Nuclease_A1 (from Qβ)

See the protocol "heat shock transformation".

The transformants were selected on LB agar plates plus Ampicilline (100µg/mL) and 0.2% glucose.

Results :

Beginning of induction with Ara and first measurement at 30min then every hour.

Spread on petri dishes from transformed cells

Second step: reading the OD using the plate spectrophotometer.

Measurement automatically of the DO every 20min during 12hours.

Results :

Transformation verification : colony PCR. For each sample : pBAD24A1.1, pBAD24A1.2, pBAD24GG.1, pBAD24GG.2 transformed in E.coli on 1st/2nd july :

PCR program (named "IGM") :

94°C/5'

30 cycles : 94°C/30''

51°C/30''

72°C/2min

72°C/10'

Electrophoresis : 100V, 10 μL sample/well, 25 minutes.

Results :

Pre-culture : From glycerol stock on 1st/2nd july (pBAD24A1.1, pBAD24A1.2, pBAD24GG.1, pBAD24GG.2 transformed in E.coli), for each sample : 2 mL LB + 100μg/mL Ampicilline + 20% glucose, incubation with agitation overnight.

60mL culture of A1 and GG. Then beginning of induction with Ara in 30mL of A1 and GG, and first measurement of Optic Density at 30min each sample, then every hour twice.

Results :

Rq : We can notice that the population of A1+Ara bacteria decreases over time until 2h30. Then, during the night bacteria develop. We can assume that bacteria mutate during the night and grow.

Box spreading from transformers' solutions.

Second step : reading the OD using the plate spectrophotometer just after induction.

Mesure automatically of the DO every 20min during 12hours.

cf "test 2 9-07-2019.xlsx"

First step (Laura_Arnaud_Abdel) : 60mL culture of A1 and GG. Then beginning of induction with Ara in 30mL of A1 and GG, and first measurement at 30min each sample, then every hour twice.

Results : see the figures bellow

Box spreading from transformers' solutions.

Second step (Marie) : reading the OD using the plate spectrophotometer just after induction (add Ara just before the measurement).

Mesure automatically of the DO every 20min during 3 hours.

keioZ1F'A1 ; keioZ1F'GG in LB + Amp + glu from glycerol stock N°1

k12Hfr in LB from an agar plate

Culture overnight at 37°C

From a over night bacterial culture of Keio pBAD24 MoClo in LB-amp-glu (0,2%), dilute in 10mL LB-amp to get an OD600=0,1

Incubate at 37°C to obtain an OD600=0,4

Add 100µL arabinose 20% in the liquid culture (i. e. 0,2% arabinose final)

Incubate 5 min at 37°C with agitation

Stick tape on slides in order to form a delimited area on the slide

Pour 200µL solid LB-amp-ara (0,2%)

Sandwich the gelose with annother slide. After dry out, remove the top-slide

Place 1µL of arabinose-inducuced-bacteria (see protocol "Culture and OD600") on the solid gelose

Dry the drop then place the cover glass above the drop

Put 1 drop of oil on the cover glass then orientate the cover glass toward the ground

Take picture every 20min for 6 hours (do it manualy because the focus doesn't work properly)

Primers : pBAD24_F and pBAD24_R

Two Tm are used for this PCR (62°C and 72°C)

For 1 PCR , Vf = 49 µL

2.5 µL per primers 10mM

1 µL template

25 µL Master Mix (Enzyme, dNTPs, Buffer)

18 µL Water

PCR Program:

98°C - 1 min

98°C - 10 s

62°C - 30 s

72°C - 165 s

Repeat from step 2 (5 times)

98°C - 10 s

72°C - 30 s

72°C - 165 s

Repeat from step 6 (25 times)

72°C - 2 min

8°C - Infinity

The products are analysed by electrophoresis

keioZ1F'A1

keioZ1F'T7

keioZ1F'Rdrp

keioZ1F'Moclo

in LB + Amp + glu 3 mL

Culture overnight at 37°C

From a over night bacterial culture of Keio pBAD24-A1 in LB-amp-glu (0,2%), dilute in 10mL LB-amp to get an OD600=0,1

Incubate at 37°C to obtain an OD600=0,4

Add 100µL arabinose 20% in the liquid culture (i. e. 0,2% arabinose final)

Incubate 5 min at 37°C with agitation

Stick tape on slides in order to form a delimited area on the slide

Pour 200µL solid LB-amp-ara (0,2%)

Sandwich the gelose with annother slide. After dryout, remove the top-slide

Place 1µL of arabinose-inducuced-bacteria (see protocol Culture and OD600) on the solid gelose

Dry the drop then place the cover glass above the drop

Put 1 drop of oil on the cover glass then orientate the cover glass toward the ground

Take picture every 20min for 6 hours (do it manualy because the focus doesn't work properly)

60mL culture of A1, T7, Moclo and Rdrp in LB + Amp after a preculture overnight in LB + amp + glucose. Liquide culture are diluted in order to have an O.D = 0.1 when starting the experiment. Beginning of induction with Ara when O.D = 0.4. At this moment, each bacterial solution are split in 2 new samples of 25mL. One with arabinose, one whitout.

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 (+1110)

When arabinose is added, another experiment is done with the bacterial culture. bacterial solutions are used for a perpetual O.D measurement with clariostar.

Results :

60mL culture of A1, T7, Moclo and Rdrp in LB + Amp after a preculture overnight in LB + amp + glucose. Liquide culture are diluted in order to have an O.D = 0.1 when starting the experiment. Beginning of induction with Ara when O.D = 0.4. At this moment, each bacterial solution are split in 2 new samples of 25mL. One with arabinose, one whitout.

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 (+1110)

When arabinose is added, another experiment is done with the bacterial culture. bacterial solutions are used for a perpetual O.D measurement with clariostar.

Results :

25 mL of LB + Amp of each culture are incubated at 37°C with shaking starting with O.D = 0.1. from precultures. Arabinose is added and 750µL is withdraw at different time point (T0; T30; T90) for cell fixation and DAPI tagging.

For cell fixation and DAPI tagging the following schedule is used :

750 µL of bacterial culture is withdrawn and poured in a 1.5 mL tube.

750 µL of PFA solution (Formaldehyde 37% : 6.8 mL ; Glutaraldhéhyde 25% : 120µL ; PBS 1X : 43 mL)

Mix for 20min at least in cold room

Centrifuge at 6000 RPM for 3 min

Throw the supernatant

Wash 2 times with 1 mL PBS 1X

Resuspend gently in 0.5 mL GTE(Glucose 50mM ; tris pH8 20mM ; EDTA 10mM)/DAPI

Incubate at room temperature for 10 min

Centrifuge at 6000 RPM 3 min and eliminate supernatant

Wash with 1 mL PBS 1X

Resuspend in 100 µL PBS 1X

Hide from light and store at around 5°C

To observe the bacteria, pour 10 µL of the solution on top of an agarose 1% in PBS 1X layer on a glass plate for microscopy. Wait for the solution to dry on the layer. Observe.

PBS 10X pH7 solution is composed of : NaCl 80g, KCl 2g, Na2HPO4.7H2O 25.6g, KH2PO4 2g, H2O qsp 1L

In 5 mL of LB with Glucose (50µL 20%) and Ampiciline (5µL) in 2 Falcons 50 mL. Incubation overnigth with shaking at 37°C

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

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 :

Most of the results are not usable because the droplet mix themself together

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 :

Cells were fixed according to the protocol "Fixation of cells and coloration with DAPI". Aliquots were taken for :

KeioZ1F'+pBAD-Moclo t0, t30min, t90min and t150min

Preculture of KeioZ1F'_A1, Moclo, T7, B.sub, Cheater T7 clone 2 (Survivor from Nuclease induction of 7/08 "Tomorrow , T7 N°2") and Cheater T7 clone 3 (Survivor from Nuclease induction of 7/08 "Tomorrow , T7 N°1") in 3 mL LB + Ampiciline + Glucose 0.2% overnight with shaking.

Culture of A1, Moclo, T7 , B.sub, T7 cheater clone 2 and clone 3 (they survided the nuclease induction from a previous induction) in LB + Amp (20mL) 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 12h28 (Cf = 0.2%)

Results :

Cells were fixed according to the protocol "Fixation of cells and coloration with DAPI". Aliquots were taken for :

Preculture of KeioZ1F'_A1, Moclo, T7, B.sub n°1 and B.sub n°2 and Cheater B.sub (Survivor from Nuclease induction of 13/08 "Tomorrow , B.sub") in 3 mL LB + Ampiciline + Glucose 0.2% overnight with shaking.

Culture of A1, Moclo, T7 , B.sub n°1 and B.sub n°2 and Cheater B.sub (they survided the nuclease induction from a previous induction (13.08 "Tomrorrow, B.sub") 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 12h20 (Cf = 0.2%)

Results :

Preculture of KeioZ1F'_A1, Moclo, Cheater A1 clone 1 and Cheater A1 clone 4 (Survivor from Nuclease induction of in 3 mL LB + Ampiciline + Glucose 0.2% overnight with shaking.

Culture of A1, Moclo,Cheater A1 clone 1 and Cheater A1 clone 4 (they survided the nuclease induction from a previous induction 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)

For each timepoint, 1mL of culture is take out to extract gDNA and rRNA.

Arabinose added at 12h35 (Cf = 0.2%)

Results :

Results : gDNA is degraded at 90min with nucleases A1 and T7 however, gDNA is present at 120min with the A1 nuclease. This might be due to the presence of "cheater bacteria".

About the rRNA, the gel is surprising. rRNA "appears" at t=30min in cells expressing the A1 nuclease but fade away at t=90min. This contrasts with the first gel obtain (from the july 18th). The presence of RNases could explain the degradation of rRNA at some timepoint.

gDNA extractions need tobe re-done in RNase-free conditions.