1. add H2O
2. add 10x T4 ligase buffer
3. add BsaI
4. add T4 ligase (NEB)
5. add differents parts of DNA

**Thermal cycling :**

6. Cycle with the following parameters:
   [42°C, 2 min.; 16°C 5 min.] x25
   60°C, 10 min.
   80°C, 20 min.
   4°C, ∞

##### Two parts assembly

open

##### Materials

| **Reagent** | **Concentration (ng/µl)** | **Volume(µl)** |
| --- | --- | --- |
| DNA Part 1 | 100 | 1 |
| DNA Part 2 | 200 | 0.5 |
| 10x T4 ligase buffer |  | 2 |
| T4 ligase (NEB) |  | 1 |
| BsaI |  | 1 |
| dH2O |  | 14.5 |
| **TOTAL:** |  | 20 |

- DNA part 1: promoter cassette
- DNA part 2: Lys2-GFP dropout multi-gene backbone

##### Procedure

**Mix:**

1. add H2O
2. add 10x T4 ligase buffer
3. add BsmBI
4. add T4 ligase (NEB)
5. add differents parts of DNA

**Thermal cycling :**

6. Cycle with the following parameters:
   [42°C, 2 min.; 16°C 5 min.] x25
   60°C, 10 min.
   80°C, 20 min.
   4°C, ∞

### Yeast

##### Yeast Transformation

open

The following protocol uses the LiAc/SS carrier DNA/PEG method for yeast transformation and is based on Gietz et al. (2007)[1](#expref1).

##### Materials

* Shaking incubator at 30°C
* Stationary incubator at 30°C
* Water bath at 42°C
* Heat block at 105°C
* SC-URA plate
* 5-FOA plate
* Centrifuge
* YPD medium
* Lithium acetate dihydrate
* PEG 3350
* Integration cassette

##### Procedure

**Day 0**

1. Pick colony from plate into 5 ml growth media for overnight growth in 30°C shaker

**Day 1**

2. Turn on water bath to 42 °C.
3. Prepare an ice-water bath.
4. Set dry-block to 105 °C.
5. Place a SC -ura plate into the 30 °C incubator.

*Cells preparation*

6. Back dilute over night culture to OD 0.175 in 5 ml YPD.
7. Grow up yeast to OD600 ~0.75 at 30°C. Takes 4-5 hours for WT in YPD.
8. Pellet at 2000 rpm for 10 min in large centrifuge.
9. Prepare lithium acetate transformation while harvesting cells (see table).

Lithium Acetate mix for one transformation

|  | **Volume (µl)** |
| PEG-3350 (50% w/v) | 260 |
| LiOAc 1.0 M | 36 |
| ssDNA (10 mg/mL) | 0.125 |
| **Total** | 306 |

Integration DNA prep

|  | **Volume (µl)** |
| Integration cassette | 20 |
| Cas9 (100ng) | 5 |
| Guide RNA (200 ng of each gRNA) | 4 |
| H2O | 25 |
| **Total** | 54 |

10. Wash with 0.5x volume H2O and repeat step 8.
11. Wash with 0.5x volume 100 mM LiOAc and repeat step 8.
12. Final suspension in 0.01x volume 100 mM LiOAc (100 μL per transformation - approx 300 μl from 15 mL culture).
13. Pellet at 8000 rpm for 1 min at 20°C on small tabletop centrifuge.

*Heat shock*

14. Resuspend in the lithium acetate transformation mix. I suggest resuspending with the plasmid DNA and ddH2O (54 μL) first, then mixing with the LiOAc+PEG+ssDNA mixture (306 μL) afterwards, as it is too viscous to easily disrupt the pellet.
15. Incubate at 30°C for 30 min.
16. Incubate for another 17 min at 42°C in water bath (or heat block).
17. Centrifuge at 8000 rpm for 2 min and remove supernatant.
18. Depending on the selection marker:
    a. Prototrophic/auxotrophy gene: Resuspend in 200 μL of H2O.
    b. Eukaryotic antibiotic: Recover in 1.0 mL YPD for 2-3 hrs at 30°C.
19. Plate desired volume on plates. Typically do half.

**Day 2**

20. If there are colonies streak them on a 5-FOA plate for curing.

##### Pesticide Assay

open

#### Pesticide Assay

The pesticide assay was carried out using a selection of the most frequently detected pesticides in groundwater (Based on the 2016 NAQUA Swiss groundwater monitoring report[2](#expref2)). Those pesticides were diluted over a range of concentrations to be tested (1000µl/L - 0.1µl/L). Yeast cells were grown with the presence of pesticides at 30°C.

##### Materials

* Pesticides
* Yeast strains (overnight cultures)
* YPD medium
* SC medium
* 96-well plate
* Breathable membrane

##### Procedure

1. Serially dilute the pesticides in deionized water to the desired concentrations
2. Dilute yeast overnight cultures to 0.05 OD600 in YPD medium (only growth measurements), or to 0.3 OD600 in SC medium (fluorescence + growth measurements).
3. Pipette 99 µl of the cultures into the wells of the 96-well plate and add 1 µl of the pesticide dilution
4. Cover the plate with a breathable membrane to prevent evaporation
5. Insert in the plate reader and run for 18 to 24 hours while measuring the OD600 and the fluorescence (excitation wavelength = 570 nm, emission wavelength = 593 nm) over this period of time.

### Bacteria

##### Bacterial Transformation

open

#### Bacterial Transformation

##### Materials

* Shaking incubator at 37°C
* Stationary incubator at 37°C
* Water bath at 42°C
* Ice bucket filled with ice
* Microcentrifuge tubes
* Glass beads
* LB agar plate (with appropriate antibiotic)
* SOC medium
* LB medium
* Competent cells
* DNA (to transform)

##### Procedure

1. Add total volume of master mix (20.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0mL tube.
2. Incubate on ice for 5 min. heat shock at 42°C for exactly 45 sec., immediately place on ice.
3. Add 950 μL sterile SOC medium.
4. Grow with shaking at 37°C for 30 min.
5. Pellet the cells at 4000g speed in a microcentrifuge for 3 min. at room temp.
6. Discard the supernatant. Resuspend the cells in 100 μL LB.
7. make a 100µl 10x dilution.
8. Plate the original and diluted cells on pre-warmed LB agar (with antibiotic) with sterile glass beads.
9. Grow overnight at 37°C.

##### DNA Extraction

open

##### Materials

* Bacterial overnight cultures
* P1 Buffer (Qiagen)
* P2 Buffer (Qiagen)
* N3 Buffer (Qiagen)
* PE Buffer (Qiagen)
* Microcentrifuge tubes
* QIAprep 2.0 spin column

##### Procedure

1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting. Centrifuge for 30–60 s and discard the flow-through
7. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through.
8. Centrifuge for 1 min to remove residual wash buffer.
9. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

### References

1. Gietz RD, Schiestl RH. Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature protocols. 2007 Jan;2(1):35.
2. OFEV (éd.) 2019 : État et évolution des eaux souterraines en Suisse. Résultats de l’Observation nationale des eaux souterraines NAQUA, état 2016. Office fédéral de l’environnement, Berne. État de l’environnement n o 1901 : 144 p.

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# Team:EPFL/Results

iGEM EPFL 2020

* Home (current)
* Context
* Wet Lab
* Hardware
* Human Practices

# Results

You can describe the results of your project and your future plans here.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)