Difference between revisions of "Team:Valencia UPV/Notebook"

Revision as of 09:57, 12 August 2016

Home

18/05/2016

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:

Plasmid	GB Code
Promoter 35s:Cas9:nopaline synthase terminator (Tnos)	GB0639
Luciferase (Luc) in pUPD2	GB0096
Tnos in pUPD2	GB0037

01/07/2016

Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
- Promoter 35s:Cas9 : Tnos

Luciferase and nopaline synthase terminator cultures haven’t succeed. Repeat Luc and Tnos cultures.

Primers IG16JUN01 and IG16JUN02 have arrived.

Finish orange DNA Genome Extraction and check DNA concentration with NanoDrop. Concentration is very low so extraction will be done again.

02/07/2016

Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
- Luc in pUPD2
- Tnos in pUPD2

Check orange DNA genome concentration with NanoDrop.

Sample	DNA concentration (ng / μL)
Clemenules1	3153.8
Clemenules 2	4527.9

Perform a PCR to bind linker with luciferase:

Reagent	Volume(μL)	Program
LuciferasepUPD	1	Temperature	Time
Buffer HF	10	98°C	5 minutes
dNTPs	2	98°C	35x	30 seconds
IG16JUN01	2.5	70°C		30 seconds
IG16JUN02	2.5	72°C		1 minute 30 seconds
Taq phusion	0.5	72°C	10 minutes
H₂O milli-Q	31.5	16°C	∞

04/07/2016

Run electrophoresis gel of Clemenules DNA (agarose 1%). 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.

Rice DNA Genome Extraction Protocol

Run electrophoresis gel of Luciferase PCR product. 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X. Voltage used is 100 V.

Ligate Reaction

Transform E. coli DH5α with it. The method that is necessary to carry out this procedure is explained in protocols

Check DNA concentration with NanoDrop.

SAMPLE	DNA Concentration(ng / μL)
Rice Gleva 1	22.8
Rice Gleva 2	17.3

05/07/2016

Run electrophoresis gel of Gleva rice DNA. We have checked that there isn’t DNA.

Repeat: Rice DNA Genome Extraction

Check DNA concentration with NanoDrop.

SAMPLE	DNA Concentration(ng / μL)
Rice Gleva 1	294.9
Rice Gleva 2	193.7

Run electrophoresis gel of Gleva rice DNA. It observed that genome extraction is correctly done.

06/07/2016

Take glycerinated cultures from Goldenbraid Collection:

GB part	Plasmid	Antibiotic	Number GB
psgRNA	pUPD	Ampicillin	0645
U6-26	pUPD	Ampicillin	1001

07/07/2016

Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
- psgRNA in pUPD2
- U6-26 in pUPD2

Primers IG16JUL01, IG16JUL02, IG16JUL03, IG16JUL04, IG16JUL05, IG16JUL06, IG16JUL07 and IG16JUL08 arrived.

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.

Primers IG16JUL09, IG16JUL10, IG16JUL11, IG16JUL12, IG16JUL13, IG16JUL14, IG16JUL15 and IG16JUL16 arrived.

Ligation using Golden Braid assembly of the next devises:
- promoter 35s:5’ region:Clemenules target control positive:Luc:Tnos
- promoter 35s:5’ region:Gleva target control positive:Luc:Tnos
- promoter 35s:5’ region:Clemenules target consensus:Luc:Tnos
- promoter 35s:5’ region:Gleva target consensus:Luc:Tnos
- promoter U6-26:sgRNA Clemenules: psgRNA (scaffold)
- promoter U6-26:sgRNA Gleva: psgRNA (scaffold)

Step 1: Annealing of oligonucleotides. Received primers are at 1uM. It is necessary taking to 10 uM.
Mix in an Eppendorf:
18 μL of H₂O milli-Q
1 μL forward primer
1 μL reverse primer

Step 2: Ligation reaction

	Reagent	Volume (μL)
	Target control positive / Target consensus	1
	promoter 35s:5’ region	1
	Luciferase	1
	Tnos	1
	α1 plasmid	1
	BSA10X	1.2
	Ligase Buffer	1.2
	BsaI	1
	T4 ligase	1
	H₂O milli-Q	2.6

	Reagent	Volume (μL)
	sgRNA Clemenules / sgRNA Gleva	1
	promoter U6 -26	1
	psgRNA (scaffold)	1
	α1 plasmid	1
	BSA10X	1.2
	Ligase Buffer	1.2
	BsaI	1
	T4 ligase	1
	H₂O milli-Q	3.6

E. coli Transformation with the devises previously explained.

E. coli plating in 6 plates (6 ligation reactions) with Kanamycin (antibiotic). Incubate overnight at 37°C.

Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
- Promoter 35s:5’ region : CL target : Luc : Tnos (3α1)
- Promoter 35s:5’ region : A target : Luc : Tnos (3α1)

Digestion of minipreps with EcoRI. Incubate 1 hour at 37°C

15/07/2016

Ligation using Golden Braid assembly of the next devises:
- Promoter 35s:Cas9:Tnos – promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos
- Promoter 35s:Cas9:Tnos – promoter 35s:5’ region:Ga20ox Rice target:Luc:Tnos

E. coli Transformation with the devises previously explained.

Run an electrophoresis gel of the products from the digestion of minipreps. The devises are:
- Promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos (3α1)
- Promoter 35s:5’ region :Ga20ox Rice target:Luc:Tnos (3α1)

Sequencing the following products:
- Promotor 35s:5’ region in pUPD2 → correct

16/07/2016

Transformations in E. coli DH5α with:
- Promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos (3α1)
- Promoter 35s:5’ region:Ga20ox Rice target:Luc:Tnos (3α1)

Plating the last devises in 2 plates with Agrobacterium C58. Incubate 2 days at 28°C.

Minipreps (2 samples for each devise) with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
- Promoter 35s:5’ region:Clemenules target control positive:Luc:Tnos
- Promoter 35s:5’ region:Gleva target control positive:Luc:Tnos
- Promoter 35s:5’ region:Clemenules target consensus:Luc:Tnos
- Promoter 35s:5’ region:Gleva target consensus:Luc:Tnos
- Promoter U6-26:sgRNA Clemenules:psgRNA (scaffold)
- Promoter U6-26:sgRNA Gleva:psgRNA (scaffold)

Ligation using Golden Braid assembly of the next devises:
- Promotor 35s:Cas9:Tnos – U6:TFL Clemenules sgRNA:psgRNA (scaffold) (Ω1)
- Promotor 35s:Cas9:Tnos – U6: Ga20ox Rice sgRNA:psgRNA (scaffold) (Ω1)

Reagent	Volume (μL)
Promotor 35s:Cas9 : Tnos	1
U6-26:TFL Clemenules sgRNA:psgRNA / U6-26:Ga20ox rice sgRNA:psgRNA	1
3Ω1	1
BSA10X	1.2
Ligase Buffer	1.2
BsmbI	1
T4 ligase	1
H₂O milli-Q	4.6

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.

17/07/2016

Transformation in DH5α E. coli with:
- Promoter 35s:Cas9:Tnos – U6:TFL Clemenules sgRNA:psgRNA (scaffold) (Ω1)
- Promoter 35s:Cas9:Tnos – U6:Ga20ox sgRNA:psgRNA (scaffold) (Ω1)

Plating E. coli transformations in plates with LB + agar + IPTG + XGal and incubate it overnight at 37°C.

Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
- Promoter U6-26:Ga20ox sgRNA:psgRNA
- Promoter 35s:5’ region:TFL Knock-out:Luc:Tnos (3α1)

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
- Promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
- Promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos
- Promoter U6-26:sgRNA Clemenules:psgRNA (scaffold)
- Promoter U6-26:sgRNA Gleva:psgRNA (scaffold)

Reagent	Volume(μL)	Reagent	Volume(μL)
TFL/Ga20ox gRNA	1	promoter 35s:5’region	1
U6-26	1	TFL/Ga20ox consensus TFL/Ga20ox knock-out	1
psgRNA	1	luciferase	1
3α1	1	Tnos	1
BSA10X	1.2	3α1	1
Ligase Buffer	1.2	BSA10X	1.2
BsaI	1	Ligase Buffer	1.2
T4 ligase	1	BsmbI	1
H₂O milli-Q	3.6	T4 ligase	1
		H₂O milli-Q	2.6

18/07/2016

Transformation in DH5α E. coli with ligation products (consensus targets and knock-out targets of TFL and Ga20ox as well as their gRNAs)

Plating E. coli transformations in plates with LB + agar + IPTG + XGal and incubate it overnight at 37°C.

Pick a single Agrobacterium C58 colony from the plates that have been incubating since 16/06/2016. The devises are:
- Promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos (3α1)
- Promoter 35s:5’ region:Ga20ox Rice target:Luc:Tnos (3α1)

Incubate it 48 hours at 28°C.

19/07/2016

Pick transformed E. coli colony from the incubated plates. The devises are:
- promoter 35s:5’ region:TFL Clemenules target control positive:Luc:Tnos
- promoter 35s:5’ region:Ga20ox Gleva target control positive:Luc:Tnos
- promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
- promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos
- promoter U6-26: sgRNA Clemenules:psgRNA (scaffold)
- promoter U6-26: sgRNA Gleva:psgRNA (scaffold)

Incubate it overnight at 37°C.

20/07/2016

Miniprep with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
- promoter 35s:5’ region:TFL Clemenules target knock-out:Luc:Tnos
- promoter 35s:5’ region:Ga20ox Gleva target control knock-out:Luc:Tnos
- promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
- promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos
- promoter U6-26:sgRNA TFL Clemenules:psgRNA (scaffold)
- promoter U6-26:sgRNA Ga20ox Gleva:psgRNA (scaffold)

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.
- promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
- Promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

21/07/2016

Transformations in E. coli DH5α with:
- promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
- promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

Incubate 2 hours at 37°C.

Plating E. coli transformation explained before and incubate it at 37°C overnight.

Agrobacterium C58 transformation with:
- promoter 35s:5’ region:TFL Clemenules target knock-out:Luc:Tnos
- promoter 35s:5’ region:Ga20ox Gleva target control knock-out:Luc:Tnos
- promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
- promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos

Incubate 48 hours at 28°C.

22/07/2016

Sequencing reaction:

Reagent	Volume (μL)
Primer in order to sequence	3
Miniprep reaction	5
H₂O milli-Q	6

Sequence	Order
TFL gRNA	210.13.201
Ga20 gRNA	210.13.202

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:
- promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
- promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

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.

Agrobacterium C58 transformations with:
- promoter 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
- promoter 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

24/07/2016

Store the next cultures at -80°C:
- promoter 35s:5’ region in pUPD2 (DH5α) number 1
- Linker: luciferase in pUPD2 (DH5α) number 2

25/07/2016

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
- promoter 35s:5’ region:TFL Clemenules target consensus:Luc:Tnos
- promoter 35s:5’ region:Ga20ox Gleva target consensus:Luc:Tnos

Digestion of minipreps with the restriction enzyme EcoRI. Incubate 1 hour at 37°C.

Run an electrophoresis gel of the digestion products.

Lanes	Samples	Verification
1	1 Kb molecular weight marker
2	Target Ga20ox consensus
3	Target Ga20ox Knock-out
4	Target TFL consensus
5	Target TFL knock-out
6	1 Kb molecular weight marker

26/07/2016

Minipreps with E.Z.N.A. ® Plasmid Mini Kit I, Q(capless) Spin of:
- promoter 35s:5’ region:TFL Clemenules target:Luc:Tnos in 3α1 plasmid from C58 Agrobacterium
- promoter 35s:5’ region:Ga20ox Gleva target:Luc:Tnos in 3α1 plasmid from C58 Agrobacterium

Digestion of minipreps with the restriction enzyme EcoRI. Incubate 1 hour at 37°C.

Run an electrophoresis gel of the digestion products:

Lanes	Samples	Verification
1	1 Kb molecular weight marker
2	promoter 35s:5’ region:Target Ga20ox Rice:Luc:Tnos
3	promoter 35s:5’ region:Target TFL Clemunules:Luc:Tnos
6	1 Kb molecular weight marker

27/07/2016

Received the necessary primers to sequence:

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:
- Promoter 35S : 5’Region : TFL consensus : Luc : Tnos
- Promoter 35S : 5’Region : TFL knockout : Luc : Tnos
- Promoter 35S : 5’Region :TFL target: Luc : Tnos
- Promoter 35S : 5’Region : Ga20 ox consensus : Luc : Tnos
- Promoter 35S : 5’Region : Ga20 ox knockout : Luc : Tnos
- Promoter 35S : 5’Region :Ga20 ox target: Luc : Tnos

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.

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

Ligate reactions of TFL target and Ga20ox target with Renilla

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:
- Promoter 35s: 5’ region: TFL PCR: Luc: Tnos - promoter35s: Renilla: Tnos
- promoter35s: 5’ region: Ga20 PCR: Luc: Tnos - promoter35s: Renilla: Tnos
- promoter35s: 5’ region: Ga20 consensus: Luc: Tnos - promoter35s: Renilla: Tnos
- promoter35s: 5’ region: Ga20 KO: Luc: Tnos - promoter35s: Renilla: Tnos

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.

Infiltration cultures:
- Promoter 35s: Luc: Tnos Laboratory controls
- Pnos: Luc: Tnos Laboratory controls
- gTS TFL KO + Cas9 - XT1:XT2 Positive controls
- gTSGa20 KO + Cas9 - XT1:XT2 Positive controls
- gTS TFL PCR + Cas9 - XT1:XT2 Negative controls
- gTS Ga20 PCR + Cas9 - XT1:XT2 Negative controls
- gTS TFL PCR + Cas9 - TFLgRNA Samples
- gTS TFL cons + Cas9 - TFLgRNA Samples
- gTS Ga20 PCR + Cas9 - Ga20gRNA Samples
- gTS Ga20 cons + Cas9 - Ga20gRNA Samples

08/08/2016

Transplant agroinfiltrated plants (Nicotiana benthamiana)

Pick up 6 disks per each plant in order to carry out the luciferase assay on 09/08

09/08/2016

Ligate reaction of:

Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
- Promoter 35s: 5’ region: TFL/Ga20 PCR: Luc: Tnos – promoter 35s: Renilla: Tnos

Transform in C58 Agrobacterium promoter 35s: Renilla : Tnos (3 α2)

Plating promoter35s: Renilla: Tnos (3α2)

Luciferase assay

Transform the products of ligation in DH5 α. Incubate it at 37°C during 2 hours.

We store at -80°C:

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)

Ligation reaction of promoter 35s: 5’ region: TFL/ Ga20: Tnos - promoter 35s: Renilla: Tnos + promoter 35s: Cas9: Tnos - U6: TFL/Ga20 gRNA: psgRNA

Devises	Volume(μL)
gTS TFL/ Ga20 - Renilla	1
Cas9 - gRNA	1
3 α 1 plasmid	1.2
Bsa 10x	1.2
Buffer ligase	1
Bsa I	1
T4 ligase	1
H20 milliQ	4.6

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

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.

Promoter35s	pNos
TFLKO	Ga20KO
TFLPCRXT1	Ga20PCRXT1
TFLPCROK	Ga20PCROK
TFLconsOK	Ga20consOK

Devises	Volume (μL)
Promoter 35s : 5’ region: Ga20/TFL KO: Luc: Tnos - promoter 35s: Renilla: Tnos - promoter 35s: hCas9: Tnos: U6: Ga20/TFL gRNA: psgRNA	1
Promoter 35s : 5’ region: Ga20/TFL cons: Luc: Tnos - promoter 35s: Renilla: Tnos - promoter 35s: hCas9: Tnos: U6: Ga20/TFL gRNA: psgRNA	1
3 α 1 plasmid	1.2
Bsa 10X	1.2
Buffer ligase	1
Bsa I	1
T4 ligase	1
H20 milliQ	4.6

Devise	Volume of culture (mL)	Volume of Agroinfiltration solution (mL)
Cas9 - TFL gRNA	0.7	9.3
Cas 9 - Ga20 gRNA	0.7	9.3
Cas 9 - XT1: XT2	0.59	9.41
TFL KO	0.67	9.33
TFL target	0.73	9.27
Ga20 consensus	0.71	9.28
Pnos	0.68	9.32
Promoter 35s : Luc	0.78	9.22
Ga20 target	0.74	9.26
TFL consensus	0.71	9.29
Ga20 - KO	0.73	9.27

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

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

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

Device	Order	Sequencing
TFL target	210.13.250	SNP in the position 652 of the target. Position 1 of the gRNA.
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

TFL consensus, TFL knockout, Ga20ox consensus and Ga20ox knockout.

@@ Line 2,377: / Line 2,377: @@
                                      </div>
                                      <table>
-                                        <tr>
-                                            <td><br></td>
-                                        </tr>
-                                        <tr>
-                                            <td><br></td>
-                                        </tr>
-                                        <tr>
-                                            <td></td>
-                                        </tr>
-                                        <tr>
-                                            <td><br></td>
-                                        </tr>
                                          <tr>
                                              <td>Devises</td>
@@ Line 2,499: / Line 2,487: @@
                                  </table>
                              </div>
-                            <table>
                                  <tr>
                                      <td>Centrifuge the cultures at 3000 rpm
@@ Line 2,508: / Line 2,496: @@
                                      hours. OD&rsquo;s measurement</td>
                                  </tr>
-                             </table>
                          </div>
                          <table>
@@ Line 2,550: / Line 2,538: @@
                          </table>
                      </div>
-                    <table>
-                        <tr>
-                            <td><br></td>
-                        </tr>
-                    </table>
-                </div>
                  <table>
                      <tr>
@@ Line 2,620: / Line 2,603: @@
                      <td>Same sequence</td>
                  </tr>
-                 <tr>
-                    <td></td>
-                </tr>
-                <tr>
-                    <td></td>
-                </tr>
-                <tr>
-                    <td><br></td>
-                </tr>
-                <tr>
-                    <td></td>
-                </tr>
-                <tr>
-                    <td><br></td>
-                </tr>
-                <tr>
-                    <td><br></td>
-                </tr>
-                <tr>
-                    <td><br></td>
-                </tr>
              </table>
          </div>