Team:GO Paris-Saclay/test2
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Cheaters
Project: Survivors
Authors: iGEM_GO_Paris_saclay
Monday, 29/7/2019
Some bacteria can escape DNA degradation. We called these survivor cells cheaters. Following assays aim to characterize the proportion of bacteria that escapes DNA cleavage and mecanisms leading to this escape.
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Principle: to characterize if this new phenotype was due to a mutation carried by the plasmid, we extract the plasmid of the cheater cells and sequence it. In the same time, we also performe CFU plus we take the OD of these cheaters.
Production of M9 medium (Colombe - Laura)
15mL of M9 salt (10x)
3 mL of Glucose 20%
1,5mL of CAA 20%
150μL of MgSo4 (1M)
1,5μL of CaCl2 (1M)
150μL of Biotin (1mg/mL)
150μL of Thiamin (1mg/mL)
1,5mL of Trace element (100x)
128,5mL of (200mL of H20 + 3,5gr of Agar)
Toxicity of B. sub / DH5α (Marie - Laura)
Indution of survivors A1 et T7
Preparation of pre-culture :
3mL of LB
30μL of Glucose
3μL of Amp
+ stamp
Different stamp :
1.
Bacilus subtilis from glycerol stock
2.
Moclo from glycerol stock
3.
A1 from glycerol stock
4.
T7 from glycerol stock
5.
T7 from the box "16/07 iGEM LB+Glu+Amp T7 - Ara Lendemain"
6.
A1 from the box "16/07 iGEM LB+Glu+Amp A1 - Ara Lendemain"
put overnight at 37°C with agitation.
Tuesday, 30/7/2019
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Toxicity of B. sub / DH5α + Induction : Marie_Laura
From a culture that has grown overnight, dilute to an OD of 0.1 in 15mL LB + Amp, then grow at 37°C with agitation to an OD of 0.4. Take OD, it's the time t0.
1.
Bacilus subtilis from DH5alpha glycerol stock = B.sub
2.
KeioZ1F' + PBAD24-Moclo from glycerol stock = Moclo
3.
KeioZ1F' + PBAD24-A1 from glycerol stock = A1
4.
KeioZ1F' + PBAD24-T7 from glycerol stock = T7
5.
KeioZ1F' + PBAD24-T7 from the box "16/07 iGEM LB+Glu+Amp T7 - Ara Lendemain" = "tricheur"T7 = TT7
6.
KeioZ1F' + PBAD24-A1 from the box "16/07 iGEM LB+Glu+Amp A1 - Ara Lendemain" = "tricheur"A1 = TA1
Add 0,2% of Arabinose. Then let it grow at 37°C and take the OD after 30min, 1h30, 2h30 and the next day. At the same time make droplets of different dilutions: from 10-1 to 10^-7.
Results :
A A | B B | C C | D D | E E | F F | G G | |
1 1 | T7 | TT7 | A1 | TA1 | Moclo | B.sub | |
2 2 | t0min | 0,53 | 0,43 | 0,54 | 0,38 | 0,72 | 0,53 |
3 3 | t30min | 0,49 | 0,62 | 0,71 | 0,65 | 0,93 | 0,68 |
4 4 | t1h30 | 0,43 | 1,06 | 0,67 | 1,12 | 1,46 | 0,88 |
5 5 | t2h30 | 0,41 | 1,47 | 0,68 | 1,69 | 1,87 | 0,85 |
6 6 | overnight | 2,7 | 3,5 | 3,1 | 4,1 | 3,8 | 5,9 |
Table2
Wednesday, 31/7/2019
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Toxicity of B. sub / DH5α + Induction: Marie_Laura
Isolation of T7 and TA1 (=cheater T7 and A1) in a new agar plate with LB+Amp+Glucose to attempt an isolate of a single colonies.
Agar plates are then incubated at 37°C overnight.
Results of the spreading boxs from transformers' solutions.
A A | B B | C C | D D | E E | F F | |
1 1 | Time | Omin | 30min | 90min | 150min | Tomorow |
2 2 | Cheater A1 | 10^-5 | 10^-5 | 10^-6 | 10^-6 | 10^-6 |
3 3 | 5 | 5 | 3 | 16 | 12 | |
4 4 | 8 | 5 | 2 | 14 | 14 | |
5 5 | 11 | 11 | 5 | 13 | 15 | |
6 6 | 8 | 8 | 10 | 16 | 14 | |
7 7 | Cheater T7 | 10^-5 | 10^-5 | 10^-6 | 10^-6 | 10^-6 |
8 8 | 8 | 11 | 4 | 9 | 20 | |
9 9 | 6 | 13 | 6 | 11 | 19 | |
10 10 | 11 | 13 | 6 | 16 | 21 | |
11 11 | 9 | 8 | 9 | 19 |
CFU in 10^8 (cell/mL)
Monday, 5/8/2019
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Preculture for nuclease characterization (CFU and O.D.) : Arnaud
4 precultures are put at 37°C o/v : keioZ1F' pBAD24_A1 ; keioZ1F' pBAD24_Moclo ; and 2 keioZ1F' pBAD24_A1 survivors from a previous nuclease induction.
Each preculture is composed of 3 mL LB + ampiciline + glucose
Tuesday, 6/8/2019
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Nucleases characterization and cheater study : Arnaud
Culture of A1, Moclo and A1 cheater (they survided the nuclease induction from a previous induction but not a single clone but many were used for the preculture) in LB + Amp (25mL) after a preculture overnight in LB + amp + glucose. Liquide preculture are diluted in order to have an O.D = 0.1 when starting the experiment. Beginning of induction by adding arabinose when O.D is around 0.4.
O.D and droplet of dilutions in plate are done at different time point compared to the moment arabinose is added (minutes) : -30 ; +30 ; +90 ; +150 ; Tomorow (around +1110)
Results :
A A | B B | C C | D D | E E | F F | G G | H H | |
1 1 | O.D | |||||||
2 2 | Time Point | 10h14 | 10h44 (-30') | 11h14 (T0) Arabinose is added | 11h44 (+30') | 12h44 (+90') | 13h44 (+150') | TOMORROW 12h15 (+1351) |
3 3 | A1 | 0.1 | 0.16 | 0.32 | 0.59 | 0.57 | 0.58 | 3.00 |
4 4 | Moclo | 0.1 | 0.18 | 0.40 | 0.66 | 1.18 | 1.64 | 2.40 |
5 5 | A1 Cheater | 0.1 | 0.20 | 0.42 | 0.69 | 1.20 | 1.66 | / |
O.D Measurement
A A | B B | C C | D D | E E | F F | |
1 1 | CFU | Time (min) | ||||
2 2 | Bacteria | -30 | 30 | 90 | 150 | 1351 |
3 3 | A1 | -6 | -2 | |||
4 4 | 1 | 5 | ||||
5 5 | 4 | |||||
6 6 | Moclo | -6 | -6 | -7 | ||
7 7 | 12 | 10 | 33 | |||
8 8 | 15 | 7 | 22 | |||
9 9 | Survivor A1 N°3 | -5 | -7 | -6 | ||
10 10 | 12 | 1 | 12 | |||
11 11 | 7 | 6 | ||||
Colony number at different time for each condition and dilution factor associated
Most of the results are not usable because the droplet mixed themself together
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Precultures of one clone pBAD24 Moclo, pBAD24 T7 and 2 survivors after induction of the nuclease A1 : Clara
Each strains were put into 15mL of LB + amp (15µL) + glucose (150µL)
The survivors/cheaters were taking from the plate 07/16 for the "day after" condition, #2 clone 5 and #1 clone4
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Culture of DH5alpha to make ultra competent cells : Laetitia
1mL from the day before preculture (05/08/2019) has been added to 250mL of LB and let grown at 20°C, 140 rpm until D0600=0.55
Wednesday, 7/8/2019
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Nucleases T7 characterization and A1 cheaters study : Arnaud_Clara
Culture of Moclo T7 (*2) and A1 cheaters clone 4 and 5 (they survided the nuclease induction from a previous induction but not a single clone but many were used for the preculture) in LB + Amp (25mL) after a preculture overnight in LB + amp + glucose. Liquide preculture are diluted in order to have an O.D = 0.1 when starting the experiment. Beginning of induction by adding arabinose when O.D is around 0.4. O.D and droplet of dilutions in plate are done at different time point compared to the moment arabinose is added (minutes) : -30 ; +30 ; +90 ; +150 ; Tomorow (around +1110)
Results :
A A | B B | C C | D D | E E | F F | G G | H H | |
1 1 | O.D. | |||||||
2 2 | Time point | 11h20 | 11h50 (-30') | 12h20 (T0) | 12h50 (+30') | 13h50 (+90') | 14h50 (+150') | TOMORROW |
3 3 | Moclo | 0.1 | 0.29 | 0.53 | 0.87 | 1.53 | 1.94 | 4.5 |
4 4 | T7 n°1 | 0.1 | 0.20 | 0.32 | 0.42 | 0.36 | 0.29 | 3.8 |
5 5 | T7 n°2 | 0.1 | 0.25 | 0.41 | 0.42 | 0.38 | 0.36 | 3.9 |
6 6 | Cheater A1 clone 4 | 0.1 | 0.31 | 0.52 | 0.85 | 1.51 | 1.87 | 3.7 |
7 7 | Cheater A1 clone 5 | 0.1 | 0.29 | 0.47 | 0.59 | 0.58 | 0.59 | 3.4 |
Table1
A A | B B | C C | D D | E E | F F | |
1 1 | CFU | Time (min) | ||||
2 2 | Bacteria | -30 | 30 | 90 | 150 | TOMORROW |
3 3 | Moclo | -5 | -5 | -6 | -5 | |
4 4 | 27 | 26 | 19 | 27 | ||
5 5 | 26 | 33 | 12 | 26 | ||
6 6 | 27 | 29 | 16 | 27 | ||
7 7 | T7 N°1 | -4 | -1 | -1 | -1 | -5 |
8 8 | 19 | 14 | 10 | 21 | 7 | |
9 9 | 10 | 7 | 6 | 29 | 4 | |
10 10 | 12 | 15 | 5 | 22 | 5 | |
11 11 | T7 N°2 | -4 | -1 | -1 | -1 | -3 |
12 12 | 18 | 15 | 17 | 25 | 16 | |
13 13 | 12 | 18 | 25 | 18 | ||
14 14 | 19 | 20 | 19 | 11 | ||
15 15 | Survivor A1 N° 4 | -4 | -5 | -5 | -6 | -5 |
16 16 | 22 | 21 | 17 | 15 | 20 | |
17 17 | 32 | 28 | 17 | 12 | 34 | |
18 18 | 14 | 22 | 12 | 9 | 28 | |
19 19 | Survivor A1 N° 5 | -4 | -2 | -1 | -1 | -2 |
20 20 | 26 | 22 | 17 | 2 | ||
21 21 | 15 | 26 | 15 | 1 | ||
22 22 | 18 | 7 | 4 |
CFU
Friday, 9/8/2019
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Plasmid extraction : Arnaud
Extraction of plasmids from :
a.
Cheater (survivor of previous nuclease induction) A1 clone 1
b.
Cheater A1 clone 4
c.
Cheater T7 clone 1
Use of Macherey-Nagel kit "DNA and RNA purification" protocol page 18 "Low copy"
Analysis with Nanodrop :
A A | B B | |
1 1 | Plasmid from | Concentration (ng/µL) |
2 2 | Cheater A1 clone 1 | 149 |
3 3 | Cheater A1 clone 4 | 169 |
4 4 | Cheater T7 clone 1 | 184 |
Nanodrop Results 9/08
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Heatshock plasmid Transformation : Arnaud
5 transformations are done : 2 are double transformations and the other are simple transformation. 1 µL per plasmid and 50 µL of competent KeioZ1F' :
I.
KeioZ1F' with plasmid from cheater A1 clone 4 (previously extracted)
II.
KeioZ1F' with plasmid from cheater T7 clone 1 (previously extracted)
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Preculture : Arnaud
Preculture of KeioZ1F'_A1, Moclo, T7, B.sub, KeioZ1F' transformed with plasmid from Cheater A1 clone 4 and KeioZ1F' transformed with plasmid from Cheater T7 clone 1 3 mL LB + Ampiciline + Glucose 0.2% overnight with shaking at 37°C.
Monday, 12/8/2019
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Nucleases caracterization and cheater study : Arnaud
Culture of A1, Moclo, T7 , B.sub, KeioZ1F' transformed with plasmid from Cheater A1 clone 4 and KeioZ1F' transformed with plasmid from Cheater T7 clone 1 in LB + Amp (50mL) after a preculture overnight in LB + amp + glucose. Liquide preculture are diluted in order to have an O.D = 0.1 when starting the experiment. Beginning of induction by adding arabinose when O.D is around 0.4.
O.D and droplet of dilutions in plate are done at different time point compared to the moment arabinose is added (minutes) : -30 ; +30 ; +90 ; +150 ; Tomorow (around +1110)
Arabinose added at 12h10 (Cf = 0.2%)
Results :
A A | B B | C C | D D | E E | F F | G G | H H | |
1 1 | Time point | 10h55 | 11h30 (t-30') | 12h00 (t0) | 12h40 (t+30') | 13h40 (t+90') | 14h40 (t+150') | Tomorrow 11h15 (t+1395') |
2 2 | A1 | 0.1 | 0.18 | 0.38 | 0.55 | 0.51 | 0.55 | 3.6 |
3 3 | T7 | 0.1 | 0.15 | 0.17 | 0.20 | 0.20 | 0.17 | 3.6 |
4 4 | Moclo | 0.1 | 0.19 | 0.34 | 0.71 | 1.31 | 1.83 | 3.6 |
5 5 | B.sub | 0.1 | 0.13 | 0.21 | 0.38 | 0.61 | 0.58 | 4.6 |
6 6 | KeioZ1F' transformed with pasmid from Cheater A1 clone 4 | 0.1 | 0.21 | 0.40 | 0.68 | 1.38 | 1.80 | 3.6 |
7 7 | KeioZ1F' transformed with pasmid from Cheater T7 clone 1 | 0.1 | 0.16 | 0.35 | 0.63 | 1.23 | 1.75 | 3.3 |
O.D. 12/08
A A | B B | C C | D D | E E | F F | |
1 1 | CFU | Time (min) | ||||
2 2 | Bacteria | -30 | 30 | 90 | 150 | 1395 |
3 3 | Moclo | -5 | -5 | -5 | -6 | |
4 4 | 12 | 8 | 27 | 8 | ||
5 5 | 2 | 9 | 13 | |||
6 6 | 5 | |||||
7 7 | B.sub | -4 | -4 | -3 | -2 | |
8 8 | 20 | 25 | 11 | 31 | ||
9 9 | 16 | 30 | 10 | 30 | ||
10 10 | 21 | 28 | 15 | 24 | ||
11 11 | T7 | -3 | -2 | -1 | -2 | |
12 12 | 4 | 19 | No colony after | 2 | ||
13 13 | 8 | 20 | 4 | |||
14 14 | 5 | 19 | ||||
15 15 | A1 | -4 | -1 | -1 | -1 | |
16 16 | 14 | No colony after | No colony after | No colony after | ||
17 17 | 16 | |||||
18 18 | ||||||
19 19 | KeioZ1F' transformed with pasmid from Cheater A1 clone 4 | -4 | -5 | -5 | -6 | |
20 20 | 10 | 12 | 28 | 7 | ||
21 21 | 15 | 14 | 29 | 15 | ||
22 22 | 13 | 11 | ||||
23 23 | KeioZ1F' transformed with pasmid from Cheater T7 clone 1 | -4 | -5 | -6 | -6 | |
24 24 | 15 | 8 | 25 | 8 | ||
25 25 | 16 | 14 | 29 | 9 | ||
26 26 | 20 | 13 | 16 | 8 | ||
CFU 12/08