Team:GO Paris-Saclay/NucleasesCharacterization/gp3

Nuclease_gp3 (From T7) · Benchling

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Nuclease_gp3 (From T7)

Project: Nucleases characterizations
Authors: iGEM_GO_Paris_saclay
Wednesday, 3/7/2019
Aim: Insert the gene of gp3 in pBD24MoClo with golden gate method in order to express gp3 nuclease under the control of the inducive promoter arabinose-dependent promoter Para.
●
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
A
B
C
D
1
Endonuclease T7Nuclease B. subtilisRdRp
2
Volume (µL ) for Golden Gate cloning 277
Table1
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, 4/7/2019
●
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
A
B
C
1
Primer namePrimer sequenceTm °C
2
2019GO-5-Seq-pBAD-F5'- TGGCTATGCCATAGCATTTTTATCC55,6
3
2019GO-6-Seq-pBAD-R5'- GGT CTG ATT TAA TCT GTA TCA GGC TG55,3
Primer sequences for checking insertion of the inserts
A
B
1
Buffer57,5µL
2
dNTP23µL
3
Oligo523µL
4
Oligo623µL
5
H2O195,5µL
6
TAQ3,45µL
PCR master mix
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, 5/7/2019
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, 8/7/2019
●
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.
A
B
1
plasmid DNA nameConcentration (µg/µL)
2
pBADMoClo T7 clone 2116
3
pBADMoClp T7 clone 166
4
pBADMoClo Rdrp clone 1 89
5
pBADMoClo Rdrp clone 456
6
 pBAD24 MOCLO clone 1 42
7
pBADMoClo T7 clone 1'16
8
pBADMoClo Rdrp clone 5'23
9
pBADMoClo Rdrp clone 2'29
10
Table2
●
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, 9/7/2019
Culture of transformed cells with pBADMoClo Rdrp clone 2' & 5' and pBADMoClo T7 clone 1' for glycerol stock tomorrow.
Thursday, 11/7/2019
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, 12/7/2019
●
Characterization of T7 nuclease and Rdrp (Clara)
A
B
C
D
E
1
TimeOD T7 nucleaseOD Rdrp
2
Without ARAWith ARAWithout ARAWith ARA
3
0 min0.430.430.650.65
4
30 min0.720.211.011.01
5
1 hour0.980.641.251.31
6
2 hour1.250.851.681.65
Table3
Monday, 15/7/2019
●
Isolation of clones keio T7 and keio Rdrp : Arnaud
Stripes from colonies are done in 2 agar plates with LB + amp + glu
Tuesday, 16/7/2019
Microscopy Keio MoClo : Hugues
AIM: As a control for the characterization of all our nucleases
○
Culture and OD600
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
○
Preparation of slides for microscopy
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)
●
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
Wednesday, 17/7/2019
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 :
A
B
C
D
E
F
G
H
I
J
K
L
1
Time-30min+30min+90min+150min+tomorrow
2
GluGlu+AmpGluGlu+AmpGluGlu+AmpGluGlu+AmpGluGlu+Amp
3
A1Ara-3-1-3-1-4-1-6-2
4
1,10E+019,00E+002,90E+018,00E+001,00E+013,40E+011,40E+011,50E+01
5
2,30E+011,90E+012,90E+011,10E+011,60E+012,50E+011,20E+012,00E+01
6
1,50E+012,00E+013,00E+011,00E+018,00E+003,10E+011,30E+011,90E+01
7
No Ara-5-5-6-5-6-6-6-6-6-6
8
5,00E+006,00E+007,00E+003,00E+014,00E+004,00E+001,40E+011,50E+012,20E+011,80E+01
9
9,00E+007,00E+001,10E+012,50E+013,00E+001,00E+011,30E+011,00E+011,40E+011,80E+01
10
7,00E+005,00E+005,00E+002,80E+016,00E+008,00E+001,10E+011,20E+011,70E+011,70E+01
11
T7Ara-1-1-2-1-2-1-6-2
12
2,50E+015,00E+001,00E+011,30E+012,80E+011,50E+011,00E+017,00E+00
13
2,60E+019,00E+001,90E+011,50E+011,70E+012,50E+011,70E+015,00E+00
14
2,10E+011,40E+011,70E+017,00E+007,00E+001,70E+011,70E+012,00E+00
15
No Ara-4-4-5-5-5-5-5-5-4
16
1,50E+012,00E+011,40E+011,20E+012,40E+011,90E+011,00E+011,30E+011,40E+01
17
1,60E+011,60E+011,70E+012,20E+012,10E+012,30E+011,10E+019,00E+001,90E+01
18
1,90E+011,70E+011,70E+012,20E+012,60E+012,30E+014,00E+001,10E+011,90E+01
19
MocloAra-5-5-6-6-6
20
2,70E+011,80E+019,00E+001,00E+012,30E+01
21
1,80E+012,80E+015,00E+001,70E+012,50E+01
22
1,90E+012,20E+011,10E+011,30E+013,00E+01
23
No Ara-5-5-5-5-6-6-6-6
24
9,00E+001,50E+012,80E+011,90E+018,00E+008,00E+001,50E+011,90E+01
25
1,60E+011,10E+012,80E+012,10E+016,00E+009,00E+009,00E+001,80E+01
26
1,10E+011,40E+012,20E+012,80E+015,00E+001,40E+011,70E+012,10E+01
27
RdrpAra-5-5-5-5-6-6-6-6
28
2,10E+012,40E+012,80E+011,40E+018,00E+009,00E+009,00E+005,00E+00
29
2,70E+011,40E+012,60E+012,70E+011,30E+018,00E+007,00E+001,00E+01
30
2,50E+012,10E+012,80E+012,40E+019,00E+006,00E+001,20E+011,00E+01
31
No Ara-4-5-5-5-6-6-6-6-6-6
32
1,70E+014,00E+003,10E+012,40E+019,00E+001,00E+011,30E+011,50E+012,10E+011,80E+01
33
1,90E+011,30E+012,80E+012,60E+018,00E+007,00E+001,50E+011,20E+011,70E+012,20E+01
34
2,40E+014,00E+002,50E+012,60E+018,00E+009,00E+001,60E+011,10E+011,90E+012,10E+01
Table4
A
B
C
D
E
F
G
1
O.DTIME
2
-3030901501110
3
A1Ara0,250,600,590,572,90
4
No ara0,250,771,701,884,20
5
T7Ara0,180,420,370,382,80
6
No ara0,180,621,091,621,00
7
MocloAra0,160,701,151,543,90
8
No ara0,160,771,381,853,90
9
RdrpAra0,180,711,201,565,50
10
No ara0,180,741,371,843,90
Table5
Thursday, 18/7/2019
●
Measurement of gDNA concentration with Nanodrop and electrophorese on an agarose gel for T7, moclo and A1 characterization assays (Kim)
A
B
C
D
1
T7MocloA1
2
0h290 ng/µl278 ng/µl283 ng/µl
3
30min250 ng/µl528 ng/µl379 ng/µl
4
90min179 ng/µl580 ng/µl324 ng/µl
5
150min148 ng/µl810 ng/µl307 ng/µl
Table6
○
Result of gDNA extractions at 0, 30, 90 and 150 min after nucleases gp3 and A1 inductions with arabinose
image.png
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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 :
A
B
C
D
E
F
G
H
I
J
K
L
1
Time-30min+30min+90min+150min+tomorrow
2
GluGlu+AmpGluGlu+AmpGluGlu+AmpGluGlu+AmpGluGlu+Amp
3
A1Ara-3-2-3-2-3
4
20944332
5
2181038
6
254934
7
No Ara-4-4-6-6-6-6-5-5
8
1617551092928