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    • Single-temperature Double Digest
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    Cheaters

    Monday, 7/29/2019Some 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.Tuesday, 7/30/2019Table2Wednesday, 7/31/2019CFU in 10^8 (cell/mL)Monday, 8/5/2019Tuesday, 8/6/2019O.D MeasurementColony number at different time for each condition and dilution factor associatedWednesday, 8/7/2019Table1CFUFriday, 8/9/2019Nanodrop Results 9/08Monday, 8/12/2019O.D. 12/08CFU 12/08Tuesday, 8/13/2019O.D. 13/08CFU 13/08Nanodrop resultsMonday, 8/19/2019Tuesday, 8/20/2019O.D. 20/08CFU 20/08Wednesday, 8/21/2019O.D. 21/08CFU 21/08Sequencing results about the mutations carried by the plasmid
    Monday, 7/29/2019
    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.
    ●
    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, 7/30/2019
    ●
    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 :
    Wednesday, 7/31/2019
    ●
    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.
    Monday, 8/5/2019
    ●
    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, 8/6/2019
    ●
    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 :
    Most of the results are not usable because the droplet mixed themself together
    ●
    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
    ●
    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, 8/7/2019
    ●
    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 :
    Friday, 8/9/2019
    ●
    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 :
    ●
    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)
    ●
    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, 8/12/2019
    ●
    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 :
    Tuesday, 8/13/2019
    ●
    Preculture : Arnaud
    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 at 37°C.
    ●
    Nucleases caracterization and cheater study : Arnaud
    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 :
    ●
    Plasmidic DNA extraction of pBAD24-T7-cheater-clone 2 and clone 3 (Clara)
    See the protocol of NucleoSpin® Plasmid / Plasmid (NoLid) kit.
    Monday, 8/19/2019
    Colony PCR
    ●
    Preculture : Natacha
    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.
    Tuesday, 8/20/2019
    ●
    Nucleases caracterization and cheater study : Arnaud
    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 :
    Survivor 1:
    Unexpectedly, upon addition of arabinose, growth of “Survivor 1” was stopped and the survival rate dropped . This experiment was performed only once and here, the drop-in survival was more efficient and faster than for the original clone carrying yqcG. This survivor was lucky to survive the first nuclease induction as yqcG appears to be still fully functional.
    ●
    Preculture : Arnaud_Hugues_Natacha
    Preculture of KeioZ1F'_A1, Moclo, Cheater A1 clone 1 and Cheater A1 clone 4 in 3 mL LB + Ampiciline + Glucose 0.2% overnight with shaking.
    Wednesday, 8/21/2019
    ●
    Nucleases caracterization and cheater study : Arnaud
    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)
    Arabinose added at 12h35 (Cf = 0.2%)
    Results :
    Sequencing results:

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