Nuclease_yqcg (From B. subtilis)

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

Golden gate of pBAD MoClo + gstock b. stubtilis

1µL plasmid

2µL mix (ligase + XbaI)

1µL buffer

7µL plasmid pBAD 24 Moclo nuclease b. stubtilis

9µL H2O

Colony PCR of transformed cells DH5alpha with first Golden gate of pBADMoClo_NucleaseB. We used the protocol called "Colony PCR".

PCR programm (3O cycles):

94°C 5min

94°C 30sec

60°C 30sec

72°C 1min/kb

72°C 10min

Each colony was cultured in liquid and solid LB with 100µg/mL Amp and 0.2% glucose

Results of transformation of July the 8th : lots of single colonies for transformed cells with first golden gate reaction for B.subtilis but none with the new one. Kim and Natacha didn't run a PCR with just the golden gate reaction so we don't know if it is the transformation or if it is the golden gate reaction which didn't work. But other transformations worked so might be the golden gate reaction which didn't.

If sequencing is good, we have to transforme keïo cells with pBAD24 moClo Rdrp clone 1 & 4 and pBAD24 moClo T7 clone 2 & 1.

Sequencing result of pBADMoClo B.subtilis nuclease is not good because no priming (apparently other people got problems too so might be a problem from the company).

So digestion with ECORV of all clones containing pBADMoClo b. subtilis nuclease :- 10µL of plasmid

- 2µL buffer R

- 1µL ECORV

- 7µL H2O

Heat shock transformation in KEÏO cells with pBAD MoClo according the the protocol " heat shock transformation".

From stripes of DH5alpha pBAD24-B. sub 05/07, collect small number of bacteria and place them into 10µL of water.

Then add : - 2,5µL green taq buffer

-1µL dNTP 5mM

-1µL primer 5

-1µL primer 6

-0,15µL Taq polymerase

-Qsp H2O for 25µL final

PCR program : 94°C 5 min

94°C 30 sec

62°C 30 sec repeat these steps 30 times

72°C 1min30

72°C 2min

PCR products are run on an electrophoresis agarose gel 1%.

The clone 1', 3', 5' and 6' posses a unique band at the correct lenght (1,600 bp).

The clone 1' and 3' are incubated in 15mL LB amp glu at 37°C O/N for miniprep 07/16.

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)

Use of protocol "Heatshock plasmid transformation" in KeioZ1F' 200µL. Warning Heatshock at 42°C for 1 min has been done two times because of user mistake. Bacteria are then spread on 3 plates LB+amp+glu (50 µL, 100 µL, Remaining)

Result : there wasn't any transmormant, must do to the 2nd heatshock

1 µL per plasmid and 50 µL of competent KeioZ1F' :

KeioZ1F' with plasmid from B.sub clone 1

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(100ug/mL) + 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 :