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RNA cell (Archived)
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RNA cell

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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