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  • HUMAN  PRACTICES

    PROJECT

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    Picto Sciences

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    W1W2W3W4W5W1W2W3W4W5W1W2W3W4W5W1W2W3W4W5

    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.


    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.





    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.




    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.



    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.



    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.


    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.


    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.


    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



    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.


    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.

    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.

    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.

    Gel slices were cut in order to extract DNA.

    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.

    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.

    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.

    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.

    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.