iGEM 2019 — Team Pasteur_Paris

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# Team:Pasteur Paris/Notebook

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- Education
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- Team
- [Team members](https://2019.igem.org/Team%3APasteur_Paris/Team)
  [Collaborations](https://2019.igem.org/Team%3APasteur_Paris/Collaborations)
  [Attributions](https://2019.igem.org/Team%3APasteur_Paris/Attributions)
- Sciences
- [Notebook](https://2019.igem.org/Team%3APasteur_Paris/Notebook)
  [Results](https://2019.igem.org/Team%3APasteur_Paris/Collaborations)
  [Attributions](https://2019.igem.org/Team%3APasteur_Paris/Attributions)
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  Us](https://2019.igem.org/Team%3APasteur_Paris/About_Us)

[[SAFETY](https://2019.igem.org/Team%3APasteur_Paris/Safety)](https://2019.igem.org/Team%3APasteur_Paris/Safety)

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HUMAN  PRACTICES

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  [Collaboration](https://2019.igem.org/Team%3APasteur_Paris/Collaborations) [Law & Ethics](https://2019.igem.org/Team%3APasteur_Paris/law_ethics)

PROJECT

- [Description](https://2019.igem.org/Team%3APasteur_Paris/Description)
  [Entrepreneurship](https://2019.igem.org/Team%3APasteur_Paris/Entrepreneurship)
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  [Improve](https://2019.igem.org/Team%3APasteur_Paris/Improve)

[[HOME](https://2019.igem.org/Team%3APasteur_Paris)](https://2019.igem.org/Team%3APasteur_Paris)

TEAM

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  [Attributions](https://2019.igem.org/Team%3APasteur_Paris/Attributions)

Picto Sciences

Sciences

- [Notebook](https://2019.igem.org/Team%3APasteur_Paris/Notebook)
  [Results](https://2019.igem.org/Team%3APasteur_Paris/Results)

[1

AWARDS

- [Medals](https://2019.igem.org/Team%3APasteur_Paris/Medals)
  [Demonstrate](https://2019.igem.org/Team%3APasteur_Paris/Demonstrate)
  [Special Prizes](https://2019.igem.org/Team%3APasteur_Paris/Special_prizes)](https://2019.igem.org/Team%3APasteur_Paris/Giant_Jamboree)

[iGEMPASTEURPARIS2019

[ABOUT  US](https://2019.igem.org/Team%3APasteur_Paris/About_Us)](https://2019.igem.org/Team%3APasteur_Paris/About_Us)

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July Week 2  :  09.09.19  to
            09.13.19

09.09.19

Bacteria culture - E. coli containing the plasmid
          PET28:GFP

In order to extract the plasmid PET28:GFP, E. coli
          was cultured on LB petri dishes containing kanamycin at 50 µg/mL. The
          cultures were incubated all night at 37°C.

Bacteria culture - E. faecium and S. aureus

E. faecium and S. aureus were grown until
          the OD reached between 0.5 and 0.6.

Digestion - EFr2 & SAr2

PCR samples from the Round 2 of SELEX were digested using
          lambda exonuclease to free the top strand. A mix was performed in order to scale-up this step
          and get enough digested material.

1. Prepare the mix as
          follows

|  |  |  |
| --- | --- | --- |
|  | 50µL reaction | Mix |
| DNA (PCR11) | 4 µg | 120 µg |
| Lambda exonuclease buffer (10X) | 5µL | µL |
| Lambda exonuclease | 1µL | 30µL |
| Nuclease-free H²0 | QSP 50 µL | QSP 1.5 mL |

2. Incubate samples for 30
          min at 37°C in a water-bath

3. Stop the reaction by
          adding EDTA to 10 mM

4. Heat at 75°C for 10
          minutes to inactivate the enzyme

Samples concentrations were checked using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Dig-Round 2 - SA | 32.6 | 1.91 | 0.27 |
| Dig - Round 2- EF | 20 | 1.92 | 0.25 |

Phenol/chloroform extraction with isopropanol - Digested
          products EFr2 & SAr2

Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| EFr2 sample 1 | 46.7 | 1.47 | 1.49 |
| EFr2 sample 2 | 42.1 | 1.48 | 1.52 |
| SAr2 sample 1 | 29.1 | 1.66 | 1.63 |
| SAr2 sample 2 | 37.3 | 1.52 | 1.55 |

Interpretation

A total quantity of 692 pmol was obtained for E.
            faecium in the Round 2, and 516 pmol for S. aureus

SELEX Round 3

The first round of SELEX was performed with 1h45 hours of
          incubation. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| SAr3 | 33.6 | 2.19 | 1.45 |
| EFr3 | 42.9 | 2.3 | 1.76 |

PCR 11 - Amplification of EFr3 and SAr3

This PCR was performed to amplified the DNA after the
          Round 3 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and held
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| PCR 11 - Round 3 - SA | 1086.6 | 1.67 | 1.20 |
| PCR 11 - Round 3 - EF | 1496 | 1.72 | 1.38 |

09.10.19

Bacteria culture - E. coli containing the plasmid
          PET28:GFP

After an overnight growth, a colony of bacteria from the
          09.09 was added into an erlenmeyer containing LB media. Then, the
          erlenmeyer was put at 37°C all night under agitation at 140 rpm.

Bacteria culture - E. faecium and S. aureus

E. faecium and S. aureus were grown until
          the OD reached between 0.5 and 0.6

Digestion - EFr3 & SAr3

PCR samples from the Round 3 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| Dig - Round 3 - SA | 36.8 | 1.92 | 0.27 |
| Dig - Round 3 - EF | 45.3 | 1.81 | 0.23 |

Phenol/chloroform extraction with isopropanol and
          Glycogen - Digested products EFr3 & SAr3

Once digested, samples were purified after digestion
          using a phenol/chloroform extraction. Glycogen for a final 0.5 μg/μL
          concentration was added in order to maximise the amount of DNA
          recovered and avoid pipetting the pellet. They were analysed with a
          Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| EFr3 sample 1 | 32.1 | 1.56 | 1.86 |
| EFr3 sample 2 | 22.7 | 1.48 | 1.89 |
| SAr3 sample 1 | 27.1 | 1.52 | 1 |
| SAr3 sample 2 | 65.4 | 1.50 | 1.24 |

Interpretation

A total quantity of 856 pmol was obtained for E.
            faecium in the Round 3, and 516 pmol for S. aureus.
          Because the purity of SAr3 sample 1 was really low, we only used SAr3
          sample 2 for the following steps.

09.11.19

Midiprep - E. coli containing the plasmid
          PET28:GFP

The liquid culture of E. coli from the 10.09 was
          centrifuged 10 minutes at 3,500 x g and the supernatant was removed.
          Then, we used a midiprep kit from Qiagen to extract the plasmid from E.
            coli. For that, the pellet was resuspended with 4 mL of P1
          containing RNAse. Then, 4 mL of P2 was added and we waited 5 minutes
          at RT. After that, 4 mL of P3 was added and the solution was mixed.
          The solution was loaded on a column and the filtrate was recovered.
          After, 4 mL of QBT Qiagen lip was added on the column and then the
          filtrate was loaded on the column. After filtration, the column was
          washed twice with QC buffer. In the filtrate, 5 mL of QF buffer and
          3.5 mL of isopropanol were added. The solution was centrifuged during
          30 minutes at 15000 x g. The supernatant was removed and the pellet
          was washed with 1 mL of ethanol 70% and centrifuged again 30 minutes
          at 15,000 x g. We removed all the supernatant and let the pellet dry.
          Then, 10 µL of TE buffer was added and the sample was vortexed.
          Finally, the sample was stored at -20°C. The sample was checked with a
          Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| pET28:GFP | 212.6 | 1.88 | 2.24 |

09.12.19

Bacteria culture - E. faecium and S. aureus

E. faecium and S. aureus were grown until
          the OD reached between 0.5 and 0.6

SELEX Round 4

The Round 4 of SELEX was performed by incubating for
          1h30. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| SAr4 | 37.1 | 2.02 | 1.40 |
| EFr4 | 21.2 | 2.25 | 1.55 |

PCR 12 - Amplification of EFr4 and SAr4

This PCR was performed to amplified the DNA after the
          Round 4 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and held
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| PCR 12 - Round 4 - SA | 1373.9 | 1.62 | 1.05 |
| PCR 12 - Round 4 - EF | 1314.2 | 1.62 | 1.06 |

09.13.19

Bacteria culture - E. faecium and S. aureus

E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6

Digestion - EFr4 & SAr4

PCR samples from the Round 4 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| Dig - Round 4 - SA sample 1 | 59.70 | 1.79 | 0.27 |
| Dig - Round 4 - SA sample 2 | 59.20 | 1.80 | 0.25 |
| Dig - Round 4 - EF sample 1 | 57.40 | 1.80 | 0.24 |
| Dig - Round 4 - EF sample 2 | 58.80 | 1.79 | 0.24 |

Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr4 & SAr4

Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| EFr4 sample 1 | 49.60 | 1.46 | 1.17 |
| EFr4 sample 2 | 34.10 | 1.51 | 1.13 |
| SAr4 sample 1 | 39.60 | 1.54 | 1.15 |
| SAr4 sample 2 | 18.70 | 1.54 | 1.33 |

Interpretation

A total quantity of 650 pmol was obtained for E.
            faecium in the Round 4, and 684 pmol for S. aureus.

SELEX Round 5

The Round 5 of SELEX was performed by incubating for
          1h15. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| SAr5 | 30.30 | 1.70 | 1.03 |
| EFr5 | 33.70 | 2.21 | 1.74 |

PCR 13 - Amplification of EFr5 and SAr5

This PCR was performed to amplified the DNA after the
          Round 5 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| PCR 13 - Round 5 - SA | 1228.20 | 1.59 | 0.99 |
| PCR 13 - Round 5 - EF | 1334.80 | 1.62 | 1.02 |

July Week 4   :   07/22   to   07/28

07.22.19

Resuspension of oligonucleotides  :

DNA library and primers were produced by Eurofins Genomics. Once received, they were resuspended according to the manufacturer’s instructions. Stock solutions of 100 µM were made by diluting the lyophilized oligonucleotides with a 10 mM Tris- 0.1 mM EDTA pH 8.0 solution (TE8.1). Concentrations were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Samples | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| DNA library | 2217.65 | 1.89 | 1.56 |
| Reverse primer | 8810.90 | 1.95 | 1.58 |

PCR 1  -  Amplification of the DNA library

In order to start the SELEX, we need to amplify our DNA library in order to get enough material to start with. We tried several PCR protocols to optimize the amplification. This test was performed in order to know if the magnesium concentration could impact the quality of the amplification, and different annealing temperatures from 44 to 54°C were tested. A mix of Taq DNA polymerase and phusion DNA polymerase was used.

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | Volume needed (μL) | | |  |  |
| Components | Final concentration | Mix 1 | Mix 2 | Mix 3 |
| Phusion HF buffer 5X | 1X | 50 | 50 | 50 |
| 25 mM dNTPs | 0.20  mM | 2 | 2 | 2 |
| Forward primer | 0.50  μM | 9.75 | 9.75 | 9.75 |
| Reverse primer | 0.50  μM | 10.10 | 10.10 | 10.10 |
| 25 mM Mg2+ | 2.5  mM | 25.0 | 50.0 | 75.0 |
| DNA library | 10.0  ng | 22.5 | 22.5 | 22.5 |
| Taq DNA polymerase | 0.10  U/μL | 0.02 | 0.02 | 0.02 |
| Phusion DNA polymerase | 1    U/μL | 0.50 | 0.50 | 0.50 |
| Nuclease-free water | / | 130.13 | 105.13 | 80.13 |
| Total volume | / | 250 | 250 | 250 |

Rq: Enzymes were added at the end as hot-start PCR protocols can optimize the yield of the desired product while limiting the likelihood of nonspecific amplification

PCR plate plan

|  |  |  |  |
| --- | --- | --- | --- |
| A (54°C) | 1.1 | 2.1 | 3.1 |
| B | / | / | / |
| C (51.9°C) | 1.2 | 2.2 | 3.2 |
| D | / | / | / |
| E (47.6°C) | 1.3 | 2.3 | 3.3 |
| F | / | / | / |
| G (44.7°C) | 1.4 | 2.4 | 3.4 |
| G | / | / | / |

PCR program used:

-    95°C             5 min

-    95°C             30 s

-    44-54°C      1 min        30 cycles

-    68°C             30 s

-    68°C             5 min

-    15°C             HOLD ALL NIGHT

PCR was run overnight. Samples were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280nm ratio | 260/230nm ratio |
| 1.1 | 465.90 | 1.39 | 0.66 |
| 1.2 | 501.7 | 1.42 | 0.69 |
| 1.3 | 480.4 | 1.43 | 0.68 |
| 1.4 | 469.9 | 1.43 | 0.65 |
| 2.10 | 498.30 | 1.40 | 0.68 |
| 2.20 | 498.70 | 1.43 | 0.70 |
| 2.30 | 532.90 | 1.42 | 0.71 |
| 2.40 | 495.20 | 1.42 | 0.69 |
| 3.10 | 497.80 | 1.43 | 0.70 |
| 3.20 | 512.20 | 1.43 | 0.70 |
| 3.30 | 505.50 | 1.42 | 0.70 |
| 3.40 | 527.20 | 1.41 | 0.70 |

Interpretations :

No special condition seemed better than others.

07.23.19

Gel electrophoresis 1  -  Amplified library

To verify that the samples were all the same with the right size, we performed a gel electrophoresis using a 3% agarose gel. Each band was cut and then kept at -4°C in order to be purified later.

![](https://static.igem.org/mediawiki/2019/9/94/T--Pasteur_Paris--NotbookImg1.jpg)

|  |  |
| --- | --- |
| 1. | Sample 1.1 (54°C, 25  μL, Mg2+) |
| 2. | Sample 1.2 (51.9°C, 25  μL, Mg2+) |
| 3. | Sample 1.3 (47.6°C, 25  μL, Mg2+) |
| 4. | Sample 1.4 (44.7°C, 25  μL, Mg2+) |
| 5. | Sample 2.1 (54°C, 50  μL, Mg2+) |
| 6. | Sample 2.2 (51.9°C, 50  μL, Mg2+) |
| 7. | Sample 2.3 (47.6°C, 50  μL, Mg2+) |
| 8. | Sample 2.4 (44.7°C, 50  μL, Mg2+) |
| 9. | Sample 3.1 (54°C, 75  μL, Mg2+) |
| 10. | Sample 3.2 (51.9°C, 75  μL, Mg2+) |
| 11. | Sample 3.3 (47.6°C, 75  μL, Mg2+) |
| 12. | Sample 3.4 (44.7°C, 75  μL, Mg2+) |

Interpretations

All samples had the same size of 120 bp approximately whereas the library size was 80 bp normally.

Conclusion

PCR amplification resulted in a shift in size of the oligonucleotides because of the double-stranded form.

Cell culture

TO COMPLETE

07.24.19

Growth curves

Bacteria were cultured and their growth was analyzed by establishing a growth curve associated with the number of colonies. Several strains were used:

- E. faecium (ref CIP 103014T)
- P. aeruginosa (ref 104116)
- A. baumanii (ref CIP 79.34T)
- S. aureus (ref CIP 103429)
- S. pneumoniae (ref CIP 104340)
- E. coli (ref 53.126)
- E. enterica (ref CIP 60.62T)

All strains were provided by the CRBIP of the Institut Pasteur.

TO COMPLETE

07.25.19

Purification of PCR products  -  Invitrogen Pure Link Quick Gel extraction kit  -  PCR 1

After PCR amplification, PCR products need to be cleaned up. Gel slices were extracted and purified using Invitrogen Pure Link Quick Gel extraction kit following the manufacturer’s instructions, and stored at -20°C.

![](https://static.igem.org/mediawiki/2019/b/b2/T--Pasteur_Paris--NotebookImg2.png)

END OF THE WEEK

July Week 5   :   07/29
            to   08/04

07.29.19

Gel electrophoresis 2  -  Purified PCR products from gel extraction kit

To verify that the samples were purified after gel extraction, we performed a gel electrophoresis using a 3% agarose gel. We used two ladders, one at 25 bp and the other at 50 bp from Promega.

![](https://static.igem.org/mediawiki/2019/5/54/T--Pasteur_Paris--NotebookImg3.png)

Interpretation

There was no band after purification by gel extraction. So, during the purification step we lost the DNA.

Digestion  -  Amplified library

This step is made to pass from a double-stranded DNA to a single-stranded one.
We performed the digestion using Lambda Exonuclease from New England Biolabs directly on the amplified library (PCR 1) without purifying it beforehand.

1.   Set-up the reaction as follows:

|  |  |
| --- | --- |
| Components | Volume for 50  μL reaction |
| DNA from PCR 1 | 5  μg |
| Lambda Exonuclease Reaction Buffer (10X) | 5 μL  (1X) |
| Lambda Exonuclease | 1 μL  (5 units) |
| Nuclease-free water | up to 50μL |

2.   Incubate at 37°C for 30 minutes.

3.   Stop reaction by adding EDTA to 10 mM.

4.   Heat at 75°C for 10 minutes.

07.31.19

PCR 2  -  Testing different conditions

To try to enhance our PCR protocol, we performed a new PCR using different conditions. These conditions enabled us to determine if the Taq DNA Polymerase has to be activated prior addition, and if both polymerases have to be added at 95°C or not. The PCR was run overnight and held at 4°C.

|  |  |  |  |
| --- | --- | --- | --- |
|  | Volume needed (in μL) | |  |
| Components | Mix 1 | Mix 2 |
| Phusion HF buffer 5X | 500 | 500 |
| 25 mM dNTPs | 20 | 250 |
| Forward primer | 97.5 | 50 |
| Reverse primer | 101 | 50 |
| 25 mM Mg2+ | 250 | 250 |
| DNA library (1/1000 dilution) | 225 | 250 |
| Taq DNA polymerase diluted 1/10 | 5 | 5 |
| Phusion DNA polymerase | 25 | 25 |
| Nuclease-free water | 1276.5 | 1135 |
| Total volume | 2500 | 2500 |

PCR plan

|  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 1A | 2A | 3A | 4A | 5A | 6A | 7A | 8A | 9A | 10A | 11A | 12A |
| B | 1B | 2B | 3B | 4B | 5B | 6B | 7B | 8B | 9B | 10B | 11B | 12B |
| C | 1C | 2C | 3C | 4C | 5C | 6C | 7C | 8C | 9C | 10C | 11C | 12C |
| D | 1D | 2D | 3D | 4D | 5D | 6D | 7D | 8D | 9D | 10D | 11D | 12D |
| E | 1E | 2E | 3E | 4E | 5E | 6E | 7E | 8E | 9E | 10E | 11E | 12E |
| F | 1F | 2F | 3F | 4F | 5F | 6F | 7F | 8F | 9F | 10F | 11F | 12F |
| G | 1G | 2G | 3G | 4G | 5G | 6G | 7G | 8G | 9G | 10G | 11G | 12G |
| H | 1H | 2H | 3H | 4H | 5H | 6H | 7H | 8H | 9H | 10H | 11H | 12H |
|  | Mix 1 | | | | | | Mix 2 | | | | | |

Legend  :

- Condition 1: Taq was activated and added with Phusion in ice
- Condition 2: Taq was not activated and added with Phusion in ice
- Condition 3: Phusion was added in ice
- Condition 4: Taq was activated and added with Phusion at 95°C
- Condition 5: Taq was not activated and added with Phusion at 95°C
- Condition 6: Phusion was added at 95°C
- Condition 7: Taq was activated and added with Phusion in ice
- Condition 8: Taq was not activated and added with Phusion in ice
- Condition 9: Phusion was added in ice
- Condition 10: Taq was activated and added with Phusion at 95°C
- Condition 11: Taq was not activated and added with Phusion at 95°C
- Condition 12: Phusion was added at 95°C

Gel electrophoresis 4  -  PCR 2

A gel containing 3% of agarose was run to determine which condition led to the best results.

![](https://static.igem.org/mediawiki/2019/a/ad/T--Pasteur_Paris--NotebookImg4.png)

Interpretation

Condition 1 provided the best results because the band is the brightest. Moreover, there was almost no difference between the samples containing activated Taq polymerase and when the polymerases were added on ice or in 95°C. Because we obtained the same results when we amplified using both Taq and phusion polymerases and only Phusion polymerase, we decided to only use Phusion polymerase for our PCR.

08.01.19

DNA precipitation  -  Product of phenol/chloroform from 29.07.19

1.   Add 1/10 volume of sodium acetate (3 M, pH 5.2) and 2.5x volume of 100% ethanol

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Sample | Pool 1 Elution 1 | Pool 1 Elution 2 | Pool 2 Elution 1 | Pool 2 Elution 2 |
| Upper aqueous phase recovered | 2000 µL | 430 µL | 1150 µL | 415 µL |
| Sodium acetate 3M to add | 200 µL | 43 µL | 115 µL | 41.5 µL |
| Volume after addition of sodium acetate | 2200 µL | 473 µL | 1265 µL | 456.5 µL |
| Ethanol 100%  to add | 5500 µL | 1182.5 µL | 3162.5 µL | 1141.2 µL |
| Total volume | 7700 µL | 1655.5 µL | 4427.5 µL | 1597.7 µL |

2.   Place the tube at -20°C overnight to precipitate the DNA from the sample. The experiment was continued on 08.02.19

08.2.19

DNA precipitation

1.    Centrifuge the sample at 4°C for 30 minutes at 16,000 × g to pellet the cDNA.
          2.    Carefully remove the supernatant without disturbing the cDNA pellet.
          3.    Add 150 μL of 70% ethanol.
          4.    Centrifuge the sample at 4°C for 15 minutes at 16,000 × g. Carefully remove the supernatant.
          5.    Dry the cDNA pellet at room temperature
          6.    Resuspend the cDNA pellet in 300 μL of TEN (10 mM pH 8.0 Tris-EDTA 0.1 mM-NaCl 50mM) buffer by pipetting up and down

Purification of PCR product  -  illustra MicroSpin G-25 Columns kit from GE Healthcare - PCR 2

Because the purification by gel extraction was not conclusive, we tested a second method of purification using the illustra MicroSpin G-25 Columns kit from GE Healthcare. The experiment was performed according to the manufacturer’s recommendation.

Gel electrophoresis 5  -  Purified products from gel extraction vs. columns

Because the purification by gel extraction was not conclusive, we tested a second method of purification using the illustra MicroSpin G-25 Columns kit from GE Healthcare. The experiment was performed according to the manufacturer’s recommendation.

![](https://static.igem.org/mediawiki/2019/e/e1/T--Pasteur_Paris--NotebookImg5.png)

Interpretation

We only observed the bands from the samples purified with the columns. Moreover, they seemed well purified because there was almost no smear.

PCR 3  -  Testing different conditions (with/without DMSO + new program)

To try to enhance our PCR protocol, we performed a new PCR using two conditions, one with DMSO and the other without . The PCR was run overnight and hold at 4°C.

|  |  |  |  |
| --- | --- | --- | --- |
|  | Volume needed (in μL) | |  |
| Components | Mix 1 (With DMSO) | Mix 2 (Without DMSO) |
| Phusion HF buffer 5X | 50 | 50 |
| 25 mM dNTPs | 5 | 5 |
| Forward primer | 10 | 10 |
| Reverse primer | 10 | 10 |
| DMSO | 7.5 | 0 |
| Template DNA | 22.5 | 22.5 |
| Phusion DNA polymerase | 2.5 | 2.5 |
| Nuclease-free water | 142.5 | 150 |
| Total volume | 250 | 250 |

August Week 1  :  08/05   to  08/11

08.05.19

Purification of PCR products  -  illustra MicroSpin G-25 Columns kit from GE Healthcare  -  PCR 3

Once again, we tried to purify our PCR products using different methods in order to compare them. This experiment was performed according to the manufacturer’s protocol.

Purification of PCR products  -  PCR clean-up kit from Quiagen  -  PCR 3

This experiment was performed according to the manufacturer’s protocol.

Gel electrophoresis 6  -  Purified products from gel extraction (30.07.19) vs. GE Healthcare vs. Quiagen

To analyze the different purification methods of our PCR products from 02.08 and the effect of DMSO, we ran the samples on an 3% agarose gel. Moreover, we compared the percentage of agarose in gel between 3 and 4% to see if the band resolution was better.

![](https://static.igem.org/mediawiki/2019/7/78/T--Pasteur_Paris--NotebookImg6.png)

1.    PCR product from 08.02 with DMSO
          2.    PCR product from 08.02 without DMSO
          3.    PCR product with DMSO purified with Qiagen kit
          4.    PCR product whithout DMSO purified with Qiagen kit
          5.    PCR product with DMSO purified with GE Healthcare column
          6.    PCR product without DMSO purified with GE Healthcare column
          7.    PCR product with DMSO purified by gel extraction
          8.    PCR product without DMSO purified by gel exctraction

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Wells | Purification method | Concentration (ng/μL) | 260/280nm ratio | 260/230nm ratio |
| 4 | PCR product with DMSO | 1157.60 | 1.64 | 0.85 |
| 5 | PCR product without DMSO | 1143.00 | 1.65 | 1.18 |
| 6 | PCR clean up kit from QIAGEN with DMSO | 7.90 | 2.26 | -0.32 |
| 7 | PCR clean up kit from QIAGEN without DMSO | 15.00 | 2.07 | -0.62 |
| 8 | GE Healthcare columns with DMSO | 34.50 | 0.96 | 0.34 |
| 9 | GE Healthcare columns without DMSO | 45.90 | 1.16 | 0.08 |
| 10 | Gel extraction kit with DMSO | 6.10 | -6.66 | 0.01 |
| 11 | Gel extraction kit without DMSO | -0.50 | 0.53 | 0 |

Interpretations

The gel containing 4% of agarose gave a better resolution of the bands. Moreover, we did not observe any difference between the amplified products with and without DMSO. Concerning the purification methods, there was still no bands after the purification by gel extraction. Concerning the purification by PCR clean-up, we did not see anything on the gel at 4% agarose and there was only one band in the well 9 on the gel with 3% of agarose. On the contrary, we observed a band on both gels  for the two products purified by the columns.

We analyzed then the same products as above but after digestion. The digestion was performed according to the manufacturer’s protocol.  This time we only did a 4% of agarose gel because we noticed with the previous experiment that we had a better resolution with this concentration.

![](https://static.igem.org/mediawiki/2019/2/25/T--Pasteur_Paris--NotebookImg7.png)

Interpretation

After digestion, there was no more bands even for the sample purified with the columns. Thus, either the lambda exonuclease had digested the two strands of DNA, or the concentration of DNA was too low to be seen on the gel. The last hypothesis was that the SYBR Green may not reveal single-stranded DNA.

Conclusion

Several parameters may change during the digestion step in order to determine if the enzyme digested the two strands of DNA.

Phenol/chloroform extraction

We performed a purification step on all samples to remove the digestion enzyme according to the protocol previously described. Samples were then analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| Wells | Purification method | Concentration (ng/μL) | 260/280nm ratio | 260/230nm ratio |
| 5 | PCR product with DMSO | 1157.60 | 1.64 | 0.85 |
| 6 | PCR product without DMSO | 1143.00 | 1.65 | 1.18 |
| 8 | PCR product clean up kit from QIAGEN with DMSO | 7.90 | 2.26 | -0.32 |
| 9 | PCR product clean up kit from QIAGEN without DMSO | 15.00 | 2.07 | -0.62 |
| 10 | GE Healthcare columns with DMSO | 34.50 | 0.96 | 0.34 |
| 11 | GE Healthcare columns without DMSO | 45.90 | 1.16 | 0.08 |
| 13 | Gel extraction kit with DMSO | 6.10 | -6.66 | 0.01 |
| 14 | Gel extraction kit without DMSO | -0.50 | 0.53 | 0.00 |

07.08.19

PCR 4  -  15 cycles vs 30 cycles

We tried to amplify our library with only 15 cycles instead of 30 to compare if it had an impact in the shift of size of our PCR product (120 bp instead of 80 bp normally). The PCR was made with the same conditions as the PCR 4 without DMSO.

Gel electrophoresis 7  -  15 cycles vs 30 cycles

The amplified products were run on a 4% agarose gel and compared with amplified products from PCR 3 and 4.

![](https://static.igem.org/mediawiki/2019/4/4b/T--Pasteur_Paris--NotebookImg8.png)

Interpretations

We observed a slight decrease of the size for products amplified with only 15 cycles compared to those amplified with 30 cycles. However, this decrease in size was not significant enough. And the bands from the amplified samples with 30 cycles were brighter than those amplified with 15 cycles.

Purification of PCR products  -  Dialysis tubing

A new purification method of our PCR products was tested which is dialysis tubing. The DNA will migrate out of the gel and into the dialysis bag. Samples were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280nm ratio | 260/230nm ratio |
| Dialysis sample 1 pool | 1.20 | 2.71 | -9.20 |
| Dialysis sample 2 pool | 2.80 | 2.22 | 0.6 |

Interpretation

Even if the ratio 260/280nm was good the concentration of DNA was too low. Thus, this technique cannot be used due to a yield too low.

Kit montage Gel Extraction

We used the Kit montage Gel extraction from Millipore to try to extract our DNA from the gel and purify it. This kit was used according to the manufacturer’s protocol. Samples were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280nm ratio | 260/230nm ratio |
| Sample 1 | 8.30 | 1.95 | 0.15 |
| Sample 2 | 6.40 | 2.13 | 0.16 |
| Sample 3 | 5.00 | 2.24 | 0.12 |
| Sample 4 | 6.90 | 1.37 | 0.14 |

Interpretation

The concentration of DNA was too low. Hence, another method of DNA purification has to be found.

August Week 2 :  07/12  to  07/18

07.12.19

Digestion  -  Testing different conditions on
          amplified library

A new digestion was carried out in order to optimize the
          yield. Two parameters were studied, the incubation time and the
          quantity of enzyme used. Here is the list of all conditions tested:

- 1A : 5 min incubation, 5 U
- 1B : 5 min incubation, 2.5 U
- 1C : 5 min incubation, 1 U
- 2A : 10 min incubation, 5 U
- 2B : 10 min incubation, 2.5 U
- 2C : 10 min incubation, 1 U
- 3A : 15 min incubation, 5 U
- 3B : 15 min incubation, 2.5 U
- 3C : 15 min incubation, 1 U
- 4A : 20 min incubation, 5 U
- 4B : 20 min incubation, 2.5 U
- 4C : 20 min incubation, 1 U

Gel electrophoresis 8  -  Digestion conditions
          comparison

![](https://static.igem.org/mediawiki/2019/9/94/T--Pasteur_Paris--NotebookImg9.png)

Samples were then checked using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280nm ratio | 260/230nm ratio |
| 1A after digestion | 121.40 | 1.52 | 0.29 |
| 1B after digestion | 117.20 | 1.52 | 0.29 |
| 1C after digestion | 118.00 | 1.55 | 0.29 |
| 2A after digestion | 69.30 | 1.64 | 0.3 |
| 2B after digestion | 116.50 | 1.49 | 0.3 |
| 2C after digestion | 117.30 | 1.51 | 0.28 |
| 3A after digestion | 119.00 | 1.55 | 0.28 |
| 3B after digestion | 110.60 | 1.54 | 0.28 |
| 3C after digestion | 117.40 | 1.57 | 0.24 |
| 4A after digestion | 120.50 | 1.52 | 0.26 |
| 4B after digestion | 118.30 | 1.56 | 0.24 |
| 4C after digestion | 130.10 | 1.52 | 0.26 |

Interpretations

There was no bands on the gel. However, the nanodrop detected DNA with a pretty good 260/280 ratio. But there was no difference between the different conditions. So, either the single-strand DNA i s not revealed with SYBR Green or the digestion enzyme does not work properly.

08.14.19

Denaturation  -  of PCR 5 products

In order to optimize the digestion step, we tried to denature our PCR product before being digested. Samples were heated at 95°C for 5 min and directly put in ice for 15 min.

Digestion  -  Testing different conditions on PCR 5 products

Different conditions of digestion were tested in order to optimize this step (buffers, DNA quantity to digest, enzyme quantity, with/without denaturation before).
          Conditions tested:- Cutsmart, 15 µg DNA, 1 U (37°C, 15 min)
- Cutsmart, 10 µg DNA, 1 U (37°C, 15 min)
- Cutsmart, 15 µg DNA, 0,5 U (37°C, 15 min)
- Cutsmart, 10 µg DNA, 0,5 U (37°C, 15 min)
- Lambda buffer, 15 µg DNA, 1 U (37°C, 15 min)
- Lambda buffer, 10 µg DNA, 1 U (37°C, 15 min)
- Lambda buffer, 15 µg DNA, 0,5 U (37°C, 15 min)
- Lambda buffer, 10 µg DNA, 0,5 U (37°C, 15 min)
- Lambda buffer, 15 µg DNA, 1 U (37°C, 15 min) + denaturation before
- Lambda buffer, 10 µg DNA, 1 U (37°C, 15 min) + denaturation before
- Lambda buffer, 15 µg DNA, 0,5 U (37°C, 15 min) + denaturation before
- Lambda buffer, 10 µg DNA, 0,5 U (37°C, 15 min) + denaturation before
- 50 bp ladder
- PCR product before digestion
- PCR product before digestion followed by heat shock

Gel electrophoresis 9  -  Digestion conditions comparison

A 4% agarose gel was made to compare all the different conditions of digestion.

![](https://static.igem.org/mediawiki/2019/3/3b/T--Pasteur_Paris--NotebookImg10.png)

Interpretations

We are looking for a band with a shift in size compared to the PCR product before digestion. Once again, bands were similar to the PCR product, it means that conditions were too restrictive and that the digestion did not worked.

08.15.19

PCR 6  -  Generation of new materials

To continue our experiments, a new part of the library was amplified in order to work with. Samples were also analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280nm ratio | 260/230nm ratio |
| Sample 1 | 1231.1 | 1.65 | 1.15 |
| Sample 2 | 1217.1 | 1.5 | 1.15 |
| Sample 3 | 1219.1 | 1.66 | 1.16 |
| Sample 4 | 1215.3 | 1.64 | 1.15 |
| Sample 5 | 1216.2 | 1.65 | 1.15 |
| Sample 6 | 1213.2 | 1.66 | 1.14 |
| Sample 7 | 1201.4 | 1.66 | 1.14 |
| Sample 8 | 1211.5 | 1.65 | 1.15 |
| Sample 9 | 1221.2 | 1.65 | 1.14 |
| Sample 10 | 1185.2 | 1.65 | 1.14 |

August Week 3  :  08/19  to
           08/21

08.19.19

PCR 7 - Generation of new materials (10 ng, 5 ng, 1
          ng)

A new PCR was performed to produce starting material. For
          this, different amounts of DNA to be amplified have been tested (10
          ng, 5 ng, 1 ng) in order to reduce nonspecific amplifications.

|  |  |  |  |
| --- | --- | --- | --- |
|  | Volume needed (in μL) | | |
| Components | Mix 1(10ng) | Mix 2(5ng) | Mix 3(1ng) |
| Phusion Buffer 10X | 100 | 100 | 100 |
| dNTPs 25 mM | 10 | 10 | 10 |
| Forward primer (1/10) | 20 | 20 | 20 |
| Reverse primer (1/10) | 20 | 20 | 20 |
| Template DNA (1/1000) | 45 | 22.5 | 5 |
| Phusion DNA polymerase | 5 | 5 | 5 |
| Nuclease-free water | 300 | 322.5 | 340 |
| Total volume | 500 | 500 | 500 |

Samples were also analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μl) | 260/280 ratio | 260/230 ratio |
| Sample pooled (10ng) | 990.6 | 1.62 | 1.05 |
| Sample pooled (5ng) | 1028.5) | 1.61 | 1.05 |
| Sample pooled (1ng) | 1028.7) | 1.61 | 1.04 |

08.20.10

Digestion - 30 min, 5 U, from 5 to 1 µg with control
          without enzyme from PCR 7

A new digestion was performed with different quantity of
          DNA to digest for each sample. An undigested sample from the PCR 7 was
          used as a negative control.

Gel electrophoresis 10 - Digestion conditions comparison

A gel with 4% of agarose was made to compare each sample.
          The staining was performed with GelRed because it is more specific of
          single-strand DNA than ethidium bromide and SYBR Green.

![](https://static.igem.org/mediawiki/2019/thumb/4/43/T--Pasteur_Paris--Week3.png/800px-T--Pasteur_Paris--Week3.png)

08.21.19

PCR 8 - Generation of new materials

A new PCR was performed to produce starting material.

          Samples were analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration(ng/µl | 260/280 ratio | 260/230 ratio |
| PCR 8 pooled | 1093.9 | 1.63 | 1.11 |

August Week 4  :   08/26  to
           09/1

08.26.19

Purification of PCR product  -  illustra
          MicroSpin G-25 Columns kit from GE Healthcare  -  PCR 9

The PCR 9 products were purified according to the
          manufacturer’s protocol. Then, samples were analyzed using a Nanodrop
          2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ratio |
| PCR 9 purified by column | 868.7 | 1.58 | 1.03 |

Digestion  -  from PCR 9

After the purification, the sample was digested
          according to the manufacturer’s instructions with 4 µg of DNA.

Phenol/chloroform extraction  -  Digested
          products from PCR 9

The sample was purified by phenol/chloroform according
          to the protocol previously described. Then, samples were analyzed
          using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ration |
| PCR 9 purified by phenol/chloroform | 17 | 1.48 | 1.34 |

Interpretation

We lose a huge quantity of DNA. Thus, the concentration
          is too low to begin the first round of SELEX.

08.27.19

Digestion - from PCR 9

We purify the digested samples using either 2.5 X volume
          of ethanol, as previously, or 1 X volume of isopropanol. The samples
          were then analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ration |
| PCR 9 purified with ethanol    1 | 9.2 | 1.6 | 0.91 |
| PCR 9 purified with ethanol    2 | 7.3 | 1.5 | 0.91 |
| PCR 9 purified with isopropanol    1 | 44.9 | 1.41 | 1.10 |
| PCR 9 purified with isopropanol    1 | 48.1 | 1.42 | 1.39 |

Interpretations

The concentration of DNA are much higher when we used
          isopropanol compared to ethanol.

Conclusion

Isopropanol is used for the next purifications by
          phenol/chloroform. These concentrations are enough to perform the
          first round of SELEX on one bacteria. However, we want to select
          aptamers for two different bacteria so we must perform this step
          again.

08.28.19

Bacteria culture  -  E. faecium and S. aureus

The strains E. faecium and S. aureus were grown on Petri
          dish on Blood agar and LB media respectively. The plates were
          incubated at 37°C during 24 hours.

![](https://static.igem.org/mediawiki/2019/c/c5/T--Pasteur_Paris--NotebookImg13.jpg)

Digestion  -  from PCR9

The PCR 9 products were digested using 4 µg of DNA
          according to the manufacturer’s protocol.

Phenol/chloroform extraction with Isopropanol
           -  Digested products from PCR 9

After digestion, the samples were purified using 1 X
          volume of isopropanol. The samples are then analysed using a Nanodrop
          2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ration |
| PCR 9 purified by phenol/chloroform                    1 | 42.1 | 1.46 | 1.40 |
| PCR 9 purified by phenol/chloroform 2 | 3.1 | 1.65 | 0.71 |
| PCR 9 purified by phenol/chloroform 3 | 3.1 | 1.55 | 1.64 |
| PCR 9 purified by phenol/chloroform 4 | 8.6 | 1.43 | 1.19 |

Interpretations

The concentration of DNA is lower than expected. We may
          have pipeted some DNA as the pellet is invisible.

Conclusion

Because of these low concentrations, the first round of
          SELEX cannot be performed.

08.29.19

Bacteria culture  -  E. faecium and S. aureus

Few colonies of E. faecium and S. aureus were taken from
          the Petri dish culture of the 28.08 and grow in their respective
          liquid media at 37°C under agitation. After 3 hours of growth, they
          are centrifuged and the pellet of bacteria is resuspended in PBS 1X.
          Dilutions were performed until the OD reached between 0.5 and 0.6.

Digestion  -  from PCR9

In order to have more DNA before starting the SELEX, we
          performed a digestion with 4 µg of DNA from the PCR 9 samples
          according to the manufacturer’s protocol.

Phenol/chloroform extraction with Isopropanol
           -  Digested products from PCR 9

The samples were purified after digestion using 1 X
          volume of isopropanol. Then, they were analysed with a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ration |
| PCR 9 purified sample 1 | 41.4 | 1.41 | 1.46 |
| PCR 9 purified sample 2 | 4.2 | 1.25 | 0.57 |
| PCR 9 purified sample 3 | 9.4 | 1.33 | 0.99 |
| PCR 9 purified sample 4 | 23.7 | 1.39 | 1.08 |
| PCR 9 purified sample 5 | 11.1 | 1.37 | 0.41 |
| PCR 9 purified sample 6 | 27.8 | 1.40 | 0.90 |

Conclusion

After the purification, we have enough DNA concentration to start the SELEX on the two bacteria.

SELEX 1 Round 1

The first round of SELEX was performed  with 2 hours of incubation. The samples are then analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ration |
| S. aureus | 60.1 | 1.61 | 0.96 |
| E. faecium | 43.6 | 1.69 | 1.09 |

PCR 10  -  Amplification of EFr1 & SAr1

This PCR was performed to amplified the DNA after the first round of SELEX. We tried two different concentrations for both samples. The PCR followed the same program as described previously. It was run overnight and hold at 4°C.

|  |  |  |
| --- | --- | --- |
|  | S aureus | |
| Components | SA 1 (1/10) | SA 2 (1/90) |
| Phusion buffer | 10 | 10 |
| dNTPs | 1 | 1 |
| Forward primer | 2 | 2 |
| Reverse primer | 2 | 2 |
| Template DNA (1/10) | 1 | 0 |
| Template DNA (1/90) | 0 | 1 |
| Phusion DNA polymerase | 0.5 | 0.5 |
| Nulease-free water | 33.5 | 33.5 |
| Total volume | 50 | 50 |

|  |  |  |
| --- | --- | --- |
|  | E. faecium | |
| Components | SA 1 (1/10) | SA 2 (1/80) |
| Phusion buffer | 10 | 10 |
| dNTPs | 1 | 1 |
| Forward primer | 2 | 2 |
| Reverse primer | 2 | 2 |
| Template DNA (1/10) | 1 | 0 |
| Template DNA (1/80) | 0 | 1 |
| Phusion DNA polymerase | 0.5 | 0.5 |
| Nulease-free water | 33.5 | 33.5 |
| Total volume | 50 | 50 |

The samples are analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ration |
| PCR 10 - Round 1 - SA    1 | 1253.9 | 1.62 | 1.05 |
| PCR 10 - Round 1 - SA    2 | 1200.4 | 1.64 | 1.09 |
| PCR 10 - Round 1 - EF    1 | 1118.9 | 1.56 | 0.89 |
| PCR 10 - Round 1 - EF    2 | 1207.9 | 1.66 | 1.13 |

08.30.19

Gel electrophoresis 12  -  PCR 10

On a 4% agarose gel, samples before and after the PCR 12 were run.

September Week 1  :  09/05  to  09/15

Digestion   -  EFr1 & SAr1

The digestion was performed with 4 µg of DNA according to the manufacturer’s protocol.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ratio |
| EFr1 digested | 27.4 | 1.87 | 0.26 |
| SAr1 digested | 30.2 | 1.89 | 0.28 |

Purification of digested products EFr1 & SAr1  -  kit Monarch from NEB

A new kit of purification was tried to purify the PCR Products. The experiment was performed according to the manufacturer’s recommendations.

The samples are checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| EFr1 purified | 7.8 | 1.27 | 0.27 |
| EFr1 wash 1 | -1.7 | 0.58 | -0.03 |
| EFr1 wash 2 | 2.5 | 5.51 | 0.27 |
| EFr1 purified | 7.8 | 1.27 | 0.27 |
| SAr1 purified | 7.2 | 1.52 | 0.58 |
| SAr1 wash 1 | 22.5 | 1.29 | 0.53 |
| SAr1 wash 2 | 2.4 | 8.31 | 0.30 |

Interpretations

The concentrations are really low. Moreover, the quantity of DNA within the sample is not high because the ratio 260/280 is not close to 2. Finally, the ratio 260/230 did not change increase before and after the purification.

Conclusion

This kit cannot be used to purify our DNA after digestion.

Purification of digested products EFr1 & SAr1  -   illustra MicroSpin G-25 Columns kit from GE Healthcare

The purification was performed according to the manufacturer’s protocol.
The samples are checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/μL) | 260/280 ratio | 260/230 ratio |
| EFr1 purified | 53.2 | 1.62 | 0.24 |
| SAr1 purified | 56.2 | 1.54 | 0.27 |

Interpretation

Even if the concentrations are quite elevated, we noticed that the ratio 260/230 did not change before and after the purification using these columns. Hence, the enzymes may not be  purified by this technique.

Conclusion

We tried several kits to purify our PCR products but none of them worked. Thus, we decided to directly continue with the digestion after the PCR without purifying. All the enzymes will be removed during the phenol/chloroform extraction just before the SELEX.

09.06.19

Bacteria culture  -  E. faecium and S. aureus

Few colonies of E. faecium and S. aureus were taken from the Petri dish culture of the 28.08 and grow in their respective liquid media at 37°C under agitation. After 3 hours of growth, they are centrifuged and the pellet of bacteria is resuspended in PBS 1X. Dilutions were performed until the OD reached between 0.5 and 0.6.

Phenol/chloroform extraction with Isopropanol  -  Digested products EFr1 & SAr1

The samples were purified after digestion. Then, they were analysed with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concetration (ng/μL) | 260/280 ratio | 260/230 ratio |
| EFr1 sample 1 | 17.2 | 1.46 | 1.29 |
| EFr1 sample 2 | 26.5 | 1.49 | 1.44 |
| EFr1 sample 3 | 29.8 | 1.49 | 1.47 |
| EFr1 sample 4 | 37.2 | 1.56 | 1.5 |
| SAr1 sample 1 | 69 | 1.51 | 1.54 |
| SAr1 sample 2 | 61.5 | 1.75 | 0.28 |

Interpretations

A total quantity of 1728 pmol was obtained for E. faecium in the Round 1, and 869 pmol for S. aureus.

SELEX Round 2

The first round of SELEX was performed  with 2 hours of incubation. The samples are then analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concetration (ng/μL) | 260/280 ratio | 260/230 ratio |
| SAr2 | 33.8 | 2.19 | 1.45 |
| EFr2 | 32 | 2.21 | 1.6 |

PCR 10  -  Amplification of EFr2 and SAr2

This PCR was performed to amplified the DNA after the second round of SELEX. The PCR followed the same program as described previously. It was run overnight and hold at 4°C.

|  |  |  |
| --- | --- | --- |
| Components | SAr1 | EFr1 |
| Phusion buffer | 200 | 200 |
| dNTPs | 20 | 20 |
| Forward primer | 40 | 40 |
| Reverse primer | 40 | 40 |
| Template DNA (1/3) | 20 | 20 |
| Phusion DNA polymerase | 10 | 10 |
| Nuclease-free water | 670 | 670 |
| Total volume | 1000 | 1000 |

The samples are analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concetration (ng/μL) | 260/280 ratio | 260/230 ratio |
| PCR 10 - Round 2 - SA | 1086.6 | 1.67 | 1.20 |
| PCR 10 - Round 2 - EF | 1243.4 | 1.66 | 1.20 |

September Week 3  :  09/16
           to  09/21

09.16.19

Bacteria culture - E. faecium and S. aureus

E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6

Digestion - EFr5 & SAr5

PCR samples from the Round 5 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Dig - Round 5 - SA sample 1 | 56.7 | 1.75 | 0.25 |
| Dig - Round 5 - SA sample 2 | 57.1 | 1.79 | 0.23 |
| Dig - Round 5 - EF sample 1 | 57.6 | 1.78 | 0.25 |
| Dig - Round 5 - EF sample 2 | 57.6 | 1.81 | 0.23 |

Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr5 & SAr5

Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| EFr5 sample 1 | 24.5 | 1.67 | 0.94 |
| EFr5 sample 2 | 21.1 | 1.67 | 0.98 |
| SAr5 sample 1 | 104.2 | 1.50 | 1.56 |
| SAr5 sample 2 | 160 | 1.54 | 1.61 |

Interpretation

A total quantity of 354 pmol was obtained for E.
            faecium in the Round 5, and 2040 pmol for S. aureus.

SELEX Round 6

The Round 6 of SELEX was performed by incubating for 1h.
          The samples are then analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SAr6 | 45.3 | 1.91 | 1.33 |
| EFr6 | 23.7 | 2.22 | 1.91 |

PCR 14 - Amplification of EFr6 and SAr6

This PCR was performed to amplified the DNA after the
          Round 6 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| PCR 14 - Round 6 - SA | 1616.9 | 1.65 | 1.20 |
| PCR 14 - Round 6 - EF | 2068.6 | 1.70 | 1.37 |

09.17.19

Bacteria culture - E. faecium and S. aureus

E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6

Digestion - EFr6 & SAr6

PCR samples from the Round 6 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Dig - Round 6 - SA sample 1 | 57.5 | 1.78 | 0.24 |
| Dig - Round 6 - SA sample 2 | 56.1 | 1.81 | 0.26 |
|  |  |  |  |
| --- | --- | --- | --- |
| Dig - Round 6 - EF sample 1 | 62.4 | 1.82 | 0.28 |
|  |  |  |  |
| --- | --- | --- | --- |
| Dig - Round 6 - EF sample 2 | 58.9 | 1.89 | 0.24 |

Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr6 & SAr6

Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| EFr6 sample 1 | 245 | 1.65 | 1.73 |
| EFr6 sample 2 | 68.4 | 1.47 | 1.30 |
| SAr6 sample 1 | 19.6 | 1.52 | 1.19 |
| SAr6 sample 2 | 15.9 | 1.60 | 1.00 |

Interpretation

A total quantity of 2440 pmol was obtained for E.
            faecium the Round 6, and 276 pmol for S. aureus.

SELEX Round 7

The Round 7 of SELEX was performed by incubating for 1h.
          The samples are then analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SAr7 | 25.4 | 2.18 | 1.41 |
| EFr7 | 37.7 | 2.08 | 1.93 |

PCR 15 - Amplification of EFr7 and SAr7

This PCR was performed to amplified the DNA after the
          Round 7 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| PCR 15 - Round 7 - SA | 1358 | 1.62 | 1.05 |
| PCR 15 - Round 7 - EF | 1572.8 | 1.65 | 1.18 |

09.18.19

Bacteria culture - E. faecium and S. aureus

E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6.

Digestion - EFr7 & SAr7

PCR samples from the Round 7 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material.

          Samples concentrations were checked using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Dig - Round 7 - SA sample 1 | 50.3 | 1.81 | 0.25 |
| Dig - Round 7 - SA sample 2 | 58.1 | 1.78 | 1.24 |
| Dig - Round 7 - EF sample 1 | 60.4 | 1.83 | 0.24 |
| Dig - Round 7 - EF sample 2 | 59.9 | 1.81 | 0.28 |

Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr7 & SAr7

Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| EFr7 sample 1 | 29.8 | 1.52 | 1.20 |
| EFr7 sample 2 | 13.4 | 1.47 | 1.10 |
| SAr7 sample 1 | 98.6 | 1.52 | 1.55 |
| SAr7 sample 2 | 247.7 | 1.72 | 1.79 |

Interpretation

A total quantity of 336 pmol was obtained for E.
            faecium in the Round 7, and 1340 pmol for S. aureus.

SELEX Round 8

The Round 8 of SELEX was performed by incubating for 1h.
          The samples are then analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Sar8 | 19.3 | 2.44 | 1.57 |
| EFr8 | 17.2 | 2.30 | 1.33 |

PCR 16 - Amplification of EFr8 and SAr8

This PCR was performed to amplified the DNA after the
          Round 8 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| PCR 16 - Round 8 - SA | 933.2 | 1.58 | 0.93 |
| PCR 16 - Round 8 - EF | 1324.2 | 1.60 | 0.98 |

09.19.19

Bacteria culture - E. faecium and S. aureus

PCR samples from the Round 8 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material.

          Samples concentrations were checked using a Nanodrop 2000
          spectrophotometer.

Digestion - EFr8 & SAr8

PCR samples from the Round 8 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material. Samples concentrations
          were checked using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Dig - Round 8 - SA sample 1 | 55.5 | 1.78 | 0.22 |
| Dig - Round 8 - SA sample 2 | 54.2 | 1.79 | 0.23 |
| Dig - Round 8 - EF sample 1 | 46 | 1.77 | 0.23 |
| Dig - Round 8 - EF sample 2 | 44.5 | 1.75 | 1.23 |

09.20.19

Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr8 & SAr8

Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| EFr8 sample 1 | 100.4 | 1.53 | 1.49 |
| EFr8 sample 2 | 123.3 | 1.56 | 1.59 |
| SAr8 sample 1 | 123.3 | 1.56 | 1.59 |
| SAr8 sample 2 | 160.5 | 1.58 | 1.75 |

SELEX Round 9

The Round 9 of SELEX was performed by incubating for 45
          min. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SAr9 | 28.1 | 2.20 | 1.47 |
| SAr8 | 18.2 | 2.32 | 1.73 |

PCR 17 - Amplification of EFr9 and SAr9

This PCR was performed to amplified the DNA after the
          Round 9 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. The samples are analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| PCR 17 - Round 9 - SA | 541.4 | 1.47 | 0.74 |
| PCR 17 - Round 9 - EF | 947.6 | 1.59 | 0.96 |

Transformation - biobrick BBa_J04450 (mRFP)

As instructed by iGEM, the biobrick was resuspended with
          10 µL of TE buffer. Then, it was transferred into a tube containing
          100 µL of E. coli DH5α competent cells and let 30 minutes in
          ice. Then, the sample was heated at 42°C for 40 seconds and, after,
          put in ice for 3 minutes. Therefore, 600 µL of SOC media was added and
          the sample was incubated at 37°C during 1 hour under agitation at 140
          rpm. Then, the bacteria are put on a LB petri dish containing
          kanamycin at 25 µg/mL and stored all night at 37°C

09.21.19

Bacteria culture - E. faecium and S. aureus

E. faecium and S. aureus were grown
          until the OD reached between 0.5 and 0.6

Digestion - EFr9 & SAr9

PCR samples from the Round 9 of SELEX were digested
          using lambda exonuclease. A mix was performed in order to scale-up
          this step and get enough digested material.

          Samples concentrations were checked using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Dig - Round 9 - SA sample 1 | 59.3 | 1.67 | 0.21 |
| Dig - Round 9 - SA sample 2 | 60.1 | 1.69 | 0.18 |
| Dig - Round 9 - EF sample 1 | 56 | 1.77 | 0.23 |
| Dig - Round 9 - EF sample 2 | 54.9 | 1.77 | 0.23 |

Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr9 & SAr9

Once digested, samples were purified after digestion.
          They were analysed with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| EFr9 sample 1 | 90.8 | 1.52 | 1.52 |
| EFr9 sample 2 | 6124 | 1.54 | 1.64 |
| SAr9 sample 1 | 203.9 | 1.57 | 1.69 |
| SAr9 sample 2 | 315.1 | 1.71 | 1.70 |

SELEX Round 10

The Round 10 of SELEX was performed by incubating for 45
          min. The samples are then analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SAr10 | 20.8 | 2.33 | 1.56 |
| EFr10 | 20.4 | 2.54 | 1.71 |

PCR 18 - Amplification of EFr10 and SAr10

This PCR was performed to amplified the DNA after the
          Round 10 of SELEX. The PCR followed the same program as described
          previously with the same concentration. It was run overnight and hold
          at 4°C. Samples were analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| PCR 18 - Round 10 - SA | 1123.6 | 1.64 | 1.13 |
| PCR 18 - Round 10 - EF | 560.7 | 1.46 | 0.73 |

September Week 4  : 09/24  to
          09/29

09.24.19

Phenol/chloroform extraction with Isopropanol and
          Glycogen - Digested products EFr10 & SAr10

In order to purify our DNA, we performed a
          phenol/chloroform extraction. Samples were analyzed using a Nanodrop
          2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SAr10 purified | 431.9 | 1.61 | 1.84 |
| EFr10 purified | 301.2 | 1.62 | 1.78 |

Double digestion with Hind-III-HF and BamHI-HF - EFr10,
          SAr10 & pET28:GFP

After being purified, DNA aptamers of EFr10 & SAr10
          as well as the pET28:GFP (from Addgene) were digested using HindIII-HF
          and BamHI-HF enzymes from New England Biolabs.

|  |  |  |
| --- | --- | --- |
| Component | 50 µL reaction | MIX |
| 1 µg DNA (SAr10) | 2.5 µL | 52.5 µL |
| HindIII-HF | 1 µL | 15 µL |
| BamHI-HF | 1 µL | 15 µL |
| Nuclease-free water | 40.5 µL | 592.5 µL |
| 10X Cutsmart Buffer | 5 µL | 75 µL |

|  |  |  |
| --- | --- | --- |
| Component | 50 µL reaction | MIX |
| 1 µg DNA (EFr10) | 2.5 µL | 37.5 µL |
| 10X Cutsmart Buffer | 5 µL | 75 µL |
| HindIII-HF | 1 µL | 15 µL |
| BamHI-HF | 1 µL | 15 µL |
| Nuclease-free water | 39.5 µL | 607.5 µL |

|  |  |  |
| --- | --- | --- |
| Component | 50 µL reaction | MIX |
| 1 µg DNA (pET28:GFP) | 5 µL | 75 µL |
| 10X Cutsmart Buffer | 5 µL | 75 µL |
| HindIII-HF | 1 µL | 15 µL |
| BamHI-HF | 1 µL | 15 µL |
| Nuclease-free water | 38 µL | 570 µL |

1. Incubate at 37°C for
          5-15 minutes 2. Incubation at 80°C for 20 min (inactivation of
          HindIII-HF) as recommended on
          https://international.neb.com/tools-and-resources/usage-guidelines/heat-inactivation

Phenol/chloroform extraction with Isopropanol and
          Glycogen - Double digested products EFr10 & SAr10 for ligation

A new purification step by phenol/chloroform was
          performed in order to get pure digested aptamers before the ligation
          step.

          Samples were analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SAr10 digested and purified | 38.8 | 1.55 | 1.46 |
| EFr10 digested and purified | 58.2 | 1.58 | 1.54 |

Gel electrophoresis 13 - Purification of the digested
          vector pET28:GFP

To purify our vector, pET28:GFP was run onto a 1%
          agarose gel using a 1 kb ladder.

![](https://static.igem.org/mediawiki/2019/thumb/3/39/T--Pasteur_Paris--W4Sept.png/800px-T--Pasteur_Paris--W4Sept.png)

Gel slices were cut in order to extract DNA.

![](https://static.igem.org/mediawiki/2019/f/ff/T--Pasteur_Paris--W3Sept2.jpeg%22)

Gel extraction purification - Kit montage Gel extraction

We used the Kit montage Gel extraction from Millipore to
          extract the plasmid and purify it. This kit was used according to the
          manufacturer’s protocol.

Gel extraction purification - QIAquick Gel Extraction
          kit

We used the QIAquick Gel Extraction kit from Qiagen to
          extract the plasmid and purify it. This kit was used according to the
          manufacturer’s protocol.

09.25.19

Ligation - DNA aptamers of EFr10 & SAr10 with
          pET28:GFP

This protocol was performed according to the
          manufacturer’s protocol. Different ratios were tested.

|  |  |  |  |
| --- | --- | --- | --- |
|  | Ratio 1:1 (in μL) | | |
| Sample | Volume |
| Vector (pET28:GFP) | 15 µL |
| Insert* (EFr10) | 0.7 µL |
| Buffer 10 X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 1.7 µL |

*The insert was diluted to 1/10

|  |  |  |  |
| --- | --- | --- | --- |
|  | Ratio 1:7 (in μL) | | |
| Sample | Volume |
| Vector (pET28:GFP) | 15 µL |
| Insert* (EFr10) | 0.7 µL |
| Buffer 10 X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | / µL |

*The insert was diluted to 1/10

|  |  |  |  |
| --- | --- | --- | --- |
|  | Ratio 1:1 (in μL) | | |
| Sample | Volume |
| Vector (pET28:GFP) | 15 µL |
| Insert* (EFr10) | 0.17 µL |
| Buffer 10 X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 2.23 µL |

*The insert was diluted to 1/10

|  |  |  |  |
| --- | --- | --- | --- |
|  | Ratio 1:3 (in μL) | | |
| Sample | Volume |
| Vector (pET28:GFP) | 15 µL |
| Insert (EFr10) | 0.51 µL |
| Buffer 10 X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 1.89 µL |

|  |  |  |  |
| --- | --- | --- | --- |
|  | Ratio 1:7 (in μL) | | |
| Sample | Volume |
| Vector (pET28:GFP) | 15 µL |
| Insert (EFr10) | 1.19 µL |
| Buffer 10 X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 1.21 µL |

09.26.19

Transformation - with E. coli DH5α competent cells.

E. coli DH5α competent cells were transformed with the
          plasmid pET28:GFP containing the aptamer candidates. Firstly, the
          competent cells were thawed on ice and then the plasmid containing the
          aptamer insert was added. The mix was incubated on ice for 30 minutes
          and then heat shocked at 42°C for 40 seconds. The bacteria were then
          put on ice for 5 minutes before adding SOC and were incubated at 37°C
          under shaking conditions at 250 rpm. Afterwards, the bacteria were
          spread on plates containing Kanamycin and incubated overnight at 37°C.

09.27.19

Isolation - Transformed E. coli DH5α

E. coli DH5α competent cells were transformed with the
          plasmid pET28:GFP containing the aptamer candidates. Firstly, the
          competent cells were thawed on ice and then the plasmid containing the
          aptamer insert was added. The mix was incubated on ice for 30 minutes
          and then heat shocked at 42°C for 40 seconds. The bacteria were then
          put on ice for 5 minutes before adding SOC and were incubated at 37°C
          under shaking conditions at 250 rpm. Afterwards, the bacteria were
          spread on plates containing Kanamycin and incubated overnight at 37°C.

09.28.19

Transformation - with E. coli DH10β competent
          cells

E. coli DH5β competent cells were transformed with the
          plasmid pET28:GFP containing the aptamer candidates. The
          transformation protocol was performed as detailed by the manufacturer
          New England Biolabs.

09.29.19

Transformation - with E. coli DH10β competent
          cells

Unfortunately, no bacteria E. coli DH10β grow
          after 24 hours of incubation. Thus, we cannot verify by fluorescence
          if the plasmid pET28:GFP was into the bacteria and we cannot sequence
          this strain.

September Week 5  : 09/30  to
          10/08

09.30.19

Sequencing - E. coli DH5α

Isolated colonies were stabbed into the PlateSeq kits
          from Eurofins Genomics and cultured overnight at 37°C before sending
          it to the company for the sequencing.

10.02.19

Transformation -biobricks BBa_K1033925 and BBa_K1073022

E. coli DH5α competent cells were transformed
          with the biobricks BBa_K1033925 and BBa_K1073022 as previously
          described. Then, transformed bacteria were cultured on LB petri dish
          containing chloramphénicol at 25 µg/mL and stored at 37°C all night.

10.03.19

Preparation of electrodes

The electrodes we will use are coated with carbon
          nanotubes, a material used for its excellent electrical conductivity.
          To fix the aptamers to the electrodes, we modified the nanotubes to
          have around 1% of their surface covered in carboxylic groups. Then we
          chemically bound the amine group at the end of the aptamers to said
          carboxylic groups, creating a strong peptide bond.

(image)

Binding the aptamer to the electrode by peptide bond.

          At first, our goal was to manufacture our carbon-nanotube electrodes
          from scratch using graphite electrodes and spray-depositing the
          nanotubes. We worked on DropSens nanotube electrodes, already
          functionalized with carboxylic groups.

          The carboxylic groups first need to be activated in order to react
          with the amine group of the aptamer.

1.Prepare 10 mL of a
          solution of EDC (100 nmol) and NHS (25 nmol) in MES buffer (50 mM,
          pH=5). 2. Deposit 40 µL of the solution as a drop unto each electrode.
          The activation lasted 30 minutes.

Meanwhile, solutions containing PBS (1 mM, pH= 7.4) and CTAB (0.2 mM),
          as well as the aptamer sequences at a concentration of 1µM, were
          prepared. We had specific aptamers for five different strains (A.
            baumanii, S. pneumoniae, S. aureus, P. aeruginosa and E. Coli),
          so five different solutions were made.

1. After 30 minutes, remove
          the COOH activation solution.

          2. Deposit 40 µL of the aptamer solution in the same manner.

          3. Leave the drops overnight to allow time for fixation.

Three electrodes for each strain were manufactured, for
          a total of fifteen functional electrodes.

Bacteria culture - E. coli containing the
          biobricks BBa_K1033925 and BBa_K1073022

After an overnight growth, a colony of bacteria from the
          02.10 was put into an erlenmeyer containing LB media. Then, the
          erlenmeyer was incubated at 37°C all night under agitation at 140 rpm.

10.04.19

Midiprep - E. coli containing the biobricks
          BBa_K1033925 and BBa_K1073022

The liquid culture of E. coli from the 03.10 was
          centrifuged for 10 minutes at 3,500 x g and the supernatant was
          removed. Then, we performed the midiprep kit from Qiagen as described
          by the manufacturer’s protocol.

          The sample was checked with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| BBa_K1033925 | 602.4 | 1.86 | 2.19 |
| BBa_K1073022 | 1157.9 | 1.87 | 2.16 |

October Week 1  : 09/30  to
          10/08

09.30.19

Sequencing - E. coli DH5α

Isolated colonies were stabbed into the PlateSeq kits
          from Eurofins Genomics and cultured overnight at 37°C before sending
          it to the company for the sequencing.

10.02.19

Transformation -biobricks BBa_K1033925 and BBa_K1073022

E. coli DH5α competent cells were transformed
          with the biobricks BBa_K1033925 and BBa_K1073022 as previously
          described. Then, transformed bacteria were cultured on LB petri dish
          containing chloramphénicol at 25 µg/mL and stored at 37°C all night.

10.03.19

Preparation of electrodes

The electrodes we will use are coated with carbon
          nanotubes, a material used for its excellent electrical conductivity.
          To fix the aptamers to the electrodes, we modified the nanotubes to
          have around 1% of their surface covered in carboxylic groups. Then we
          chemically bound the amine group at the end of the aptamers to said
          carboxylic groups, creating a strong peptide bond.

(image)

Binding the aptamer to the electrode by peptide bond.

          At first, our goal was to manufacture our carbon-nanotube electrodes
          from scratch using graphite electrodes and spray-depositing the
          nanotubes. We worked on DropSens nanotube electrodes, already
          functionalized with carboxylic groups.

          The carboxylic groups first need to be activated in order to react
          with the amine group of the aptamer.

1.Prepare 10 mL of a
          solution of EDC (100 nmol) and NHS (25 nmol) in MES buffer (50 mM,
          pH=5). 2. Deposit 40 µL of the solution as a drop unto each electrode.
          The activation lasted 30 minutes.

Meanwhile, solutions containing PBS (1 mM, pH= 7.4) and CTAB (0.2 mM),
          as well as the aptamer sequences at a concentration of 1µM, were
          prepared. We had specific aptamers for five different strains (A.
            baumanii, S. pneumoniae, S. aureus, P. aeruginosa and E. Coli),
          so five different solutions were made.

1. After 30 minutes, remove
          the COOH activation solution.

          2. Deposit 40 µL of the aptamer solution in the same manner.

          3. Leave the drops overnight to allow time for fixation.

Three electrodes for each strain were manufactured, for
          a total of fifteen functional electrodes.

Bacteria culture - E. coli containing the
          biobricks BBa_K1033925 and BBa_K1073022

After an overnight growth, a colony of bacteria from the
          02.10 was put into an erlenmeyer containing LB media. Then, the
          erlenmeyer was incubated at 37°C all night under agitation at 140 rpm.

10.04.19

Midiprep - E. coli containing the biobricks
          BBa_K1033925 and BBa_K1073022

The liquid culture of E. coli from the 03.10 was
          centrifuged for 10 minutes at 3,500 x g and the supernatant was
          removed. Then, we performed the midiprep kit from Qiagen as described
          by the manufacturer’s protocol.

          The sample was checked with a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| BBa_K1033925 | 602.4 | 1.86 | 2.19 |
| BBa_K1073022 | 1157.9 | 1.87 | 2.16 |

October Week 2  : 10.09  to
          10.13

10.09.19

Making competent Staphyloccocus aureus cells

Recombinant plasmids were then transformed first into S.
            aureus (RN4220 strain), a mutagenized strain that accepts
          foreign DNA.

1. Inoculate 150 mL of TSB
          with 1 mL of overnight culture

          2. Incubate at 37°C at 170 rpm, until OD 600 = 0.4 to 0.5 (about 5x108
          cells/mL)

          3. Let the culture at 4°C for 30 min or more

          4. Centrifuge 10 min about 7,000 rpm at 4°C

          5. Resuspend the pellet in 80 mL of saccharose 0.5 M at 4°C

          6. Centrifuge 10 min about 7,000 rpm at 4°C

          7. Repeat steps 4 to 6, 3 times

          8. Resuspend the pellet in 0.75 mL of saccharose 0.5 M at 4°C

          9. Aliquot in 100 µL samples

          10. Freeze the cells in tubes containing 100 µL of suspension
          immediately by using liquid nitrogen or a mix of dry ice and ethanol.

          11. Store the tubes at -80°C.

Bacteria culture - Pseudomonas aeruginosa

In order to make competent P. aeruginosa cells,
          a 10 mL culture was prepared from frozen cells and incubated overnight
          at 37°C at 160 rpm.

Bacteria culture - E. coli

In order to extract the plasmid pPMK4prot, a 25 mL
          culture of competent E. coli DH5α was prepared and incubated
          overnight at 37°C at 160 rpm.

10.10.19

DNA precipitation of biobricks BBa_K1033925 and
          BBa_K1073022

DNA precipitation by ethanol was used to concentrate our
          plasmids and to remove certain contaminants that can influence the
          transformation.

1.Dilute at least 500 ng
          of plasmid in 50 µL of DNAse-free water.

• 1 µL of BBa_k1033925 =
          600 ng

          • 1 µL of BBa_k1073022 = 1100 ng

2. Add 1/10 volume of
          Sodium Acetate (2,8 M, pH 4.8).

          3. Add 2.5-3.0X volume (calculated after addition of sodium acetate)
          of at least 95% ethanol.

          4. Vortex the sample

          5. Incubate 10 min on ice

          6. Centrifuge 25 min at 13,000 rpm at room temperature

          7. Discard the supernatant carefully using a pipet on the opposite
          side of the pellet

          8. Rinse with 500 µL of cold 70% EtOH

          9. Invert gently the tubes to mix

          10. Centrifuge 5 min at 13,000 rpm at room temperature

          11. Discard the supernatant carefully using a pipet on the opposite
          side of the pellet

          12. Dry the pellet

Making competent Pseudomonas aeruginosa cells

In order to transform Pseudomonas aeruginosa with
          BBa_k1033925 and BBa_k1073022 biobricks, we need to make them
          competent. We prepared 2 aliquots of competent bacteria for each
          biobrick tested.

1. Prepare 4x aliquots of 1
          mL of an overnight bacterial culture.

          Remark: The number of aliquots depends on the number of
            transformations needed.

          2. Centrifuge for 2 min at 10,000 rpm

          3. Throw away the supernatant by flipping the tube. Eliminate the last
          drop of supernatant by dabbing gently on absorbent paper

          4. Resuspend in 1 mL of 300 mM sucrose, centrifuge for 2 min at 100
          rpm and throw away the supernatant

          5. Repeat the process twice to carry out 3 successive washes

          6. After the last wash, resuspend the pellet with 50 µL of 300 mM
          sucrose

Transformation of competent Pseudomonas aeruginosa
          cells with BBa_K1033925 and BBa_K1073022 biobricks

1. Transfer the 50 µL of competent cells to the dry pellet of plasmids
          and resuspend

          2. Incubate 5 min at room temperature

          3. Add cell/DNA mixture to the 0,2 mm electroporation cuvette. Tap the
          cuvette gently on the counter to move cells to the bottom.

          4. Place cuvette in electroporator. Close lid.

          5. Press the “Pulse” button on the electroporator to shock cells. (the
          program was the following one: voltage at 250 V, capacitance at 25 µF,
          resistance at 200 Ohm and cuvettes had 2mm of depth)

          6. Remove cuvette from the chamber and immediately transfer cells to 1
          mL of pre-heated LB to prevent cells from dying off

          7. Incubate tube in 37°C shaker for 1h20 to permit expression of
          antibiotic
          resistance gene.

          8. Plate 100 µL transformation onto prewarmed LB-agar plate

          9. Short-spin the cells and resuspend the cell pellet with 100 µL of
          LB and plate it onto prewarmed LB-agar plate.

          Remark: This step is optional but if bacteria survival is low, it
            will increase the chance to get more colonies.

          10. Incubate plate overnight at 37°C.

Midiprep - E. coli containing the plasmid pPMK4Pprot

This protocol was performed according to the
          manufacturer’s recommendations. The sample was checked with a Nanodrop
          2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Plasmid pMK4 | 2141.9 | 1.84 | 2.29 |

10.12.19

Resuspension of primers

Primers were produced by Eurofins Genomics. Once
          received, they were resuspended according to the manufacturer’s
          instructions. Stock solutions of 100 µM were made by diluting the
          lyophilized oligonucleotides with a 10 mM Tris-EDTA pH 8.0 solution.

• 160 µL were added to
          mRFPPrimerFD

          • 147 µL were added to mRFPPrimerRV

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| mRFPPrimerFD | 1418.2 | 1.87 | 1.44 |
| mRFPPrimerRV | 1663.2 | 2.08 | 1.36 |

Concentration of the biobrick BBa_J04450

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| J04450 biobrick | 74.2 | 1.87 | 2.10 |

PCR 19 - Adding BamHI and SalI restriction sites to
          BBa_J04450 biobrick

In order to insert the BBa_J04450 biobrick (mRFP) into
          the pMK4Pprot plasmid, a PCR was performed using specific primers
          containing the BamHI and SalI restriction sites.

|  |  |  |
| --- | --- | --- |
| Components | Final concentration (ng/µl) | Volume for the mix |
| 5X Phusion HF buffer | 1X | 100 |
| 25 µM dNTPs | 0.20 mM | 10 |
| mRFPPrimerFD | 0.5 µM | 25 |
| mRFPPrimerRV | 0.5 µM | 25 |
| J04450 biobrick | 10ng | 10 |
| Phusion DNA polymerase | 1 U/µl | 5 |
| Nuclease-free water | / | 325 |
| Total volume | / | 500 |

The samples are then analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| mRFP after PCR | 1561.4 | 1.66 | 1.25 |

Transformation of competent Escherichia coli
          DH5α cells with BBa_K3176020 plasmids

1.Thaw a tube of 250 µL of
          DH5α competent E. coli cells on ice for 10 minutes.

          2. Add 20 µl of plasmid DNA to the cell mixture. Carefully flick the
          tube 4-5 times to mix cells and DNA. Do not vortex.

          3. Place the mixture on ice for 30 minutes. Do not mix.

          4. Heat shock at exactly 42°C for exactly 40 seconds. Do not mix.

          5. Place on ice for 5 minutes. Do not mix.

          6. Add 225 µl of SOC into the mixture.

          7. Place the mixture at 37°C for 60 minutes. Shake vigorously (250
          rpm) or rotate.

          8. Warm selection plates to 37°C during 20 minutes.

          9. Plate the mixture into chloramphenicol LB plates and wait 5 minutes
          at RT

          10. Incubate 16-24 hours at 37°C

October Week 3  : 10/14  to  10/21

10.14.19

PCR 20 - Amplification of the two last rounds for SA and
          EF

In order to run a gel with all the rounds of aptamers,
          we reamplified the rounds 9 and 10 for both S. aureus and E.
            faecium.

|  |  |
| --- | --- |
| Components | Volume for the mix |
| 5X Phusion HF buffer | 10 |
| 25 mM dNTPs | 1 |
| Primer P1 | 2 |
| Primer P2 | 2 |
| DNA EFr9 | 1 |
| Phusion DNA polymerase | 0.5 |
| Nuclease-free water | 33.5 |
| Total volume | 50 |

|  |  |
| --- | --- |
| Components | Volume for the mix |
| 5X Phusion HF buffer | 10 |
| 25 mM dNTPs | 1 |
| Primer P1 | 2 |
| Primer P2 | 2 |
| DNA EFr9 | 1 |
| Phusion DNA polymerase | 0.5 |
| Nuclease-free water | 33.5 |
| Total volume | 50 |

|  |  |
| --- | --- |
| Components | Volume for the mix |
| 5X Phusion HF buffer | 10 |
| 25 mM dNTPs | 1 |
| Primer P1 | 2 |
| Primer P2 | 2 |
| DNA SAr9 | 1 |
| Phusion DNA polymerase | 0.5 |
| Nuclease-free water | 33.5 |
| Total volume | 50 |

|  |  |
| --- | --- |
| Components | Volume for the mix |
| 5X Phusion HF buffer | 10 |
| 25 mM dNTPs | 1 |
| Primer P1 | 2 |
| Primer P2 | 2 |
| DNA SAr10 | 1 |
| Phusion DNA polymerase | 0.5 |
| Nuclease-free water | 33.5 |
| Total volume | 50 |

Phenol/chloroform extraction with Isopropanol and
          Glycogen - preparation of mRFP insert

A new purification step by phenol/chloroform was
          performed in order to get a pure insert before the ligation step.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| mRFP purified | 1404.5 | 1.60 | 2.01 |

Double digestion with SalI-HF and BamHI-HF - mRFP &
          pMK4

After being purified, mRFP as well as pMK4 were digested
          using SalI-HF and BamHI-HF enzymes from New England Biolabs.

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | MIX |
| 1 µg DNA (mRFP) | 0.7 µL | 10.5 µL |
| 10X Cutsmart Buffer | 5 µL | 75 µL |
| Sall-HF | 1 µL | 15 µL |
| BamHI-HF | 1 µL | 15 µL |
| Nuclease-free water | 24.3 µL | 634.3 µL |

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | MIX |
| 1 µg DNA (pMK4) | 0.5 µL | 7.5 µL |
| 10X Cutsmart Buffer | 5 µL | 75 µL |
| Sall-HF | 1 µL | 15 µL |
| BamHI-HF | 1 µL | 15 µL |
| Nuclease-free water | 42.5 µL | 637.5 µL |

1. Incubate at 37°C for 15
          minutes

          2.Incubation at 65°C for 20 min (inactivation of SalI-HF) as
          recommended on
          https://international.neb.com/tools-and-resources/usage-guidelines/heat-inactivation

Phenol/chloroform extraction with Isopropanol and
          Glycogen - preparation of mRFP insert

This step was performed as previously described. The
          samples are then analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| mRFP digested & purified | 275.7 | 1.59 | 1.41 |

Gel electrophoresis 14 - Plasmid pMK4Pprot

After the double digestion, the plasmid pMK4Pprot was
          loaded onto a gel of 1% agarose during 1 hour at 120 volts in order
          to, after, extract and purify it.

![](https://static.igem.org/mediawiki/2019/0/03/T--Pasteur_Paris--18.png)

Gel extraction purification - Plasmid pMK4Pprot

We used the Kit montage Gel extraction from Millipore to
          extract the plasmid and purify it. This kit was used according to the
          manufacturer’s protocol. The samples are then analyzed using a
          Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| 1 | 6.2 | 1.45 | 0.12 |
| 2 | 10.3 | 1.47 | 0.14 |
| 3 | 16.3 | 1.77 | 0.24 |
| 4 | 96.8 | 2.20 | 1.31 |
| 5 | 3.6 | 1.41 | 0.08 |
| 6 | 6.3 | 1.59 | 0.11 |
| 7 | 16.7 | 1.50 | 0.26 |
| 8 | 4.8 | 1.89 | 0.10 |
| 9 | 12.2 | 1.41 | 0.17 |
| 10 | 7.9 | 1.39 | 0.15 |
| 11 | 8.0 | 1.98 | 0.16 |
| 12 | 7.2 | 1.51 | 0.15 |

Ligation - BBa_J04450 in pMK4Pprot vector

In order to insert the biobrick BBa_J04450 into the
          vector pMK4Pprot, we used the T4 DNA ligase from Promega. This
          experiment was performed accordingly to the manufacturer’s protocol.

|  |  |  |  |
| --- | --- | --- | --- |
| Ratio | 1:1 | 1:3 | 1:7 |
| Vector | 5 (475 ng) | 5 (475 ng) | 5 (475 ng) |
| Insert (1:100)* | ".6 (10 ng) | 10.8 | 25.2 |
| 10X ligase buffer | 2 | 2 | 2 |
| T4 DNA ligase | 0.6 | 0.6 | 0.6 |
| DNase-free water | 8.8 | 1.6 | 0 |

*Insert 1:100 -> 1 µL of insert into 99 µL of TE
            buffe

          The samples are analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Ratio 1:1 | 1488.6 | 2.99 | 2.93 |
| Ratio 1:3 | 986.8 | 3.01 | 3.01 |
| Ratio 1:7 | 590.3 | 2.97 | 2.80 |

10.17.19

Cyclic Voltammetry

For this experiment, we went to the lab directed by
          Sophie Griveau in one of our chemistry schools, Chimie Paristech,
          where we were given free access to a potentiostat, software and
          electrode adaptor.

          A solution of ferrocyanate [Fe(CN)₆]⁴⁻ was used for the redox couple.
          The electrolyte was simply PBS, which contains enough NaCl to act as
          such. The potentiostat was from Gamry Instruments, and the
          corresponding Gamry software was used for data treatment.

          A solution of ferrocyanate (5mM) in PBS was prepared and 100 µL of it
          was deposited onto the electrode. The extrema of the potential sweep
          were -0.2 V and 0.6 V with a scan rate of 10 mV/s.

          First, six sweeps were done on two different carbon-nanotubes-only
          electrodes. This gave us a baseline to compare with the aptamer
          electrodes. Then, the solution was deposited on two different
          electrodes per strain and two measurements were taken for each strain,
          for a total of four curves per strain.

Gel electrophoresis 15 - pMK4Pprot

For new experiments, the plasmid pMK4Pprot was run again
          on a gel containing 1% of agarose during 1 hour at 120 volts.

//IMAGE//

Gel extraction purification - Plasmid pMK4Pprot

We used the Kit montage Gel extraction from Millipore to
          extract the plasmid and purify it. This kit was used according to the
          manufacturer’s protocol. The samples are then analyzed using a
          Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| Slice 1 | 14.3 | 1.69 | 0.20 |
| Slice 2 | 12 | 1.51 | 0.20 |
| Slice 3 | 11.3 | 1.61 | 0.17 |
| Slice 4 | 14.8 | 1.64 | 0.2 |
| Slice 5 | 6.3 | 2.41 | 0.12 |
| Slice 6 | 9.8 | 1.69 | 0.16 |

10.18.19

Resuspension of primers - Biobricks from BBa_K3176000 to
          BBa_K3176003

Primers were produced by Eurofins Genomics. Once
          received, they were resuspended according to the manufacturer’s
          instructions. Stock solutions of 100 µM were made by diluting the
          lyophilized oligonucleotides with a 10 mM Tris-EDTA pH 8.0 solution.

|  |  |
| --- | --- |
| Sample | TE |
| P1 - SarAP1 - P2 | 178 µL |
| P2 - SarARBS - P3 | 163 µL |
| P2 - hldRBS - P3 | 161 µL |
| P2 - sodARBS - P3 | 170 µL |
| P3 - mRFP | 175 µL |
| P4 - SarAP1 - P2 | 111 µL |
| mRFP - P1 | 152 µL |

The samples were then analysed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| P1 - SarAP1 - P2 | 1778.5 | 1.75 | 1.41 |
| P2 - SarARBS - P3 | 1943.3 | 1.89 | 1.38 |
| P2 - hldRBS - P3 | 2036.5 | 1.89 | 1:37 |
| P2 - sodARBS - P3 | 1935.2 | 1.88 | 1.47 |
| P3 - mRFP | 1574.5 | 1.85 | 1.33 |
| P4 - SarAP1 - P2 | 1768.7 | 1.75 | 1.45 |
| mRFP - P1 | 1691.8 | 2.13 | 1.29 |

Simple digestion with EcorI- Biobricks from BBa_K3176000
          to BBa_K3176003

1. Purify your PCR products
          by phenol/chloroform extraction

          2. Set up reaction as follows:

|  |  |
| --- | --- |
| Components | 50 µL reaction |
| DNA insert | 1 µL |
| CutSmart buffer 10X | 5 µL (1X) |
| EcorI-HF | 1 µL (20 units) |
| Nuclease-free water | up to 50 µL |

3. Incubate at 37°C for
          5-15 minutes

Incubation at 80°C for 20 min (inactivation of Hind-III
          HF) as recommended on
https://international.neb.com/tools-and-resources/usage-guidelines/heat-inactivation.

Phenol/chloroform extraction- Biobricks from
          BBa_K3176000 to BBa_K3176003

We performed a phenol/chloroform extraction as
          previously described to remove the enzymes and the buffer. The samples
          were analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| P1 - SarAP1 - P2 | 313.7 | 1.6 | 1.68 |
| P2 - SarARBS - P3 | 442 | 1.71 | 1.82 |
| P2 - hldRBS - P3 | 297.4 | 1.61 | 1:70 |
| P2 - sodARBS - P3 | 282 | 1.60 | 1.76 |
| P4 - SarAP1 - P2 | 341.7 | 1.60 | 1.69 |

Ligation - Biobricks from BBa_K3176000 to BBa_K3176003

A ligation was performed as detailed in the protocol of
          the manufacturer.

|  |  |
| --- | --- |
| Sample | Volume |
| P1 - SarAP1 - P2 | 1 µL |
| P1 - SarAP1 - P2 | 1 µL |
| P2 - SarARBS - P3 | 0,7 µL |
| Buffer 10X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 15.7 µL |

|  |  |
| --- | --- |
| Sample | Volume |
| P1 - SarAP1 - P2 | 1 µL |
| P2 - hldRBS - P3 | 1 µL |
| Buffer 10X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 15.4 µL |

|  |  |
| --- | --- |
| Sample | Volume |
| P4 - SarAP1 - P2 | 0.9 µL |
| P2 - hldRBS - P3 | 1 µL |
| Buffer 10X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 15.5 µL |

|  |  |
| --- | --- |
| Sample | Volume |
| P4 - SarAP1 - P2 | 0.9 µL |
| P2 - SarARBS - P3 | 0.7 µL |
| Buffer 10X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 15.8 µL |

|  |  |
| --- | --- |
| Sample | Volume |
| P4 - SarAP1 - P2 | 0.9 µL |
| P2 - sodARBS - P3 | 1 µL |
| Buffer 10X | 2 µL |
| T4 DNA ligase | 0.6 µL |
| DNAse-free water | 15.5 µL |

The samples were analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| 1 - 1PC + 2PC | 602.5 | 5.43 | 9.41 |
| 2 - 1PC + 3PC | 481.6 | 6.16 | 22.3 |
| 3 - 1PC + 4PC | 684.8 | 1.90 | 2:39 |
| 4 - 5PC + 2PC | 593.4 | 5:38 | 9:49 |
| 5 - 5PC + 3PC | 615.9 | 5.12 | 8.29 |
| 6 - 5PC + 4PC | 483.6 | 6.05 | 20.77 |

10.19.19

Bacterial culture - BBa_K1073022

In order to analyze the fluorescence in E. coli
          DH5ɑ expressed by the biobrick BBa_K1073022, a two culture was made
          (from the 19.10 to the 21.10).

PCR 21 - mRFP insert

We performed

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SH1 | 1870 | 1.67 | 1.21 |
| SH2 | 1689.9 | 1.63 | 1.11 |
| HS1 | 875 | 1.57 | 0:93 |
| HS2 | 850 | 1:55 | 0.9 |

Simple digestion with HindIII-mRFP- Biobricks from
          BBa_K3176000 to BBa_K3176003

1. Set up reaction as
          follows:

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | 750 µL reaction |
| DNA insert (SH) | 0.6 µL (1 µg) | 9 µL |
| CutSmart buffer 10X | 5 µL (1X) | 75 µL |
| HindIII-HF | 1 µL (20 units) | 15 µL (1 µg) |
| Nuclease-free water | 43.4 µL | 651 µL |

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | 750 µL reaction |
| DNA insert (HS) | 1.2 µL (1 µg) | 18 µL |
| CutSmart buffer 10X | 5 µL (1X) | 75 µL |
| HindIII-HF | 1 µL (20 units) | 15 µL (1 µg) |
| Nuclease-free water | 42.8 µL | 642 µL |

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | 500 µL reaction |
| DNA insert (1) | 1.7 µL (1 µg) | 17 µL |
| CutSmart buffer 10X | 5 µL (1X) | 50 µL |
| HindIII-HF | 1 µL (20 units) | 10 µL |
| Nuclease-free water | up to 50 µL | 423 µL |

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | 500 µL reaction |
| DNA insert (2) | 2.1 µL (1 µg) | 21 µL |
| CutSmart buffer 10X | 5 µL (1X) | 50 µL |
| HindIII-HF | 1 µL (20 units) | 10 µL |
| Nuclease-free water | up to 50 µL | 419 µL |

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | 500 µL reaction |
| DNA insert (3) | 1.5 µL (1 µg) | 15 µL |
| CutSmart buffer 10X | 5 µL (1X) | 50 µL |
| HindIII-HF | 1 µL (20 units) | 10 µL |
| Nuclease-free water | up to 50 µL | 425 µL |

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | 500 µL reaction |
| DNA insert (4) | 1.7 µL (1 µg) | 17 µL |
| CutSmart buffer 10X | 5 µL (1X) | 50 µL |
| HindIII-HF | 1 µL (20 units) | 10 µL |
| Nuclease-free water | up to 50 µL | 423 µL |

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | 500 µL reaction |
| DNA insert (5) | 1.6 µL (1 µg) | 16 µL |
| CutSmart buffer 10X | 5 µL (1X) | 50 µL |
| HindIII-HF | 1 µL (20 units) | 10 µL |
| Nuclease-free water | up to 50 µL | 424 µL |

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | 500 µL reaction |
| DNA insert (6) | 2 µL (1 µg) | 20 µL |
| CutSmart buffer 10X | 5 µL (1X) | 50 µL |
| HindIII-HF | 1 µL (20 units) | 10 µL |
| Nuclease-free water | up to 50 µL | 420 µL |

2. Incubate at 37°C for
          5-15 minutes

          3. Incubation at 80°C for 20 min (inactivation of Hind-III HF) as
          recommended on
          https://international.neb.com/tools-and-resources/usage-guidelines/heat-inactivation

Phenol/chloroform extraction- Biobricks from
          BBa_K3176000 to BBa_K3176003

We performed a phenol/chloroform extraction as
          previously described to remove the enzymes and the buffer. The samples
          were analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SH PC | 117.0 | 1.44 | 1.54 |
| HS PC | 231.7 | 1.53/td> | 1.3 |
| 3 PC | 79.9 | 1.39 | 1.26 |
| 4 PC | 89.7 | 1.39 | 1.26 |
| 5 PC | 155.7 | 1.46 | 1.44 |
| 6 PC | 89.4 | 1.39 | 1.31 |
| PC 7 | 80.9 | 1.37 | 1.28 |
| PC 8 | 115.6 | 1.40 | 1.38 |

Ligation - Biobricks from BBa_K3176000 to BBa_K3176003

A ligation was performed as detailed in the protocol of
          the manufacturer.

|  |  |
| --- | --- |
| Sample | Volume |
| HS | 2.6 |
| 3 | 7.6 |
| Buffer 10X | 4 |
| T4 DNA ligase | 1.2 |
| DNAase-free water | 24.6 |

|  |  |
| --- | --- |
| Sample | Volume |
| HS | 2.6 |
| 4 | 6.6 |
| Buffer 10X | 4 |
| T4 DNA ligase | 1.2 |
| DNAase-free water | 25.6 |

|  |  |
| --- | --- |
| Sample | Volume |
| HS | 2.6 |
| 5 | 3.8 |
| Buffer 10X | 4 |
| T4 DNA ligase | 1.2 |
| DNAase-free water | 28.4 |

|  |  |
| --- | --- |
| Sample | Volume |
| SH | 5.2 |
| 6 | 6.8 |
| Buffer 10X | 4 |
| T4 DNA ligase | 1.2 |
| DNAase-free water | 22.8 |

|  |  |
| --- | --- |
| Sample | Volume |
| SH | 5.2 |
| 7 | 7.4 |
| Buffer 10X | 4 |
| T4 DNA ligase | 1.2 |
| DNAase-free water | 22.2 |

|  |  |
| --- | --- |
| Sample | Volume |
| SH | 5.2 |
| 6 | 6.8 |
| Buffer 10X | 4 |
| T4 DNA ligase | 1.2 |
| DNAase-free water | 22.8 |

The samples were analyzed using a Nanodrop 2000
          spectrophotometer.

|  |  |
| --- | --- |
| Sample | Volume |
| SH | 5.2 |
| 8 | 5.2 |
| Buffer 10X | 4 |
| T4 DNA ligase | 1.2 |
| DNAase-free water | 24.4 |

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SH-6 | 1083.6 | 2.97 | 3 |
| SH-7 | 986.8 | 3.19 | 3.16 |
| SH-8 | 1015.3 | 2.86 | 2.61 |
| HS-3 | 917.6 | 3.34 | 3.24 |
| HS-4 | 914.4 | 3.34 | 3.20 |
| HS-5 | 895.2 | 3.39 | 3.29 |

Double digestion with BamHI and SalI - Biobricks from
          BBa_K3176000 to BBa_K3176003

After being purified, the insert built using SarAP1
          promoter, mRFP1 and a RBS (either SarA, hld and sodA) was digested
          using SalI-HF and BamHI-HF enzymes from New England Biolabs

Purification with kit Macherey-Nagel - Biobricks from
          BBa_K3176000 to BBa_K3176003

After the digestion, we had to purify our insert before
          the ligation step. We performed a purification using the kit
          NucleoSpin Gel and PCR Clean-up of Macherey-Nagel, following the
          manufacturer’s instructions.

          The samples were analyzed using a Nanodrop 2000 spectrophotometer.

|  |  |  |
| --- | --- | --- |
| Components | 50 µL reaction | MIX |
| 1 µg DNA (mRFP) | 0.7 µL | 10.5 µL |
| 1 µg DNA (mRFP) | 0.7 µL | 10.5 µL |
| 10X Cutsmart Buffer | 5 µL | 75 µL |
| Sall-HF | 1 µL | 15 µL |
| BamHI-HF | 1 µL | 15 µL |
| Nuclease-free water | 24.3 µL | 634.3 µL |

|  |  |  |  |
| --- | --- | --- | --- |
| Sample | Concentration (ng/µl) | 260/280 ratio | 260/230 ratio |
| SH-6 dig + purif | 7.8 | 2.44 | 0.02 |
| SH-6 dig + purif bis | 4.1 | 2.19 | 0.014 |
| SH-7 dig + purif | 6.0 | 2.03 | 0.06 |
| SH-7 dig + purif bis | 12.5 | 1.83 | 0.05 |
| SH-8 dig + purif | 4.9 | 1.61 | 0.11 |
| SH-8 dig + purif bis | 3.6 | 2.66 | 0.03 |
| HS-3 dig + purif | 4.0 | 2.35 | 0.07 |
| HS-3 dig + purif bis | 2.1 | 5.84 | 0.05 |
| HS-4 dig + purif | 12.1 | 2.44 | 0.02 |
| HS-4 dig + purif bis | 5.3 | 2.22 | 0.04 |
| HS-5 dig + purif | 4.7 | 2.1 | 0.04 |
| HS-5 dig + purif bis | 4.0 | 2.38 | 0.07 |

We cannot make the transformation of S. aureus due to a
          too low concentration of the plasmid.

10.20.19

Bacterial culture - BBa_K1073022

In order to analyze the fluorescence in E. coli DH5ɑ expressed by the
          biobrick BBa_K1073022, an overnight culture was made.

Gel electrophoresis 16 - Results parts

In order to organize our results page, we ran two 4%
          agarose gels containing the 10 rounds of both E. faecium
          (first gel) and S. aureus (second gel). Both gels were run at
          120 volts during 1 hour approximately.

![](https://static.igem.org/mediawiki/2019/4/48/T--Pasteur_Paris--19.png)

![](https://static.igem.org/mediawiki/2019/0/08/T--Pasteur_Paris--20.png)

10.21.19

Bacterial culture - BBa_K1073022

In order to analyze the fluorescence in E. coli DH5ɑ
          expressed by the biobrick BBa_K1073022, a culture was made. This
          culture will serve as a time 0h reference.

Fluorescence microscopy - BBa_K1073022

In order to determine if the fluorescence is expressed
          by the biobrick BBa_K1073022, we analysed transformed E. coli with the
          fluorescence microscopy Evos life techno. Three types of samples were
          analysed, a negative control containing non transformed E. coli, E.
          coli transformed and grew during 12 hours and E. coli transformed and
          grew during 30 hours. This experiment was performed for us by Dr.
          Tobias Sahr from the team Biology of Intracellular bacteria from the
          Pasteur Institute.

![](https://static.igem.org/mediawiki/2019/f/f9/T--Pasteur_Paris--22.png)

![](https://static.igem.org/mediawiki/2019/5/5a/T--Pasteur_Paris--23.png)

Interpretation

RFP fluorescence is not expressed by all bacteria cells.
          On merged image, we can see that less than half of bacteria show red
          fluorescence.

Flow cytometry - BBa_K1073022

In order to determine if the fluorescence expressed by
          the biobrick BBa_K1073022 stay stable over time we analysed
          transformed E. coli with a flow cytometer Moflo astrios EQ (Beckman
          Coulter). Four types of samples were analysed, a negative control
          containing non transformed E. coli (sample ctrl neg), E. coli
          transformed and grew during 2 hours (sample 1), E. coli transformed
          and grew during 12 hours (sample 2) and E. coli transformed and grew
          during 30 hours (sample 3). This experiment was performed for us by
          Pierre-Henri Commere from the Cytometry and Biomarkers platform.

![](https://static.igem.org/mediawiki/2019/e/ed/T--Pasteur_Paris--24.jpeg)

Interpretation

The sample 2 was our reference because the bacteria grew
          during 12 hours which is the appropriate time of an overnight culture.
          We observed that its fluorescent peak corresponds to around 103 and
          104. Moreover, only 40% of bacteria were fluorescent. The sample 1 had
          a slight shift toward the left side but 50% of the bacteria population
          were fluorescent. Finally, the sample 3 emitted fluorescence in the
          same range as the sample 2 but fewer bacteria were expressing
          fluorescence.
          Thus, we may conclude that along time the bacteria may lose their
          fluorescence properties. It can be due to the fact that bacteria
          express less the chromoprotein along time.

10.21.19

Bacterial detection with aptamers/carbon-nanotube
            electrodes

As a proof of concept, we used our arduino as a
            voltmeter, to record the response profile of our electrodes
            following the addition of bacteria at different time points. The
            objective was to demonstrate the sensitivity and specificity of our
            own aptamers designed for E. faecium.

For each test, the same protocol was performed, as
            following :

1.50 µL of 1X PBS are
            added on the electrode.

            2.After stabilization of the potential (2-3mins), 25 µL of bacterial
            solution are deposited on the PBS droplet and mix up and down.

            3.Change in potential is recorded.

            4. After 100 seconds, 25 µL of bacterial solution are added once
            again.

After each test, the same washing protocol was
            performed, as following :

1. Tested solution is
            removed.

            2. The electrode is washed twice using 50 µL of 5M NaCl solution.

            3. The electrode is gently dried.

            4. 50 µL of 1X PBS are added on the electrode to maintain it in
            solution.

![]()https://static.igem.org/mediawiki/2019/thumb/3/36/T--Pasteur_Paris--electrodes_aptameres.jpeg/450px-T--Pasteur_Paris--electrodes_aptameres.jpeg

![]()https://static.igem.org/mediawiki/2019/thumb/3/3e/T--Pasteur_Paris--goutte.jpeg/800px-T--Pasteur_Paris--goutte.jpeg"

Four conditions were tested.

As a negative control, PBS was added on the surface of
            the electrode, following the previous protocol. Two pics are
            observed perfectly corresponding to the moment when the drop is
            added. This environment disturbance induces a temporary change in
            potential, before quickly returning to the basic potential.

            Regarding the bacterial solutions, E. faecium was first
            added, following the previous protocol. The staircase trend in the
            curve shows that bacteria are well able to bind to the aptamers,
            inducing a change in their conformation.

In a second time, the specificity of the aptamers was
            tested by adding a solution of S. pneumoniae and one of E. coli,
            following the previous protocol. The curves do not show any
            staircase potential jumps as observed for E. faecium. Indeed their
            profiles are similar to the PBS control : with two pics when the
            drop is added, before quickly returning to the basic potential.
```


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# Team:Pasteur Paris/Experiments

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# 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.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

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### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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# Team:Pasteur Paris/Results

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Picto Sciences

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[1

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ecr-v-1

ecr-p-2

ecr-v-1

ecr-p-2

ecr-p-2

ecr-p-2

ecr-v-1

ecr-p-2

ecr-p-2

ecr-v-1

ecr-p-2

ecr-v-1

ecr-p-2

ecr-p-2

ecr-v-1

ecr-p-2

ecr-v-1

ecr-p-2

RESULTS BIO

- SELEX

Main results :

Our project is based on aptamers which are
single-stranded DNA able to recognize a specific target with a high
affinity. In order to find potential aptamers, we choose 2 strains of
bacteria, *S. aureus* and *E. faecium* and we performed 10 rounds of SELEX
(Systematic Evolution of Ligands by EXponential enrichment) on both
bacteria.

After these 10 rounds of SELEX, gel electrophoresis were
made in order to verify that a high concentration of potential
aptamers are still present.

![](https://static.igem.org/mediawiki/2019/4/48/T--Pasteur_Paris--19.png)

Figure 1: Gel electrophoresis of 10 rounds of SELEX for
*E. faecium* The gel is a 4% agarose one and the ladder used was a 50 bp
ladder from Promega. The gel ran at 120 volts during 1 hour
approximately.

We can observe that for the candidate aptamers selected
against *E. faecium* we always had a single band throughout the rounds
of SELEX (Figure 1). This gel demonstrates that a selection was
applied on the initial DNA library because the bands are less brighter
at the rounds 9 and 10 than the firsts rounds. This suggests that the
most part of non-specific aptamers of *E. faecium* were removed during
the washes made during the SELEX and that the specificity of the
potential aptamers for *E. faecium* may have increased over the rounds.

![](https://static.igem.org/mediawiki/2019/0/08/T--Pasteur_Paris--20.png)

Figure 2: Gel electrophoresis of 10 rounds of SELEX for
*S. aureus* The gel is a 4% agarose one and the ladder used was a 50 bp
ladder from Promega. The gel ran at 120 volts during 1 hour
approximately.

As the previous one, the figure 2 demonstrates that the
potential aptamers against *S. aureus* were selected because the bands
loses their brightness over the rounds. However, on the round 9, we
can notice 3 bands instead of one. It may be due to non specific
amplification of DNA during the PCR. Despite this, it seems that these
non specific amplifications were removed during the washes because
they are not revealed during the round 10. So, we may think it will
not interfere with the candidate aptamers.

![](https://static.igem.org/mediawiki/parts/9/9a/T--Pasteur_Paris--plasmide_pET28_GFP.png)

Figure 3: Gel electrophoresis of the plasmid pET28:GFP
at different step of the cloning experiment. The gel is a 1% agarose
one and the ladder used was a 1 kbp ladder from Thermo Fischer. The
gel ran at 120 volts during 1 hour approximately.

On the figure 3, we can observe the different steps of
the cloning experiment. First of all, the plasmid pET28:GFP was
digested using the restriction enzymes HindIII and BamHI. We can see a
decrease on its size around 5 kbp which is a sign that the digestion
went well. Then, the insert, either aptamers against *S. aureus* or
those against *E. faecium* were ligated into the plasmid in order to
create the biobrick BBa\_K3176020. Thus, on the gel, we can see that after this step there is two bands for both samples, proving that the ligation was well performed.

Sequencing

10 rounds of SELEX were performed in order to select
potential aptamer candidates. These aptamers were cloned into
pET28:GFP in order to be sequenced as bacterial clones. A total of 15
bacterial clones for *E. faecium* and 39 for *S. aureus* were sequenced.
Unfortunately, the majority of the sequences obtained did not
correspond to an aptamer sequence. Only two bacterial clones contained
the expected 40 nucleotides of our initial library. These two
sequences were found for *E. faecium* strain, none was found for
S.aureus. Here is an histogram showing the sequence for one of these
two aptamers.

![](https://static.igem.org/mediawiki/2019/6/6c/T--Pasteur_Paris--sqc.png)

Figure 4: Sequencing profile of the aptamer selected
against *E. faecium*
The sequencing was performed by Eurofins genomics after we send them
the transformed and isolated clone.

Characterization of BBa\_K1073022

We chose to characterize BBa\_K1073022, the eforRed
chromoprotein, and focus on its fluorescence expression using two
techniques: flow cytometry and fluorescence microscopy.

Flow cytometry

Throughout the flow cytometry, we would like to
determine the transformation efficiency as well as the stability of
the fluorescence expression. For that, four samples were analysed, a
negative control containing non transformed *E. coli*, *E. coli*
transformed and grew during 2 hours, *E. coli* transformed and grew
during 12 hours and *E. coli* transformed and grew during 30 hours.

![](https://static.igem.org/mediawiki/2019/8/81/T--Pasteur_Paris--Flow_cyto.png)

Figure 5: Flow cytometry of *E. coli* transformed with BBa\_K1073022

On the left, the figure represents the number of bacteria which were analyzed.

On the top right, the figure represents the emission fluorescence of bacteria. Sample 3: *E. coli* transformed and grew during 30 hours, sample 2: *E. coli* transformed and grew during 12 hours, sample 1: *E. coli* transformed and grew during 2 hours, ctrl neg: negative control containing non transformed *E. coli*.

The sample 2 was our reference because the bacteria grew during 12 hours which is the appropriate time of an overnight culture. We observed that its fluorescent peak corresponds to around 103 and 104. Moreover, only 40% of bacteria were fluorescent. The sample 1 had a slight shift toward the left side but 50% of the bacteria population were fluorescent. Finally, the sample 3 emitted fluorescence in the same range as the sample 2 but fewer bacteria were expressing fluorescence.

Hence, due to the low number of bacteria expressing fluorescence only 2 hours of incubation, it seems that the transformation efficiency is low.

When the selective pressure was removed, the bacteria continued to lose expression of the chromoprotein as wild type bacteria out-competed those with the plasmid. Moreover, due to the fact that the expression decreases over time, we may conclude that along time the bacteria may lose their fluorescence properties. It can be due to the fact that bacteria express fluorescence less over time.

Fluorescent microscopy

For this experiment, the expression of eforRed was characterized. Three types of samples were analysed, a negative control containing non-transformed *E. coli*, *E. coli* transformed and grew during 12 hours and *E. coli* transformed and grew during 30 hours. As we can clearly see, transformed *E. coli* express red fluorescence when excited at 580 nm.

![](https://static.igem.org/mediawiki/2019/9/9d/T--Pasteur_Paris--RFP.png)

Figure 6: RFP fluorescence of the eforRed chromoprotein

**(A)** *E. coli* DH5α before being transformed. **(B)** *E. coli* DH5α after being transformed using BBa\_K1073022. Scale bars represents 10µm.

However, when we merged brightfield and mRFP images, we observed that the fluorescence is not expressed by all of the bacteria. This result is consistent with the percentages established during the flow cytometry experiment.

![](https://static.igem.org/mediawiki/2019/1/1b/T--Pasteur_Paris--RFPLIGHT.png)

Figure 7: RFP fluorescence efficiency

Chemistry

Cyclic voltammetry

If the aptamers were correctly bound to the electrodes,
they should act as a protective layer around the electrodes. When they
are not in contact with bacteria, a potential corresponding to PBS is
therefore measured. When bacteria are added, the aptamers change
conformation and allow the ions in solution easier access to the
electrode: therefore, the overall potential increases.

![](https://static.igem.org/mediawiki/2019/b/b0/T--Pasteur_Paris--scheme_aptamers.png)

Figure 8: Explanation of the increase of potential in presence of bacteria

**Cyclic voltammetry** is an analytical chemistry technique, which involves the use of a redox couple. A solution containing electrochemical species are deposited onto the electrode. Then, using a potentiostat, a potential is applied to the electrode in a sweep: starting from low potentials to higher ones, then back again. The response in current is measured and plotted against the potential.

![](https://static.igem.org/mediawiki/2019/1/11/T--Pasteur_Paris--duck_volta.png)

Figure 9: Cyclic voltammetry *ref: Edmund Dickinson, Modeling Electroanalysis: Cyclic Voltammetry, COMSOL Blog*

The above graph is the usual waveform of a cyclic
voltammetry analysis. When sweeping towards higher potentials, the
electrochemical species are oxidized, which translates into a peek in
current. When sweeping back, the oxidized species are reduced,
theoretically yielding an equivalent peak, but in negative current.
We conducted the experiments both with nanotubes-only electrodes, and
with aptamer-functionalized electrodes. If we correctly deposited
aptamers, they should form a protective layer around the electrode
like mentioned previously: therefore, the electrochemical species
should have a harder time getting close to the electrode to be
oxidized and reduced, and the peaks should be lower in intensity.

While no difference was noted in the oxidation peak, it is quite clear
that the reduction peak is more intense and slightly shifted towards
the lower potentials. This modification occurs on all electrodes for
all the strains: it was not predicted by the theory, which states that
if the binding was correct, both the oxidation and the reduction peak
should be less intense.

The only conclusion we may draw from this experiment is that we have
indeed modified the electrodes, all in the same way, but we do not
exactly know the state of the surface at this point.

The most likely explanation for this inconclusive experiment is that
the fixation of the aptamers wasn’t complete. This might be explained
by the fact that the drops of the fixating solution evaporated very
quickly: some aptamers may therefore not have had time to bind
correctly and instead adsorbed unto the nanotubes. The protective
layer we had hoped to detect using cyclic voltammetry was therefore
not thick enough for us to notice a difference.

Bacterial detection with aptamers/carbon-nanotube
electrodes

Once the aptamers are attached to the carbon-nanotube electrodes, pathogens can be selectively detected by potential change. Using our arduino as a voltmeter, we have recorded the response profile of our electrodes following the addition of bacteria at different time points. Graphs represent potential (mV) on y-axis and time (s) on x-axis.

For each test, the same protocol was performed, as following :
50 µL of 1X PBS are added on the electrode.
After stabilization of the potential (2-3mins), 25 µL of bacterial
solution are deposited on the PBS droplet and mix up and down.
Change in potential is recorded.
After 100 seconds, 25 µL of bacterial solution are added once again.

Five conditions were tested.

As a negative control, PBS has been added on the surface of the electrode coated with our own aptamers designed for *E. faecium*: **BBa\_K3176020**. On Figure 10, two peaks are observed perfectly corresponding to the moment when the drop is added. This environment disturbance induces a temporary change in potential, before quickly returning to the basic potential.

![](https://static.igem.org/mediawiki/2019/0/07/T--Pasteur_Paris--pbsef.png)

Figure 10: Negative control with PBS

We then detected a signal in the presence of the targeted bacteria *E. faecium*. Figure 11 shows a high change in potential with a staircase profile. Bacteria are well able to bind to the aptamers, inducing a change in their conformation.

![](https://static.igem.org/mediawiki/2019/5/53/T--Pasteur_Paris--efef.png)

Figure 11: *E. faecium* injected in a drop on an electrod with aptamers selected for *E. faecium*

We finally could observe that our electrodes coated with *E. faecium* aptamers are only specific to this strain. Figure 12 shows that a solution of *E. coli* does not induce any staircase potential jumps as observed for *E. faecium*. Indeed the profile of the graph is similar to the PBS control : with two peaks when the drop is added, before quickly returning to the basic potential. This observation proves that our aptamers designed for *E. faecium* are specific to this strain.

![](https://static.igem.org/mediawiki/2019/5/58/T--Pasteur_Paris--ecef.png)

Figure 12: *E. coli* injected in a drop on an electrod with aptamers selected for *E. faecium*

Finally,
*E. faecium* bacteria were added within a negative urine control (SurineTM), simulating analysis conditions. Once again, a staircase trend in the curve showed that bacteria are well able to bind to the aptamers.