Nuclease_gp3 (From T7)

The cloning needs a molar ratio insert 2:1 plasmid.

1µL plasmid (PBAD24GG)

+2µL mix (enzymes ligase/XbaI)

+1µL buffer Golden Gate

+ x µL plasmid (for molarity ratio 2:1 cf table1)

Up to 20µL with water

Heat at 37°C for 15min.

2µL pBAD24-Moclo +insert + 50µL competent DH5alpha on ice for 30min

There are colonies on each Petri dishes (for each cloning) but just a few for the nuclease of B. subtilis.

dNTP diluted 1/2

Primers diluted 1/10 : oligo 5: 557µL

oligo 6: 617 µL

6 colonies for each condition + control: Ligation gblock (endonuclease T7 or nuclease B. subtilis or RdRp) and pBAD 24 GG

Each colonies was striped on LB + AMP (100µg/mL)+ Glucose 0,2% before using it for the colony PCR and after was sown into liquid culture of LB + AMP + glucose 0.2%

Clara Natacha and Hugues:

Stripes of 6 transformed cells with pBAD24-MOCLO, and liquid culture with LB and 100µg/mL of Amp and 0,2% glucose

Result of the colony PCR of transformed cells with pBAD MoClo Rdrp and T7

Cultures of DH5alpha cells containing pBAD24 MOCLO clone 1 & 4 and pBAD MoClo Rdrp clone 2' & 5' and pBAD MoClo T7 clone 1' were cultures. Then plasmid DNA extraction was done using NucleoSpin® Plasmid / Plasmid (NoLid) kit. Culture of bacteria containing pBAD24 MOCLO clone 1' to make stock tomorrow are done. Have to do the same for the other transformed cells pBAD MoClo Rdrp clone 2' & 5' and pBAD MoClo T7 clone 1' tomorrow.

Plasmid DNA extraction of pBADMoClo Rdrp clone 1 & 4 and pBADMoClo T7 clone 2 & 1. All DNAs' concentrations were measured with nanodrop.

Sent to sequencing pBADMoClo Rdrp clone 1 & 4 and pBADMoClo T7 clone 2 & 1.

Transformed cells DH5alpha with new golden gate of pBADMoClo nuclease of B. subtilis and the first one. 50 µL of bacteria were used for 1 µL of plasmid. => Tomorrow: Colony PCR and second selection

Culture of transformed cells with pBADMoClo Rdrp clone 2' & 5' and pBADMoClo T7 clone 1' for glycerol stock tomorrow.

Sequencing result: pBADMoClo T7 clone 1' & RDRP clone 2' great :D

Heat shock transformation in Keïo with T7 clone 1' & RDRP clone 2' (see protocol "Heatshock plasmid transformation")

Stripes from colonies are done in 2 agar plates with LB + amp + glu

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)

keioZ1F'A1

keioZ1F'T7

keioZ1F'Rdrp

keioZ1F'Moclo

in LB + Amp + glu 3 mL

Culture overnight at 37°C

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

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

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

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 :

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 :