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    RNA cell (Archived)
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    • Gibson Assembly® Protocol (E5510)
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    RNA cell

    Wednesday, 7/3/2019AIM: As a first step in proving the RNA World concept, we wanted to create an “RNA cell”, in which replicating RNA, rather than DNA, would be the support of genetic information.Table1Thursday, 7/4/2019Primer sequences for checking insertion of the inserts PCR master mixFriday, 7/5/2019WhatsApp Image 2019-07-05 at 17.27.04.jpegMonday, 7/8/2019Table3Tuesday, 7/9/2019Thursday, 7/11/2019Friday, 7/12/2019Table4Monday, 7/15/2019Tuesday, 7/16/2019Wednesday, 7/17/2019Table5Table6Thursday, 7/18/2019Table7Table8Monday, 7/22/2019Table9Table10image.pngTable11Tuesday, 7/23/2019Wednesday, 7/24/2019Thursday, 7/25/2019Primer sequences for checking pZA31-MoClo-asGFPCapture d’écran 2019-07-29 à 11.30.11.pngTuesday, 7/30/2019Table12Wednesday, 7/31/2019Primers detailsTable13Thursday, 8/1/2019Table14Table15Gel pBAD Rdrp : Rdrpbis : A1 : A1bis.pngGel pBAD Rdrp : Rdrpbis : A1 : A1bis ZOOM.pngTable16Friday, 8/2/2019Monday, 8/5/2019Primer sequences for checking the insertion of our cassettesTuesday, 8/6/2019Colony PCR pbad24A1rbsrdrp.pdfTable17Wednesday, 8/7/2019Table18Thursday, 8/8/2019Friday, 8/9/2019Monday, 8/19/2019Friday, 8/23/2019Wednesday, 8/28/2019Thursday, 8/29/2019Table2
    Wednesday, 7/3/2019
    AIM: As a first step in proving the RNA World concept, we wanted to create an “RNA cell”, in which replicating RNA, rather than DNA, would be the support of genetic information.
    Principle: In order to carry out this substitution of DNA, we clone and expresse 3 nucleases: A1, gp3 and yqcg and see if they can efficiently degrade bacterial DNA. In addition to these nucleases, we integrate a plasmid containing The Rna dependent Rna polymerase 5a viral protein which amplifies RNA°. We are going to replicate an anti-sens-GFP-RNA to produce a sens GFP-RNA ,so that RNA can be translated.
    ●
    Cloning Gblocks of T7, B. subtilis and RdRp into pBAD24-MoClo (empty)
    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.
    ●
    Transform pBAD24-Moclo + insert into DH5alpha (see protocol "heat shock plasmid transformation")
    2µL pBAD24-Moclo +insert + 50µL competent DH5alpha on ice for 30min
    Heatshock at 42°C for 45 seconds
    Cool down on ice for 5min then spread on LB+glucose (0.2%) +Amp (100µL/mL)
    37°C over night
    Thursday, 7/4/2019
    ●
    Results of transformation from July the third [pBAD24-Moclo+insert (endonuclease T7 or nuclease B. subtilis or RdRp)] : Hugues-Natacha-Clara
    There are colonies on each Petri dishes (for each cloning) but just a few for the nuclease of B. subtilis.
    ●
    Colony PCR of transformed cells [pBAD24-Moclo+insert (endonuclease T7 or nuclease B. subtilis or RdRp)]:
    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%
    Friday, 7/5/2019
    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
    WhatsApp Image 2019-07-05 at 17.27.04.jpeg
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    All expected signlals have correct sizes even for the golden gate for B. subtilis, so last time we took too much genetic material during the colony PCR
    Monday, 7/8/2019
    ●
    gblock of Rdrp, T7 in plasmid pBAD24 MoClo (Clara)
    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
    Tuesday, 7/9/2019
    Culture of transformed cells with pBADMoClo Rdrp clone 2' & 5' and pBADMoClo T7 clone 1' for glycerol stock tomorrow.
    Thursday, 7/11/2019
    gblock in plasmid pBAD24MoClo (Clara)
    ●
    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")
    Friday, 7/12/2019
    ●
    Characterization of T7 nuclease and Rdrp (Clara)
    Monday, 7/15/2019
    ●
    Isolation of clones keio T7 and keio Rdrp : Arnaud
    Stripes from colonies are done in 2 agar plates with LB + amp + glu
    Tuesday, 7/16/2019
    ●
    Bacterial cultures for CFU and OD : Arnaud
    keioZ1F'A1
    keioZ1F'T7
    keioZ1F'Rdrp
    keioZ1F'Moclo
    in LB + Amp + glu 3 mL
    Culture overnight at 37°C
    ●
    Glucose 20% solution : Arnaud
    A solution of 500 mL is made with 100 g of Glucose anhydre and is filtrated.
    Small solution of 100 mL are made with the big one to reduce contamination
    Wednesday, 7/17/2019
    Culture of isolated clones (Results) : Abdel_Arnaud
    ●
    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 :
    Thursday, 7/18/2019
    Culture of isolated clones (Results) : Abdel_Arnaud
    ●
    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 :
    Monday, 7/22/2019
    pZA131 PCR to transform it into MoClo + GFPanti sens (Colombe & Clara)
    All the electrophoreses were performed at 110 volts for 30 min with an 1% agarose gel
    Result:
    image.png
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    4 other bands => impurities?
    Then DNA extraction from agarose gel with gel extraction's kit of NucleoSpinR Gel and PCR clean-up, macherey-nagel
    Seen NucleoSpin Macherey-Nagel protocol
    DNA concentration was measured with NanoDropTM
    Tuesday, 7/23/2019
    pZA131 PCR to transform it into MoClo + GFPanti sens (Clara)
    DpnI digestion
    Add 1 µL of DpnI and 2µl of tango buffer to the 20µl of plasmid
    Incubate at 37°C 1 hour
    20 minutes at 80°C
    Stop on ice
    PCR clean up
    See NucleoSpin Macherey-Nagel protocol p18 for low copy plasmid
    Nanodrop pZA31 linearized and digested by DpnI after PCR clean-up
    Quantification of plasmid linearized and gigested by DpnI and after PCR clean-up with the nanodrop:
    ●
    30 ng/µL
    ●
    0.6 A260
    ●
    0.28 260/230
    ●
    1.91 260/280
    Golden gate to introduce gblock GFP anti sens into pZA31
    See the protocol "golden gate caloning"
    2µl plasmid and 7.1 µl gBlock GFP as
    Ligation of pZA31 to make pZA31 MoClo
    Add 7µL of water into eppendorf
    +2µL of ligase buffer (10X)
    +1µL ligase (Promega)
    +10µL pZA31 (empty)
    Incubate the mix at 4°C over night
    Transformation into DH5alpha of Golden Gate product of pZA31 and gblock GFP anti sens
    See the protocol "Heatshock plasmid transformation"
    Wednesday, 7/24/2019
    pZA131 PCR to transform it into MoClo + GFPanti sens (Clara)
    ●
    Stripes of 6 colonies of cell transformed into DH5alpha from yesterday on new media => 2nd selection
    ●
    Transformation of ligation from yesterday of pZA131 MoClo => tomorrow (forgot in the freezer)
    Thursday, 7/25/2019
    pZA131 PCR to transform it into MoClo + GFPanti sens (Clara)
    ●
    Transformation of ligation from yesterday of pZA131 MoClo, see the protocol "heat shock plasmid transformation"
    ●
    PCR colony of the 2nd selection, see the protocol "COLONY PCR", see the program "ecoligem" with oligos 13 & 14
    Capture d’écran 2019-07-29 à 11.30.11.png
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    Tuesday, 7/30/2019
    ●
    Plasmid extraction of PZA31 GFPas : Colombe_Arnaud
    Use of Macherey-Nagel kit "DNA and RNA purification" protocol page 18 "Low copy"
    2 extractions per plasmids pZA31 (clones 2 & 4)
    Analysis with Nanodrop :
    Wednesday, 7/31/2019
    ●
    PCR Rdrp and PCR A1 -> golden gates with pBAD-Moclo = pBAD-A1-Rdrp: Marie_Laura
    PCR protocols:
    Mix Rdrp :
    2,5 μL oligo 16
    2,5 μL oligo 17
    1 μL pBAD-Rdrp
    25 μL Master Mix
    19 μL H2O mQ
    -> in total 50 μL
    Mix A1 :
    2,5 μL oligo 18
    2,5 μL oligo 19
    1 μL pBAD-A1
    25 μL Master Mix
    19 μL H2O mQ
    -> in total 50 μL
    PCR program :
    98°C/30sec
    25 cycles : 98°C/10sec
    56°C/30sec
    72°C/2min
    72°C/2min
    Agarose gel 1%, migration 30min at 100V.
    Result :
    Thursday, 8/1/2019
    ●
    PCR Rdrp and PCR A1 -> golden gates with pBAD-Moclo = pBAD-A1-Rdrp: Marie_Laura
    PCR protocols:
    Mix Rdrp :
    2,5 μL oligo 16
    2,5 μL oligo 17
    1 μL pBAD-Rdrp
    25 μL Master Mix
    19 μL H2O mQ
    -> in total 50 μL
    Mix A1 :
    2,5 μL oligo 18
    2,5 μL oligo 19
    1 μL pBAD-A1
    25 μL Master Mix
    19 μL H2O mQ
    -> in total 50 μL
    PCR program :
    98°C/30sec
    25 cycles : 98°C/10sec
    Tm°C/30sec
    72°C/1min
    72°C/10min
    1 bis : Tm = 55°C,
    1 : Tm = 51°C,
    2 : Tm = 51°C,
    2 bis: Tm = 55°C.
    Agarose gel 1%, migration 30min at 100V.
    Result:
    Gel pBAD Rdrp : Rdrpbis : A1 : A1bis.png
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    Gel pBAD Rdrp : Rdrpbis : A1 : A1bis ZOOM.png
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    ●
    PCR Clean up of A1, RBS_Rdrp (1 and 1bis ; 2 and 2bis) : Arnaud_Lucas
    Use of Macherey-Nagel kit , protocole "PCR Clean up" page 18. Elution with 15 µL H2O
    Analysis with Nanodrop :
    Friday, 8/2/2019
    ●
    Extraction of plasmid DNA pZA31-moclo from DH5alpha : Laura_Marie
    Follow the protocol: made from 4 mL of "pZA31 Moclo DH5aplha 01/08/19 clone 1" and "pZA31 Moclo DH5aplha 01/08/19 clone 2" culture. Eluate with 50µL H2O mQ.
    Nanodrop :
    ○
    clone 1 : concentration = 6 ng/µL ; A260 = 0,1214 ; 260/230 = 0,96 ; 260/280 = 1,94.
    ○
    clone 2 : concentration = 11,8 ng/µL ; A260 = 0,2363 ; 260/230 = 1,30 ; 260/280 = 1,97.
    ●
    Golden Gate of pBADMoclo with A1 and RBS_Rdrp : Arnaud
    2 golden gate clonings are done. One is a negative controle with only pBADMoclo
    Each tube contains :
    1.
    pBADMoclo 1 µL
    2.
    PCR Product A1 1.1 µL (Not for control)
    3.
    PCR Product RBS-Rdrp 1.5 µL (Not for control)
    4.
    NEB Golden Gate assembly mix 1 µL
    5.
    Buffer 10X 2 µL
    6.
    H2O 14.4 µL
    Then let it for 20 minutes at room temperature and store in ice for transformation later on.
    Monday, 8/5/2019
    ●
    Colony PCR of pBAD24 A1 RBS Rdrp from 5 clones : Clara
    Dream Taq was used for a TM of 62°C and elogation time of 4 min for 25 cycles.
    Primers 5 and 6 were used.
    See the protocol Colony PCR
    Clones were also streaped on LB + amp (20µL) + glucose (200µL) and added into 15mL of LB and amp (15µL) & glucose (150µL)
    Tuesday, 8/6/2019
    ●
    Result of Colony PCR of pBAD24 A1 RBS Rdrp from 5 clones : Clara
    Colony PCR pbad24A1rbsrdrp.pdf
    Just the clone 5' had the expected size.
    ●
    Plasmid extraction of pBAD24_A1_RBS_Rdrp clone 5' : Arnaud & Clara
    Use of Macherey-Nagel kit "DNA and RNA purification" protocol page 18 "Low copy"
    Analysis with Nanodrop :
    ●
    Precultures of pZA31-MoClo for plasmid DNA extraction : Clara
    2 clones (5' and 6') were put into 15mL of LB + cm (10µL)
    Wednesday, 8/7/2019
    ●
    Extraction of Plasmid DNA of pZA31 MoClo clones 5' and 6' : Clara
    Use of Macherey-Nagel kit "DNA and RNA purification" protocol page 18 "Low copy"
    Analysis with Nanodrop :
    Thursday, 8/8/2019
    ●
    Heatshock double plasmid transformation of KeioZ1F' with various plamids : Arnaud
    4 double transformations were done with different plasmids :
    ■
    pBAD_RdRp + pZA31_GFPas clone 4
    ■
    pBAD_Moclo + pZA31_GFPas clone 4
    ■
    pBAD_RdRp + pZA31_Moclo clone 6
    ■
    pBAD_A1_RBS_RdRp clone 5 + pZA31_GFPas clone 4
    One µL of plasmid was mixed with 50 µL of competent cells.
    Cells stayed 10 min on ice and then 1 min at 42°C and finally 2 min on ice. One mL of LB is added and tubes are put at 37°C for 1 hour. After that, bacteria are spread on plates
    Results: Just double transformations for the plasmids pBAD24-RDRP + pZA31-MoClo and pBAD24-RDRP + pZA31-GFP-as worked. Streaps of 4 transformants from each of these two double transformations were realised August the 9th.
    Friday, 8/9/2019
    ●
    Heatshock double plasmid transformation of KeioZ1F' with various plamids : Arnaud
    4 double transformations were done with different plasmids :
    ■
    pBAD_Moclo + pZA31_GFPas clone 4
    ■
    pBAD_A1_RBS_RdRp clone 5 + pZA31_GFPas clone 4
    One µL of plasmid was mixed with 50 µL of competent cells.
    Cells stayed 10 min on ice and then 1 min at 42°C and finally 2 min on ice. One mL of LB is added and tubes are put at 37°C for 1 hour. After that, bacteria are spread on plates
    Monday, 8/19/2019
    ●
    Preculture for GFP quantification with FACS
    Incubate KeioZ1F' pBAD-A1-RdRp + pZA31 GFP
    KeioZ1F' pBAD-RdRp + pZA31 GFP
    KeioZ1F' pBAD-MoClo + pZA31 GFP
    KeioZ1F' pBAD-RdRp + pZA31 Moclo
    Incubate single colony in 3 mL LB+glu(0,2%)+amp(100ug/ml)+chl(20ug/ml) at 37°C over night.
    Friday, 8/23/2019
    ●
    Heatshock plasmid transformation : Arnaud
    Transformation of KeioZ1F' with pZA_GFPas + pBAD_Moclo, KeioZ1F' with pBAD_T7 clone 1' + pZA_GFPas. The solutions are spread on plates with LB + AMP + Chloramphénicol.
    Wednesday, 8/28/2019
    Preculture of KeioZ1, KeioZ1 F' pBAD-MoClo + pZA31-GFPas, KeioZ1 F' pBAD-T7 + pZA31-GFPas in LB + amp + cm + glu
    Thursday, 8/29/2019
    Translate the GFP anti-sense RNA by infecting the cell with MS2 phage
    MS2 phage brings RdRp which recognizes the GFP anti-sens RNA.
    Each preculture is diluted at OD600=0,1 in 15mL LB + amp + cm + Atc (inducer of the transcription of GFPas)(1mM) + CaCl2 (1mM) + MgCl2 (1mM), incubate at 37°C.
    When OD600 reach 0,4, add arabinose 0,2% final and incubate at 37°C for 5min.
    Add MS2 phage at MOI=5.
    A each timepoint (t=0, 30min, 60min, 90min and 120min), take 500uL, dilute 100 times in PBS1X, process the sample with the FACS.
    We couldn't monitor fluorescence, we gave up that part of the project (results not showed)

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