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 infecte the cells by the MS2 phage and produce new virions, which would result from the replication of RNA and its translation into proteins, since MS2 is an positive-sense single-stranded RNA phage.
●
MS2 phage titration : Hugues
Incubate K12 Hfr in 5mL LB-amp (0,2%) initial OD600=0,1 at 37°C
Cultivate with agitation to OD600=0,8
Melt agar, add CaCl2 and MgCl2 (1mM final), keep the mix at 42°C
Pour 3mL agar-CaCl2-MgCl2 in sterile glass tubes
Add 100µL bacteria (K12 Hfr) + 100µL MS2 phage
Pour the 3mL agar(CaCl2-MgCl2-bacteria-phage on the LB plate, spread the mix
Incubate at 37°C
Thursday, 18/7/2019
MS2 infection : Hugues
KeioZ1, KeioZ1 F' MoClo, KeioZ1 F' A1, KeioZ1 F' T7
Dilute these 4 strains in LB (amp if necessary) in 12 mL LB at 0D600=0,1
Add CaCl2 and MgCl2 (1 mM final)
Incubate at 37°C with agitation (200rpm) to OD=0,4
40min after the incubation of the 4 strains, dilute K12 Hfr strain in 12mL LB at OD=0,1 (no CaCl2, MgCl2)
When Keio strains are at OD=0,4, add arabinose (0,2% final) and incubate at 37°C for 30min
Mesurate OD600 then add phage volume equals to : V(µL) = [5*vol culture bacteria (in mL)*OD600*5*10^8]/phage concentration
-> OD600 30min after arabinose
A
A
B
B
C
C
1
1
KeioZ1
0,65
466,5µL
2
2
KeioZ1 F' MoClo
0,78
559,8
3
3
KeioZ1 F' A1
0,49
551,7
4
4
KeioZ1 F' T7
0,56
401,9
Table7
Take off 0,5mL of bacteria at time 0, 1 hour, 2h and 18h. Take OD600 for each culture
A
A
B
B
C
C
D
D
E
E
1
1
t0
t1h
t2h
t18h
2
2
KeioZ1
0,65
2,6
4,5
3
3
KeioZ1 F' MoClo
0,78
0,47
2,7
4
4
KeioZ1 F' A1
0,49
0,51
1,6
5
5
KeioZ1 F' T7
0,56
0,40
0,6
Table8
With the 0,5mL, centrifugate at 13000rpm 5 min
Take off 180µL of the supernatant, put in a microplaque
Dilute 10 fold in series the supernatant (20 in 180) (No dilution to 10^-9)
Prepare 3mL soft agar (3mL for each LB plate needed)(50% solid LB + 50% liquid LB + 1mM CaCl2 + 1mM MgCl2, 100µL K12 Hfr (at OD=0,4))
Pour the 3mL onto LB plate
Once soft agar dry out, drop off 5µL of each dilution (+ND) on LB + soft agar plate. 37°C
Friday, 19/7/2019
MS2 phage stock : Hugues
Add 10mL phage buffer to a phage titration plate with lysis range. Incubate the Petri dish at room temperature 24h then repipette the phage buffer, filtrate it then stock the MS2 phage stock at 4°C.
The MS2 phage stock has to be titrated.
Tuesday, 23/7/2019
MS2 phage infection : Hugues
Start a culture of KeioZ1, Z1 F' MoClo, Z1 F' A1, Z1 F' T7 in 12mL LB (+amp if necessary) + 1mM CaCl2 + 1mM MgCl2 at OD600=0,1 then incubate at 37°C
Start the culture of K12 Hfr in 12 mL LB 30/40min after the first culture
Wednesday, 31/7/2019
See the protocol "Infection assay" below.
Protocol-phage-infection1.docx
Precultures of LB plus 10µL of ampicilline and 100µL of glucose (except for KEÏO Z1 and Hfr) were used to inoculate 10 mL of each of our strains. 583 µL of preculture A1 was put into 15mL of LB, 600µL of MoClo, 588µL of T7 and 619µL of Keio Z1.
The CACL2, MgCl2 were added after 30min of culture.
The final OD600nm used for the infection were:
A
A
B
B
C
C
D
D
E
E
1
1
Strains names
KEIO Z1
MoClo
A1
T7
2
2
OD 600nm
0.5
0.35
0.42
0.32
Table1
According to the formula from the protocol, the amount of phages used was with a concentration of 4.18*10^5 PFU/mL:
○
Keio Z1 : 281 µL in 10ml medium plus bacteria
○
MoClo: 263 µL
○
A1: 275 µL
○
T7: 233 µL
Hfr cells had an OD600nm of 0,79 when they were poured into soft Agar containing 1mM of CaCl2 and MgCl2.
Serial dilutions started from 10-1 to 10-9, 20µL of undiluted supernatant was put into 180µL of phage buffer.
The ODs were taken for 0min, 60 min and 120 min:
A
A
B
B
C
C
D
D
E
E
1
1
Strains
OD 600nm Keïo Z1
OD 600nm Keïo Z1 F' pBAD24-MoClo1
OD 600nm Keïo Z1 F' pBAD24-A1
OD 600nm Keïo Z1 F' pBAD24-T7
2
2
T=0min
0.28
0.28
0.28
0.2
3
3
T=60min
0.8
0.7
0.58
0.29
4
4
T=120min
1.4
0.69
0.55
0.21
5
5
T= Day after
3.1
2
0.9
2.16
OD600nm every 1 hour for MS2 infection and the day after
HFR stayed all night long on table and then mixed with soft agar for the last time point (day after).
Bacteria and phages mix wasn't put at 37°C in LB + ARA directly, stayed in ice
Results: the point "day after" wasn't good, couldn't count anything, maybe because of the HFR stayed all night long on the bench.
Thursday, 8/8/2019
See the protocol "Infection assay"
Precultures of LB plus 20µL of ampicilline and 200µL of glucose (except for KEÏO Z1 and Hfr) were used to inoculate 20 mL of each of our strains. 769 µL of preculture A1 and MoClo was put into 15mL of LB, 741µL of T7 and 667µL of Keio Z1.
The CACL2, MgCl2 were added at a final concentration of 1mM.
The final OD600nm used for the infection were:
A
A
B
B
C
C
D
D
E
E
1
1
Strains name
KEIO Z1
MoClo
A1
T7
2
2
OD 600nm
0,41
0,49
0,42
0,38
Table2
According to the formula from the protocol, the amount of phages used was with a concentration of 4.18*10^5 PFU/mL:
○
Keio Z1 : 294 µL in 20ml medium plus bacteria
○
MoClo: 352 µL
○
A1: 301 µL
○
T7: 273 µL
After 1hour in ice, LB + 0,2% arabinose and 100 µg/µL of ampicilline was added to the mix of phages and bacteria. Normally, the strain KEIO Z1 shouldn't have received Ampicilline since it is sensitive to it.
Hfr cells had an OD600nm of 0,67 when they were poured into soft Agar containing 1mM of CaCl2 and MgCl2.
Serial dilutions started from 10-1 to 10-9, 20µL of undiluted supernatant was put into 180µL of phage buffer.
The ODs were taken for 0min, 20min, 40min,60 min, 80 min, 100 min and 120 min:
A
A
B
B
C
C
D
D
E
E
1
1
Strains
OD 600nm Keïo Z1
OD 600nm Keïo Z1 F' pBAD24-MoClo1
OD 600nm Keïo Z1 F' pBAD24-A1
OD 600nm Keïo Z1 F' pBAD24-T7
2
2
T=-60min
0.41
0.49
0.42
0.38
3
3
T=20 min
0.54
0.64
0.58
0.33
4
4
T=40min
0.38
0.87
0.56
0.23
5
5
T=60min
0.25
0.94
0.55
0.15
6
6
T=80min
0.22
0.5
0.59
0.15
7
7
T=100min
0.15
0.27
0.59
0.11
8
8
T=120min
0.14
0.23
0.57
0.1
Table3
Results of the infection assay :
Phage titration & infection.xlsx
For the ODs 600nm, these results weren't expected, we don't know what went wrong --> look for it.
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Titration of Ms2 phage (Laetitia)
Phage has been titrated by folowing Titration phage protocol.
100 μL of a 1 mL 10-5 to 10-9 dilution has been plated.
220 mL of soft agar without MgCl2 and CaCl2 was prepared and put at 60°C overnight
Hfr and KeïoZ1 strains were precultured into 3ml of LB.
Keïo Z1 F' cells containing the following plasmids were precultured into 3 mL of LB plus ampicilline (Cf=100µg/µm) and 0.2% glucose
-pBAD-A1
-pBAD-T7
-pBAD-B.subtilis
-pBAD-MoClo
Wednesday, 14/8/2019
The soft agar which was made the day before had a little thing inside, then we were suspicious about contamination. So we made another one.
See the protocol "Infection assay" for more precise information.
Precultures of LB plus 20µL of ampicilline and 200µL of glucose (except for KEÏO Z1 and Hfr) were used to inoculate 20 mL of each of our strains. 541 µL of preculture A1 and 435 µL of preculture MoClo , 741µL of T7, 667µL of Keio Z1 and 667 µL of B. subtiliswere put into 20mL of LB.
The CACL2, MgCl2 were added at a final concentration of 1mM.
The final OD600nm used for the infection were:
A
A
B
B
C
C
D
D
E
E
F
F
1
1
Strains name
KEIO Z1
MoClo
A1
T7
B. subtilis
2
2
OD 600nm
0,4
0,48
0,50
0,34
0.26
Table5
According to the formula from the protocol, the amount of phages used was for a concentration of 8.8*10^9 PFU/mL :
○
Keio Z1 : 409 µL in 20ml medium plus bacteria
○
MoClo: 464 µL
○
A1: 511 µL
○
T7: 348 µL
○
B. subtilis: 266 µL
After 30 min in ice, LB + 0,2% arabinose + 100 µg/µL was added to the mix of phages and bacteria. Normally, the strain KEIO Z1 shouldn't have received Ampicilline since it is sensitive to it.
Hfr cells had an OD600nm of 0.42 when they were poured into soft Agar containing 1mM of CaCl2 and MgCl2.
Serial dilutions started from 10-1 to 10-9, 20µL of undiluted supernatant was put into 180µL of phage buffer. In the same time, 400 µL of the remaining supernatant was put into another tube with 4 µL of chloroform in order to spread it on another plate. This way, we can spread more than a 5 µL drop and get better results for PFU/mL.
The ODs were taken for 0min, 30 min, 60min, 90 min and 120 min:
A
A
B
B
C
C
D
D
E
E
F
F
1
1
Strains
OD 600nm Keïo Z1
OD 600nm Keïo Z1 F' pBAD24-MoClo1
OD 600nm Keïo Z1 F' pBAD24-A1
OD 600nm Keïo Z1 F' pBAD24-T7
OD 600nm Keïo Z1 F' pBAD24-B. subtilis
2
2
T=0min
Unexploitable results
0.29
0.34
0.22
0.18
3
3
T=30 min
0.66
0.59
0.22
0.29
4
4
T=60min
0.85
0.6
0.16
0.25
5
5
T=90min
0.31
0.58
0.11
0.26
6
6
T=120min
0.31
0.6
0.13
0.25
Table6
Results of the infection assay :
A
A
B
B
C
C
D
D
E
E
F
F
1
1
Time (min)
2
2
Strain name
0
30
60
90
120
3
3
MoClo
10^-5
10-4
10^-7
10^-7
10^-7
4
4
A1
10-4
10-4
10^-5/ 10^-6
5
5
T7
10-4
10-4
10^-6
10^-5/ 10^-6
10^-6/10^-7
6
6
B.subtilis
10-4/10-5
10^-5
10^-7
10^-5/ 10^-6
10^-5/ 10^-6
Dilutions where we can count lysis ranges
We can generally count lysis ranges between dilution 10^-4 and 10^-7.
OD results:
Phage titration & infection.xlsx
We can see that the optic density for pBAD-MoClo drops after 60min of infection, like the previous experiment. While OD 600nm for pBAD-T7 & A1 increase for 30 min of infection and then remain stable. Finally, pBAD-B.subtilis OD 600nm remain stable for 30 min of infection and after start to decrease slowly.
Tuesday, 20/8/2019
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Preculture of colonies for phage infection
Incubate Keio Z1F, K12 Hfr in 5mL LB at 37°C. Incubate Keio Z1F' pBAD-MoClo, KeioZ1F' pBAD-A1, KeioZ1F' pBAD-T7, Keio Z1F' pBAD-B. sub in 5mL LB+amp(100ug/ml)+glu(0,2%) at 37°C.
Wednesday, 21/8/2019
●
Phage infection
Dilute each strain (K12 Hfr included) at OD600 = 0,1 in LB +CaCl2(1uL/mL) + MgCl2(1uL/mL) (+amp if necessary), incubate at 37°C.
During bacterial growth, prepare soft agar (50% solid agar + 50% liquid agar + CaCl2(1uL/mL) + MgCl2(1uL/mL)).
Once the indicator strain K12 Hfr reaches an OD=0,4-0,8, add 100µL of K12 Hfr into the soft agar.
Pour 3mL of soft agar+K12 Hfr on top of LB plates.
Once the culture reach to OD600=0,4, add arabinose (0,2% final), incubate at 37°C for 5 minutes.
Add the volume of phage equals to : V(mL) = (5* volume (mL) of bacteria culture * OD * 5*10^8)/phage concentration.
Extract immediately 0,5mL of culture for phage titration, this correspond to the timepoint 0, mesure the OD of the culture and reincubate the culture at 37°C.
Extract 0,5mL of culture at t=30min, t=60min and t=120min.
A
A
B
B
C
C
D
D
E
E
F
F
G
G
1
1
Time (min)
KeioZ1
KeioZ1 F' pBAD_MoClo
KEIOZ1 F' pBAD_A1
KeioZ1 F' pBAD_T7
KeioZ1 F' pBAD_B.sub
2
2
0
0.55
0.51
0.4
0.38
0.5
3
3
120
1.84
1.34
0.75
0.3
0.69
4
4
Volume of phages to add (MOI = 5)
394,7
366,0
287,1
272,7
358,9
5
5
6
6
7
7
OD600 + volume of phage for infection assay
For each timepoint, centrifugate the 0,5mL of culture at max speed for 5min.
Prepare 10-fold serial dilutions of the supernatant in phage buffer (10^-1 to 10^-9).
Add 4µL of chloroform to remaining supernatant, stock phages in chloroform at 4°C.
Spot 5uL of the undiluted supernatant and its serial dilutions onto a plate with top agar + K12 Hfr.
Incubate every plate at 37°C.
Phage titration infection 21082019.xlsx
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Preculture of colonies for phage infection
Incubate K12 Hfr in 5mL LB at 37°C.
Thursday, 22/8/2019
Identify the maximum dilutions which permit to get lysis ground.
A
A
B
B
C
C
1
1
Replicate
1
2
2
2
KeioZ1
10^-5
3
3
KeioZ1 F' MoClo
10^-5
10^-5
4
4
KeioZ1 F' A1
10^-5
10^-5
5
5
KeioZ1 F' T7
10^-6
10^-6
6
6
KeioZ1 F' B. subtilis
10^-6
10^-6
7
7
t=0
Prepare soft agar with CaCl2 (1mM final) and MgCl2 (1mM final).
Dilute K12 Hfr at a OD600=0,1 in LB, wait until OD600=0,5.
Add 100uL of K12 Hfr and 100uL of phages in 3mL soft agar. Each phage dilutions were stocked in chloroform (August, 21th).
Phages come from the chloroform-conserved-phages .
●
Preculture of colonies for phage infection
Incubate Keio Z1F, K12 Hfr in 5mL LB at 37°C. Incubate Keio Z1F' pBAD-MoClo, KeioZ1F' pBAD-A1, KeioZ1F' pBAD-T7, Keio Z1F' pBAD-B. sub in 5mL LB+amp(100ug/ml)+glu(0,2%) at 37°C.
Friday, 23/8/2019
●
Phage infection : Hugues_Natacha
Dilute each strain (K12 Hfr included) at OD600 = 0,1 in LB +CaCl2(1uL/mL) + MgCl2(1uL/mL) (+amp if necessary), incubate at 37°C.
During bacterial growth, prepare soft agar (50% solid agar + 50% liquid agar + CaCl2(1uL/mL) + MgCl2(1uL/mL)).
Once the indicator strain K12 Hfr reaches an OD=0,4-0,8, add 100µL of K12 Hfr into the soft agar.
Pour 3mL of soft agar+K12 Hfr on top of LB plates.
Once the culture reach to OD600=0,4, add arabinose (0,2% final), incubate at 37°C for 15 minutes.
Add the volume of phage equals to : V(mL) = (5* volume (mL) of bacteria culture * OD * 5*10^8)/phage concentration.
Extract immediately 0,5mL of culture for phage titration and 1mL of culture for gDNA extraction. This correspond to the timepoint 0, mesure the OD of the culture and reincubate the culture at 37°C.
Extract 0,5mL of culture at t=30min, t=90min, t=120min and t=150min.
For each timepoint, centrifugate the 0,5mL of culture at max speed for 5min.
Prepare 10-fold serial dilutions of the supernatant in phage buffer (10^-1 to 10^-9).
Add 4µL of chloroform to remaining supernatant, stock phages in chloroform at 4°C.
Spot 5uL of the supernatant serial dilutions onto a plate with top agar + K12 Hfr.
Incubate every plate at 37°C.
●
Genomic DNAs of bacteria infected by phages
At different time points (t0, t30, t90, t120, t150) of the phage infection, 1ml of bacterial culture with phage is withdraw. This is done for the four conditions : A1, T7, Moclo and B.sub. The kit used to quantify the genomics DNA of infected bacteria is "QIAamp DNA Blood Mini Kit (50)" from Qiagen. The protocole is "DNA Purification from Tissues" page 32. We started at step 3. Step 1, 2(a, b, c) and 5a are not done. The elution step is done only one time with 200µL of buffer AE. The final solutions are then analyzed with Nanodrops and put on an electrophoresis gel
