Team:Pasteur Paris/Notebook
Education
Law
Events
Design
Results
Entrepreneurship
Demonstrate
Chalenges
Improve
Collaborations
Attributions
Results Attributions
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.