Take glycerinated cultures of C58 Agrobacterium with dsRED from GoldenBraid Collection. Prepare broth culture (5 mL LB + 5 μL kanamycin + 5 μL Rifampin) 1:1000 and incubate overnight at 28°C.
19/05/2016
Refresh previously made culture by inoculating 10 μL in a new culture medium.
20/05/2016
Agroinfiltration in Nicotiana benthamiana of C58 with dsRED.
06/06/2016
Take from the glycerinates of Goldenbraid Collection:
Plasmid
GB Code
pD6B3 α1
GB0015
pD6B3 α2
GB0017
pD6B3 Ω1
GB0019
pD6B3 Ω2
GB0021
pUPD2
GB0307
Prepare liquid culture (3 mL LB + 3 μL antibiotic) 1:1000. Incubate at 37°C overnight.
Experiment with snails:
Let both N. benthamiana leafs with snails overnight at room temperature in separated boxes. We will observe if snails eat the leafs and if appears fluorescence.
15/06/2016
Experiment is over due to snails haven’t eaten leafs enough so we have not been able to see fluorescence.
30/06/2016
Orange DNA Genome Extraction protocol
Take from the glycerinates of GoldenBraid Collection:
gBlocks of - promoter 35s:5’ region - have arrived.
We perform a PCR of orange and rice Genome Extraction following the protocol
Sample
Initial concentration(ng/μL)
Final concentration(ng/μL)
Initial volume(μL)
Final volume (μL)
Clemenules 1
3153.8
150
4.756
100
Clemenules 2
4527.9
150
3.31
100
Gleva 1
294.9
150
50.8647
100
Gleva 2
193.7
150
77.44
100
Reagent
Volume(μL)
Program
Clemenules DNA 1
1
Temperature
Time
Buffer HF
10
98°C
5 minutes
dNTPs
2
98°C
35x
30 seconds
IG16JUL01 (TFL_For)
2.5
64°C
30 seconds
IG16JUL02 (TFL_Rev)
2.5
72°C
30 seconds
Taq phusion
0.5
72°C
10 minutes
H2O milli-Q
31.5
16°C
∞
Reagent
Volume(μL)
Program
Gleva DNA
1
Temperature
Time
Buffer HF
10
98°C
5 minutes
dNTPs
2
98°C
35x
30 seconds
IG16JUL03 (Ga20_for)
2.5
72°C
30 seconds
IG16JUL02 (Ga20_rev)
2.5
72°C
30 seconds
Taq phusion
0.5
72°C
10 minutes
H2O milli-Q
31.5
16°C
∞
Ligate reaction of promoter 35s:5’ region in pUPD2. Following ligation protocol , BsmbI enzyme is used in this reaction.
08/07/2016
Run electrophoresis gel of Clemenules and Gleva PCR products. 45 mL agarose gel at 1 % 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 120 V.
Transform E. coli with the next devise: promoter 35s:5’ region (electroporation 2.5KV). Plating it and incubate overnight at 37°C.
Take glycerinated culture for Georgia collaboration. The devise is promoter 35s:GFP:Tnos (α1 and kanamycin)
09/07/2016
Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
promoter 35s:GFP:Tnos
No colonies have grown in the devise promoter 35s:5’ region petri dishes. Repeat transformation procedure, plating again and incubate overnight at 37°C.
11/07/2016
Pick a single E. coli DH5α (promoter 35s:5’ region in pUPD2) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of chloramphenicol in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.
Check Georgia miniprep concentration with NanoDrop (promoter 35s:GFP : Tnos)
Sample
DNA Concentration(ng / μL)
DNA Concentration(ng)
1
105.2
5035.2
2
104.6
5035.2
Targets ligations in pUPD2 (Orange Clemenules and Rice Gleva). Following ligation protocol , BsmbI enzyme is used in this reaction.
12/07/2016
Pick a single E. coli DH5α (target Gleva in pUPD2 and target Clemenules in pUPD2) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of chloramphenicol in a 50 ml tube with the colony and incubate it 16 hours at 37°C.
Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
Promoter 35s:5’region in pUPD2
Digestion of minipreps with NotI. Incubate 1 hour at 37°C
Run electrophoresis gel of the following devise: promoter 35s:5’ region in pUPD2. We remain the samples 1 and 3.
Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
Rice Gleva target in pUPD2
Orange Clemenules target in pUPD2
Digestion of minipreps with NotI. Incubate 1 hour at 37°C.
Run electrophoresis gel of the same devise:
Rice Gleva target in pUPD2
Orange Clemenules target in pUPD2
Ligation using Golden Braid assembly of the next devise:
Promoter 35s:5’ region:Target:Luc:Tnos in α1 plasmid.
13/07/2016
E. coli Transformation with the devise:
Promoter 35s:5’ region:Target:Luc:Tnos in α1 plasmid.
E. coli plating in plates with Kanamycin (antibiotic). Incubate overnight at 37°C
14/07/2016
Pick a single E. coli DH5α (promoter 35s:5’ region) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of chloramphenicol in a 50 ml tube with the colony and incubate it 16 hours at 37°C.
Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37°C. There is a total of twelve minipreps. It has been followed the Miniprep digestion protocol .
Run electrophoresis gel of the digestion products.
Lanes
Samples
Verification
1
1 Kb molecular weight marker
2
gRNA Ga20ox 1
3
gRNA Ga20ox 2
4
Ga20ox consensus 1
5
Ga20ox consensus 2
6
Ga20ox knock-out 1
7
Ga20ox knock-out 2
8
TFL consensus 1
9
TFL consensus 2
10
TFL knock-out 1
11
TFL knock-out 2
12
TFL gRNA 1
13
TFL gRNA 2
14
1 Kb molecular weight marker
Pick a single E. coli DH5α colony from the plates that have been incubated overnight. The devises are:
Promoter 35s:TFL Knock-out:Luc:Tnos (4 samples)
Promoter U6-26:Ga20ox sgRNA:psgRNA (2 samples)
Inoculate a starter culture of 4 ml of LB medium with 4 μL of kanamycin in a 50 ml tube with the colony and incubate it 16 hours at 37°C.
Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37°C. There is a total of six minipreps. It has been followed the Miniprep digestion protocol .
Run an electrophoresis gel of the digestion products.
Lanes
Samples
Verification
1
1 Kb molecular weight marker
2
gRNA Ga20ox 1
3
gRNA Ga20ox 2
4
gRNA Ga20ox 3
5
gRNA Ga20ox 4
6
TFL knock-out 1
7
TFL knock-out 2
8
1 Kb molecular weight marker
However, despite the fact that electrophoresis gel have correctly run, we have decided to repeat the ligation reactions. We have not get the expected results for a gRNA.
Ligation using Golden Braid assembly of the next devises:
Promoter 35s:5’ region:TFL Clemenules target control positive:Luc:Tnos
Promoter 35s:5’ region:Ga20ox Gleva target control positive:Luc:Tnos
Digestion of the minipreps with EcoRI which have been done before. Incubate 1 hour at 37°C. There is a total of six minipreps. It has been followed the Miniprep digestion protocol .
Run an electrophoresis gel of the digestion products. We will keep the minipreps with “1”.
Lanes
Samples
Verification
1
1 Kb molecular weight marker
2
gRNA TFL 1
3
gRNA TFL 2
4
TFL consensus 1
5
TFL consensus 2
6
TFL knock-out 1
7
TFL knock-out 2
8
1 Kb molecular weight marker
9
gRNA Ga20ox 1
10
gRNA Ga20ox 2
11
Ga20ox consensus 1
12
Ga20ox consensus 2
13
Ga20ox knock-out 1
14
Ga20ox knock-out 2
15
1 Kb molecular weight marker
Ligation using Golden Braid assembly of the next devises. Is used the restriction enzyme BsmbI.
Ordered the necessary primers to sequence: TFL consensus, TFL knockout, Ga20ox consensus and Ga20ox knockout.
E. coli transformations with:
Promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA: psgRNA
Plating the last devise in plates with Agrobacterium C58. Plates contain spec + IPTG + X-Gal. Incubate 2 days at 28°C.
Pick a single E. coli DH5α (promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA and promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA) colony from the plates that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of spectinomycin in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.
23/07/2016
Minipreps (4 samples for each devise) with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
Digestion of minipreps with BamHI following digestion protocol . Incubate 1 hour at 37°C.
Run an electrophoresis gel of the digestion products. We will keep the minipreps with the sample number 4 for TFL sgRNA and the sample number 2 for Ga20ox sgRNA.
Pick a single Agrobacterium C58 (promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA and promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA ) colony from the plates that has been incubated overnight. Inoculate a starter culture of 5 ml of LB medium with 5 μL of spectinomycin and kanamycin in a falcon tube with the colony and incubate it overnight at 28°C with shaking.
Pick a single Agrobacterium C58 (promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA in 3Ω1 and promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA in 3Ω1 ) colony from the plates that has been incubated overnight. Inoculate a starter culture of 5 ml of LB medium with 5 μL of spectinomycin and kanamycin in a falcon tube with the colony and incubate it overnight at 28°C with shaking.
Incubate it 48 hours at 28°C
Minipreps of Agrobacterium with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
promoter 35s:5’ region:TFL Clemenules target control positive:Luc:Tnos
promoter 35s:5’ region:Ga20ox Gleva target control positive:Luc:Tnos
Digestion of minipreps with the restriction enzyme EcoRI. Incubate 1 hour at 37°C.
Run electrophoresis gel of the digestion products.
Lanes
Samples
Verification
1
1 Kb molecular weight marker
2
TFL target
3
Ga20 ox target
27/07/2016
Received the necessary primers to sequence:
TFL consensus, TFL knockout, Ga20ox consensus and Ga20ox knockout.
Prepare Agrobacterium cultures: Inoculate a starter culture of 5 ml of LB medium with 5 μL of Kanamycin and 5 μL of rifampin in a 50 ml tube with the colony and incubate it 48 hours at 28°C with shaking. The devises are:
Sequencing the last devises to check if these are correct.
28/07/2016
Refresh Agrobacterium tumefaciens cultures with the aim of infiltrating in N.benthamiana on 29/07
Refresh cultures of the devises promoter 35s: 5’ region: TFL target : Luc : Tnos and promoter 35s : 5’ region: Ga20ox target : Luc : Tnos in order to prepare the more Miniprep reaction.
Sequencing results have arrived.
Device
Order
Sequencing
TFL target
210.13.250
SNP in the position 652 of the target. Position 1 of the gRNA. GA
Ga20 ox Target
210.13.253
Same sequence
TFL consensus
210.13.251
Same sequence
TFL KO
210.13.252
Same sequence
Ga20 ox Consensus
210.13.254
Same sequence
Ga20 ox KO
210.13.255
Same sequence
29/07/2016
Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
Promoter 35s: TFL Target : Luc : Tnos
Promoter 35s : Ga20 ox Target : Luc : Tnos
Digestion of minipreps with EcoRI following digestion protocol. Incubate 1 hour at 37°C.
Run electrophoresis gel of the digestion products. We will discard this culture because the gel result doesn’t correspond with what we expect.
Agroinfiltration procedure with 9 plants of N.benthamiana following the Agroinfiltration protocol.
01/08/2016
Pick up the samples of infiltrated plants. We keep 3 disks per plant in a Eppendorf tube and we take 2 samples of each plant.
Luciferase assay is made following the correct protocol. After analyzing all the data obtained, it seems that the system works but we must optimized it to increase the signal range. In this way, we will be able to distinguish signal from noise.
Conclusions luciferase assay:
Eliminate 5’ region of the devise
Change linker sequence
Add renilla in luciferase assay
Change reporter to +1
Use pless as control
Use a Wild Type as control
Devises in cis
Design a consensus target as longer as the amplified.
02/08/2016
Culture refresh of C58 Agrobacterium to infiltrate on Friday.
Pick a single E. coli DH5α (promoter 35s:5’region:TFL target:Luc:Tnos in α1 and promoter 35s:5’region:Ga20 target: Luc: Tnos in α1) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of kanamycin in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.
Targets ligations with Renilla reporters.
Reagent
Volume(μL)
TFL KO/Ga20KO/TFL cons / Ga20 cons
1
Promoter35s: Renilla: Tnos
1
pUPD2
1
BSA10X
1.2
Ligase Buffer
1.2
BsmbI
1
T4 ligase
1
H2O milli-Q
4.6
03/08/2016
DH5α E. coli transformation with the devises:
Promoter 35S : 5’ region : TFL KO : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
Promoter 35S : 5’ region : TFL consensus : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
Promoter 35S : 5’ region : Ga20ox KO : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
Promoter 35S : 5’ region : Ga20ox consensus : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
E. coli plating in plates with Kanamycin (antibiotic). Incubate 16 hours at 37°C
Minipreps with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
promoter 35s:5’region:TFL target:Luc:Tnos in α1
promoter 35s:5’region:Ga20 target: Luc: Tnos in α1
Pick a single E. coli DH5α (TMV CDS) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of kanamycin in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.
Digestion of minipreps with EcoRI. Incubate 1 hour at 37°C
Run electrophoresis gel of the devises. We remain the samples 1.
Store at -80°C the devises of gTS PCR
1
35S:5’
pUPD2
CAM
2
Linker SAGTI:Luc
pUPD2
CAM
3
35s:5’:TFLPCR:Luc:Tnos
3α1
KAN
4
35s:5’:Ga20PCR:Luc:Tnos
3α1
KAN
Ligate reactions of TFL target and Ga20ox target with Renilla
Reagent
Volume(μL)
35s:5’region: TFL/Ga20 Target: Luc: Tnos
1
Promoter35s: Renilla: Tnos
1
pUPD2
1
BSA10X
1.2
Ligase Buffer
1.2
BsmbI
1
T4 ligase
1
H2O milli-Q
4.6
04/08/2016
Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of TMV CDS
Transform DH5 α E. coli with the devise:
promoter 35s : 5’ region: PCR target: Luc: Tnos - promoter 35s: Renilla: Tnos. After incubating 2 hours, it must be plated.
Refresh C58 Agrobacterium tumefaciens cultures with the aim of infiltrating in N.benthamiana on 05/08.
Pick a single E. coli DH5α (promoter 35S : 5’ region: KO/cons target: Luc:Tnos - promoter35s: renilla: Tnos) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of spectinomycin in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.Sequencing TMV empty vector with the primer D09OCT01 (10 uM). 5μL of Miniprep and 9μL of primer (dilution 1:3). Sequencing code of 210.13.300 and 210.13.301.
05/08/2016
Pick a single E. coli DH5α (promoter35s: 5’ region: TFL/Ga20 PCR: Luc: Tnos - promoter35s: Renilla: Tnos ) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of kanamycin in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.
Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
Digestion of minipreps with EcoRV. Incubate 1 hour at 37°C
Run electrophoresis gel of the digestion products: TFLK01, TFLK02, TFLcons01, TFLcons02, GAK01, GAK02, GAcons01, GAcons02.
Prepare the Agroinfiltration with the correct protocol.
Centrifuge the cultures at 3000 rpm during 15 minutes. We discard the supernatant and it is necessary to resuspend in 5mL of Agroinfiltration solution. Let shaking it a RT during 2 hours. OD’s measurement
U6:Ga20 gRNA: psgRNA - promoter 35s: Cas9: Tnos - Miniprep number 2 was empty. We have resuspended it with 40 μL of H20 milliQ and we have checked the DNA concentration with the Nanodrop. The results show us that the DNA concentration in the Eppendorf was 140 ng/ μL so we have used it.
Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
Transform the products of ligation in DH5 α. Incubate it at 37°C during 2 hours.
We store at -80°C:
5
35s:5’:Ga20cons:Luc:Tnos
3α1
KAN
6
35s:5’:Ga20KO:Luc:Tnos
3α1
KAN
7
35s:5’:TFLcons:Luc:Tnos
3α1
KAN
8
35s:5’:TFLKO:Luc:Tnos
3α1
KAN
9
35s:5’:Ga20cons:Luc:Tnos-35s:Ren:Tnos
3Ω2
SPEC
10
35s:5’:Ga20KO:Luc:Tnos-35s:Ren:Tnos
3Ω2
SPEC
11
35s:5’:Ga20PCR:Luc:Tnos-35s:Ren:Tnos
3Ω2
SPEC
12
35s:5’:TFLcons:Luc:Tnos-35s:Ren:Tnos
3Ω2
SPEC
13
35s:5’:TFLKO:Luc:Tnos-35s:Ren:Tnos
3Ω2
SPEC
14
35s:5’:TFLPCR:Luc:Tnos-35s:Ren:Tnos
3Ω2
SPEC
15
U6:Ga20sgRNA:psgRNA
3α1
KAN
16
U6:TFLsgRNA:psgRNA
3α1
KAN
17
35s:Cas9:Tnos-U6:Ga20gRNA:psgRNA
3Ω1
SPEC
18
35s:Cas9:Tnos-U6:TFLgRNA:psgRNA
3Ω1
SPEC
19
Ga20PCR
pUPD2
CAM
20
TFLPCR
pUPD2
CAM
Run electrophoresis gel of: promoter 35s: 5’ region: TFL/Ga20 PCR: Luc: Tnos – promoter 35s: Renilla: Tnos. We keep the Miniprep number 1.
Refresh the cultures of TFL PCR (pUPD2) and Ga20 PCR (pUPD2) because we suspect that these cultures are the correct ones but we are not sure so we want to check them. The cultures that we use to refresh were made on 12/07/2016. It is important to remember that we need them to assemble the gTS with the new linkers.
10/08/2016
Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
TFL / Ga20 PCR in pUPD2
Digestion of minipreps with NotI. Incubate 1 hour at 37°C
Run electrophoresis gel of Miniprep products. We have made a small gel so we have mix 0.45 g of Agarose with 45 mL of TAE 1X. Voltage used is 100V. Both samples are correct.
Pick a single E. coli DH5α (promoter35s: 5’ region: TFL/Ga20 cons: Tnos - promoter35s: Renilla: Tnos - promoter 35s: Cas9: Tnos - U6: TFL/Ga20 gRNA: psgRNA) colony from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of chloramphenicol in a 50 ml tube with the colony and incubate it overnight at 37°C with shaking.
We throw out from Golden Braid collection the glycerinate number 1107 (Cas9 - XT1gRNA) and 0549 (promoter 35s: TEV: Tnos)
