Team:Valencia UPV/Notebook

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Notebook

18May

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.

19May

Refresh previously made culture by inoculating 10 μL in a new culture medium.

20May

Agroinfiltration in Nicotiana benthamiana of C58 with dsRED.

06Jun

• 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:
• • Two experiments: one with infiltrated Nicotiana benthamiana and another with not infiltrated N.benthamiana (negative control). Lettuce leafs haven’t been correctly infiltrated and it seems that the expression level is low.
  • 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.

15Jun

Experiment is over due to snails haven’t eaten leafs enough so we have not been able to see fluorescence.

30Jun

Orange Clemenules DNA Genome Extraction protocol

• Take from the glycerinates of GoldenBraid Collection:

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

01Jul

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

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.

02Jul

• 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)
TFL 1	3153.8
TFL 2	4527.9

Perform a PCR to bind linker SAGTI with luciferase:

Reagent	Volume(μL)	Program
Luciferase pUPD2	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	∞

04Jul

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.

Gleva 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.

Ligation Reaction of SAGTI:Luciferase into a pUPD2.

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

05Jul

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

Repeat: Rice DNA Genome Extraction Protocol

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.

06Jul

Take glycerinated cultures from Goldenbraid Collection:

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

07Jul

• 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 - 35s:5’ region - have arrived.

We perform a PCR of Clemenules Orange and Gleva Rice Genome Extraction following the protocol. We want to obtain a fragment of the gene TFL of the orange DNA extraction and the gene Ga20ox of the rice DNA extraction.

Sample	Initial concentration(ng/μL)	Final concentration(ng/μL)	Initial volume(μL)	Final volume (μL)
TFL 1	3153.8	150	4.756	100
TFL 2	4527.9	150	3.31	100
Ga20ox 1	294.9	150	50.8647	100
Ga20ox 2	193.7	150	77.44	100

Reagent	Volume(μL)	Program
TFL DNA	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
Ga20ox DNA	1	Temperature	Time
Buffer HF	10	98°C	5 minutes
dNTPs	2	98°C	35x	30 seconds
IG16JUL03 (Ga20_For)	2.5	64°C		30 seconds
IG16JUL04 (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 35s:5’ region in pUPD2. Following ligation protocol, BsmbI enzyme is used in this reaction.

08Jul

Run electrophoresis gel of TFL and Ga20ox 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 part: 35s:5’ region (electroporation 2.5KV). Plating it and incubate overnight at 37°C.

Take glycerinated culture for Georgia collaboration. The part is 35s:GFP:Tnos (α1 and kanamycin).

09Jul

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

No colonies have grown in the 35s:5’ region petri dishes. Repeat transformation procedure, plating again and incubate overnight at 37°C.

11Jul

Pick a single E. coli DH5α (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 (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 TFL and Rice Ga20ox). Following ligation protocol, BsmbI enzyme is used in this reaction.

12Jul

Pick a single E. coli DH5α (target Ga20ox in pUPD2 and target TFL 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 35s:5’region in pUPD2

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

Run electrophoresis gel of the following part: 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:
• • Ga20ox PCR in pUPD2
  • TFL PCR in pUPD2

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

• Run electrophoresis gel of the same part:
• • Ga20ox PCR in pUPD2
  • TFL PCR in pUPD2

Ligation using Golden Braid assembly of the device 35s:5’region:TFL PCR:Luc:Tnos and 35s:5’region:Ga20oxPCR:Luc:Tnos in α1 plasmid.

13Jul

E. coli Transformation with the device 35s:5’region:TFL PCR:Luc:Tnos and 35s:5’region:Ga20oxPCR:Luc:Tnos in α1 plasmid.

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

14Jul

Pick a single E. coli DH5α (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 devices:
• • 35s:5’ region:TFL PCR control positive:Luc:Tnos
  • 35s:5’ region:Ga20oxPCR control positive:Luc:Tnos
  • 35s:5’ region:TFL PCR consensus:Luc:Tnos
  • 35s:5’ region:Ga20ox PCR consensus:Luc:Tnos
  • U6-26:sgRNA TFL: psgRNA (scaffold)
  • U6-26:sgRNA Ga20ox: psgRNA (scaffold)

• Step 1: Annealing of oligonucleotides. Received primers are at 1uM. It is necessary taking to 10 uM.
• • Mix in an Eppendorf:
  • • 8 μL of H₂O milli-Q
    • 1 μL forward primer
    • 1 μL reverse primer
• Step 2: Ligation reaction

Reagent	Volume (μL)
TFL target control + / Ga20ox target control + / TFL target consensus / Ga20ox target consensus	1
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 TFL / sgRNA Ga20ox	1
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 devices 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:
• • 35s:5’ region : TFL PCR : Luc : Tnos (3α1)
  • 35s:5’ region : Ga20ox PCR : Luc : Tnos (3α1)

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

15Jul

• Ligation using Golden Braid assembly of the next devices:
• • 35s:Cas9:Tnos – 35s:5’ region:TFL PCR:Luc:Tnos
  • 35s:Cas9:Tnos – 35s:5’ region:Ga20ox PCR:Luc:Tnos

E. coli Transformation with the devices previously explained.

• Run an electrophoresis gel of the products from the digestion of minipreps. The devices are:
• • 35s:5’ region:TFL PCR:Luc:Tnos (3α1)
  • 35s:5’ region :Ga20ox PCR:Luc:Tnos (3α1)

Sequencing 35s:5’region in pUPD2 → correct

16Jul

• Transformations in E. coli DH5α with:
• • 35s:5’ region:TFL PCR:Luc:Tnos (3α1)
  • 35s:5’ region:Ga20ox PCR:Luc:Tnos (3α1)

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

• Minipreps (2 samples for each device) with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
• • 35s:5’ region:TFL target control positive:Luc:Tnos
  • 35s:5’ region:Ga20ox target control positive:Luc:Tnos
  • 35s:5’ region:TFL target consensus:Luc:Tnos
  • 35s:5’ region:Ga20ox target consensus:Luc:Tnos
  • U6-26:sgRNA TFL:psgRNA (scaffold)
  • U6-26:sgRNA Ga20ox:psgRNA (scaffold)

• Ligation using Golden Braid assembly of the next devices:
• • 35s:Cas9:Tnos – U6:TFL sgRNA:psgRNA (scaffold) (Ω1)
  • 35s:Cas9:Tnos – U6: Ga20ox sgRNA:psgRNA (scaffold) (Ω1)

Reagent	Volume (μL)
Promotor 35s:Cas9 : Tnos	1
U6-26:TFL sgRNA:psgRNA / U6-26:Ga20ox 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	correct
2	gRNA Ga20ox 1	correct
3	gRNA Ga20ox 2	correct
4	Ga20ox consensus 1	correct
5	Ga20ox consensus 2	correct
6	Ga20ox knock-out 1	-
7	Ga20ox knock-out 2	correct
8	TFL consensus 1	-
9	TFL consensus 2	-
10	TFL knock-out 1	correct
11	TFL knock-out 2	correct
12	TFL gRNA 1	correct
13	TFL gRNA 2	correct
14	1 Kb molecular weight marker	correct

• Pick a single E. coli DH5α colony from the plates that have been incubated overnight. The devices are:
• • 35s:TFL Knock-out:Luc:Tnos (4 samples)
  • 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.

17Jul

• Transformation in DH5α E. coli with:
• • 35s:Cas9:Tnos – U6:TFL sgRNA:psgRNA (scaffold) (Ω1)
  • 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:
• • U6-26:Ga20ox sgRNA:psgRNA
  • 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	correct
3	gRNA Ga20ox 2	correct
4	gRNA Ga20ox 3	correct
5	gRNA Ga20ox 4	correct
6	TFL knock-out 1	correct
7	TFL knock-out 2	correct
8	1 Kb molecular weight marker	correct

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 devices:
• • 35s:5’ region:TFL target control positive:Luc:Tnos
  • 35s:5’ region:Ga20ox target control positive:Luc:Tnos
  • 35s:5’ region:TFL target consensus:Luc:Tnos
  • 35s:5’ region:Ga20ox target consensus:Luc:Tnos
  • U6-26:sgRNA TFL:psgRNA (scaffold)
  • U6-26:sgRNA Ga20ox:psgRNA (scaffold)

Reagent	Volume(μL)	Reagent	Volume(μL)
TFL/Ga20ox gRNA	1	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

18Jul

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 devices are:
• • 35s:5’ region:TFL PCR:Luc:Tnos (3α1)
  • 35s:5’ region:Ga20ox PCR:Luc:Tnos (3α1)

Incubate it 48 hours at 28°C.

19Jul

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

Incubate it overnight at 37°C.

20Jul

• Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
• • 35s:5’ region:TFL target knock-out:Luc:Tnos
  • 35s:5’ region:Ga20ox target control knock-out:Luc:Tnos
  • 35s:5’ region:TFL target consensus:Luc:Tnos
  • 35s:5’ region:Ga20ox target consensus:Luc:Tnos
  • U6-26:sgRNA TFL:psgRNA (scaffold)
  • U6-26:sgRNA Ga20ox: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	correct
2	gRNA TFL 1	correct
3	gRNA TFL 2	correct
4	TFL consensus 1	correct
5	TFL consensus 2	correct
6	TFL knock-out 1	correct
7	TFL knock-out 2	correct
8	1 Kb molecular weight marker	correct
9	gRNA Ga20ox 1	correct
10	gRNA Ga20ox 2	correct
11	Ga20ox consensus 1	correct
12	Ga20ox consensus 2	-
13	Ga20ox knock-out 1	correct
14	Ga20ox knock-out 2	correct
15	1 Kb molecular weight marker	correct

• Ligation using Golden Braid assembly of the next devices. Is used the restriction enzyme BsmbI.
• • 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
  • 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

21Jul

• Transformations in E. coli DH5α with:
• • 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
  • 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:
• • 35s:5’ region:TFL target knock-out:Luc:Tnos
  • 35s:5’ region:Ga20ox target control knock-out:Luc:Tnos
  • 35s:5’ region:TFL target consensus:Luc:Tnos
  • 35s:5’ region:Ga20ox target consensus:Luc:Tnos

Incubate 48 hours at 28°C.

22Jul

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
Ga20oxgRNA	210.13.202

Ordered the necessary primers to sequence: TFL consensus, TFL knockout, Ga20ox consensus and Ga20ox knockout.

E. coli transformations with 35s:Cas 9:Tnos – U6-26:TFL sgRNA: psgRNA

Plating the last device in plates with Agrobacterium C58. Plates contain spec + IPTG + X-Gal. Incubate 2 days at 28°C.

Pick a single E. coli DH5α (35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA and 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.

23Jul

• Minipreps (4 samples for each device) with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
• • 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
  • 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 (35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA and 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:
• • 35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA
  • 35s:Cas 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA

24Jul

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

25Jul

Pick a single Agrobacterium C58 (35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA in 3Ω1 and 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:
• • 35s:5’ region:TFL target control positive:Luc:Tnos
  • 35s:5’ region:Ga20ox target control positive:Luc:Tnos
  • 35s:5’ region:TFL target consensus:Luc:Tnos
  • 35s:5’ region:Ga20ox 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	correct
2	Target Ga20ox consensus	correct
3	Target Ga20ox Knock-out	correct
4	Target TFL consensus	correct
5	Target TFL knock-out	correct
6	1 Kb molecular weight marker	correct

26Jul

• Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
• • 35s:5’ region:TFL PCR:Luc:Tnos in 3α1 plasmid from C58 Agrobacterium
  • 35s:5’ region:Ga20ox PCR: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	correct
2	35s:5’ region:PCR Ga20ox :Luc:Tnos	correct
3	35s:5’ region:PCR TFL Clemunules:Luc:Tnos	correct
6	1 Kb molecular weight marker	correct

26Jul

• Sequencing products have arrived:
• • U6-26:Ga20ox gRNA:psgRNA in α1 - correct
  • U6-26:TFL gRNA:psgRNA in α1 - correct

• Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
• • 35s:5’ region: Ga20ox PCR : Luc : Tnos in α1
  • 35s:5’ region: TFL PCR : Luc : Tnos in α1

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

Run electrophoresis gel of the digestion products.

Lanes	Samples	Verification
1	1 Kb molecular weight marker
2	TFL PCR
3	Ga20ox PCR

27Jul

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 devices are:
• • 35S : 5’Region : TFL consensus : Luc : Tnos
  • 35S : 5’Region : TFL knockout : Luc : Tnos
  • 35S : 5’Region :TFL PCR: Luc : Tnos
  • 35S : 5’Region : Ga20ox consensus : Luc : Tnos
  • 35S : 5’Region : Ga20ox knockout : Luc : Tnos
  • 35S : 5’Region :Ga20ox PCR: Luc : Tnos

Sequencing the last devices to check if these are correct.

28Jul

Refresh Agrobacterium tumefaciens cultures with the aim of infiltrating in N.benthamiana on 29/07

Refresh cultures of the devices 35s: 5’ region: TFL PCR : Luc : Tnos and 35s : 5’ region: Ga20ox PCR : Luc : Tnos in order to prepare the more Miniprep reaction.

Sequencing results have arrived.

Device	Order	Sequencing
TFL PCR	210.13.250	SNP in the position 652 of the target. Position 1 of the gRNA. GA
Ga20ox PCR	210.13.253	Same sequence
TFL consensus	210.13.251	Same sequence
TFL KO	210.13.252	Same sequence
Ga20ox Consensus	210.13.254	Same sequence
Ga20ox KO	210.13.255	Same sequence

29Jul

• Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
• • 35s: TFL PCR : Luc : Tnos
  • 35s : Ga20ox PCR : Luc : Tnos

Digestion of minipreps with EcoRI following digestion protocol. Incubate 1 hour at 37°.

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.

01Aug

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 device
  • Change linker sequence
  • Add renilla in luciferase assay
  • Change reporter to +1
  • Use pLess as control
  • Use a Wild Type as control
  • Insert devices in cis
  • Design a consensus target as longer as the amplified.

02Aug

Culture refresh of C58 Agrobacterium to infiltrate on Friday.

Pick a single E. coli DH5α (35s:5’region:TFL PCR:Luc:Tnos in α1 and 35s:5’region:Ga20oxPCR: 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 / Ga20oxcons	1
Promoter35s: Renilla: Tnos	1
pUPD2	1
BSA10X	1.2
Ligase Buffer	1.2
BsmbI	1
T4 ligase	1
H2O milli-Q	4.6

03Aug

• DH5α E. coli transformation with the devices:
• • 35S : 5’ region : TFL KO : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
  • 35S : 5’ region : TFL consensus : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
  • 35S : 5’ region : Ga20ox KO : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
  • 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°

• Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
• • 35s:5’region:TFL PCR:Luc:Tnos in α1
  • 35s:5’region:Ga20oxPCR: 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°

Run electrophoresis gel of the devices. We remain the samples 1.

Store at -80°C the devices of gTS PCR

1	35S:5’	pUPD2	CAM
2	SAGTI:Luc	pUPD2	CAM
3	35s:5’:TFLPCR:Luc:Tnos	3α1	KAN
4	35s:5’:Ga20PCR:Luc:Tnos	3α1	KAN

Ligate reactions of TFL PCR and Ga20ox PCR with Renilla

Reagent	Volume(μL)
35s:5’region: TFL/Ga20oxPCR: 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

04Aug

Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of TMV CDS

Transform DH5 α E. coli with the device 35s : 5’ region: PCR PCR: Luc: Tnos - 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 (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.

05Aug

Pick a single E. coli DH5α (promoter35s: 5’ region: TFL/Ga20oxPCR: 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:
• • 35s: 5’ region: TFL PCR: Luc: Tnos - promoter35s: Renilla: Tnos
  • 35s: 5’ region: Ga20oxPCR: Luc: Tnos - promoter35s: Renilla: Tnos
  • 35s: 5’ region: Ga20oxconsensus: Luc: Tnos - promoter35s: Renilla: Tnos
  • 35s: 5’ region: Ga20oxKO: Luc: Tnos - promoter35s: Renilla: Tnos

Digestion of minipreps with EcoRV. Incubate 1 hour at 37°

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.

device	Volume of culture (mL)	Volume of Agroinfiltration solution (mL)
Cas9 - TFL gRNA	0.7	9.3
Cas 9 - Ga20ox gRNA	0.7	9.3
Cas 9 - XT1: XT2	0.59	9.41
TFL KO	0.67	9.33
TFL PCR	0.73	9.27
Ga20ox consensus	0.71	9.28
Pnos	0.68	9.32
35s : Luc	0.78	9.22
Ga20ox PCR	0.74	9.26
TFL consensus	0.71	9.29
Ga20ox- KO	0.73	9.27

• Infiltration cultures:
• • 35s: Luc: Tnos Laboratory controls
  • Pnos: Luc: Tnos Laboratory controls
  • gTS TFL KO + Cas9 - XT1:XT2 Positive controls
  • gTSGa20oxKO + Cas9 - XT1:XT2 Positive controls
  • gTS TFL PCR + Cas9 - XT1:XT2 Negative controls
  • gTS Ga20oxPCR + Cas9 - XT1:XT2 Negative controls
  • gTS TFL PCR + Cas9 - TFLgRNA Samples
  • gTS TFL cons + Cas9 - TFLgRNA Samples
  • gTS Ga20oxPCR + Cas9 - Ga20gRNA Samples
  • gTS Ga20oxcons + Cas9 - Ga20gRNA Samples

08Aug

Transplant agroinfiltrated plants (Nicotiana benthamiana)

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

09Aug

Ligate reaction of:

devices	Volume (μL)
35s : 5’ region: Ga20/TFL KO: Luc: Tnos - 35s: Renilla: Tnos - 35s: hCas9: Tnos: U6: Ga20/TFL gRNA: psgRNA	1
35s : 5’ region: Ga20/TFL cons: Luc: Tnos - 35s: Renilla: Tnos - 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

U6:Ga20oxgRNA: psgRNA - 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 35s: 5’ region: TFL/Ga20oxPCR: Luc: Tnos – 35s: Renilla: Tnos

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

Plating promoter35s: Renilla: Tnos (3α2)

Luciferase assay

Promoter35s	pNos
TFLKO	Ga20KO
TFLPCRXT1	Ga20PCRXT1
TFLPCROK	Ga20PCROK
TFLconsOK	Ga20consOK

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

We store at -80°:

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: 35s: 5’ region: TFL/Ga20oxPCR: Luc: Tnos – 35s: Renilla: Tnos. We keep the Miniprep number 1.

Refresh the cultures of TFL PCR (pUPD2) and Ga20oxPCR (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.

10Aug

Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of TFL / Ga20oxPCR in pUPD2

Digestion of minipreps with NotI. Incubate 1 hour at 37°

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/Ga20oxcons: Tnos - promoter35s: Renilla: Tnos - 35s: Cas9: Tnos - U6: TFL/Ga20oxgRNA: 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 (35s: TEV: Tnos)

Ligation reaction of 35s: 5’ region: TFL/ Ga20: Tnos - 35s: Renilla: Tnos + 35s: Cas9: Tnos - U6: TFL/Ga20oxgRNA: psgRNA

devices	Volume (μL)
gTS TFL/ Ga20ox- 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

11Aug

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

• Promoter 35s: 5’region: TFL/Ga20 cons: Luc: Tnos – p35s:Renilla:Tnos – p35s:Cas9:Tnos – U6-26: XT1:XT2 gRNA: psgRNA
• Promoter35s: 5’region: Cas9: Tnos – U6-26: XT1:XT2 gRNA: psgRNA
• Promoter 35s: protease NiaTEV: Tnos

Ligation reaction of:

• promoter 35s: 5’ region: TFL/ Ga20 KO: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: XT1:XT2 gRNA: psgRNA
• promoter 35s: 5’ region: TFL/ Ga20 PCR: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: XT1:XT2 gRNA: psgRNA

Digestion of minipreps TFL/Ga20 cons with EcoRI. Incubate 2 hour at 28ºC.

Run electrophoresis gel of Miniprep products. They have not gone well and maybe, we have picked up blue colonies. This hypothesis explains that the digestion is the same that 3α1 plasmids without any insert.

Transform in C58 Agrobacterium culture by electroporation using 1440 kV during 5 mS:

• Promoter35s: protease NiaTEV
• Promoter35s:Renilla:Luc (3α2) due to the last time, culture was platting on the wrong plate.

Incubate 2 hours at 28ºC and plating it. Incubate during 48 hours at 28ºC.

Transform in DH5α E. coli culture by electroporation using 1500kV during 5mS:

• promoter 35s: 5’ region: TFL/ Ga20 PCR: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: OK gRNA: psgRNA

12Aug

Primers have arrived: IG16AG01, IG16AG02 and IG16AG03.

Perform a PCR to bind the new linkers with luciferase. We are going to try with AEAAAKA linker, with RSIATlinker and RSIAT+NiaTEV(protease) linker.

REAGENT	VOLUME(μL)	PROGRAM
Luciferase pUPD2	1	TEMPERATURE	TIME
Buffer HF	10	98ºC	5 minutes
dNTPs	2	98ºC	35x	30 seconds
IG16JUL18/19/20	2.5	63ºC		30 seconds
IG16JUN02	2.5	72ºC		1minute
Taq phusion	0.5	72ºC	10 minutes
H2O milli-Q	31.5	16ºC	∞

Run electrophoresis gel of Miniprep products. The PCR product with IG16JUL02 primer has amplified correctly. However, the other ones cannot be observed. We will try at 65ºC and 67ºC (temperature of amplification) using the same times in the program explain before.

13/08/2016
The linker RSIAT has amplified at 65ºC

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

• gTS (TFL / Ga20) – Renilla – Cas9 – TFL / Ga20 gRNA (3α1)

Digestion of minipreps with EcoRI. Incubate 1 hour at 37ºC

Run electrophoresis gel of Miniprep products. Both of them have not gone correctly so we have decided to repeat them.

Pick a single E. coli DH5α (gTS – Renilla - Cas9 - XT1:XT2 gRNA in DH5α) 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 10 ml tube with the colony and incubate it 16 hours at 37ºC with shaking.

Pick a single Agrobacterium C58 (promoter35s: NiaTEV protease: Tnos and promoter35s: Renilla: Luc) colony from the plate that has been incubated overnight. Inoculate a starter culture of 5 ml of LB medium with 5 μL of kanamycin and rifampin in a 50 ml tube with the colony and incubate it overnight at 28ºC with shaking.

DH5α E. coli transformations with PCR products

• linker (AEK / RSIAT / RSIAT+TEV) : Luciferase in pUPD2

Perform a PCR of the linker RSIAT+TEV in order to get the optimal temperature. We are going to try at 68, 69 and 70ºC.

REAGENT	VOLUME(μL)	PROGRAM
Luciferase pUPD2	1	TEMPERATURE	TIME
Buffer HF	10	98ºC	5 minutes
dNTPs	2	98ºC	35x	30 seconds
IG16JUL20	2.5	63ºC		30 seconds
IG16JUN02	2.5	72ºC		1minute
Taq phusion	0.5	72ºC	10 minutes
H2O milli-Q	31.5	16ºC	∞

14Aug

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

• gTS (TFL /Ga20) – Renilla – Cas9 – XT1:XT2 gRNA

Digestion of minipreps with EcoRI. Incubate 1 hour at 37ºC

Run electrophoresis gel of Miniprep products. We cannot interpret the results. We have decided to repeat the digestion again.

Digestion of minipreps with EcoRI. Incubate 1 hour at 37ºC.

Run electrophoresis gel of these new Miniprep products.

Run electrophoresis gel of the PCR which was made on 13-08-2016. The fragment do not amplified with any selected temperature.

15Aug

Digestion of gTS PCR/cons: Renilla : Cas9 : OK/XT1 gRNA with the restriction enzyme NdeI in order to explain the bands that we see in the electrophoresis gel.

Run electrophoresis gel of this last digestion. We have decide to repeat the ligate reaction because of appearing an unexpected band.

Ga20 cons – Ren – Cas9 – Ga20 gRNA x4	Ga20 PCR – Ren – Cas9 – XT1 gRNA x4
Ga20 PCR – Ren – Cas9 – Ga20 gRNA x4	TFL PCR – Ren – Cas9 – XT1 gRNA x4
TFL cons – Ren – Cas9 – TFL gRNA x4	TFL KO – Ren – Cas9 – XT1 gRNA x4
TFL cons – Ren – Cas9 – TFL gRNA x4	Ga20 KO – Ren – Cas9 – XT1 gRNA x4

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

• Promoter35s : Renilla : Tnos in C58
• Promoter35s : NiaTEV : Tnos in C58

Digestion of these last Minipreps with HindIII. Incubate 1 hour at 37ºC.

Run electrophoresis gel of digestion products. We cannot distinguish anything in the gel so we have decided to pick up more colonies from the plate. Inoculate a starter culture of 5 ml of LB medium with 5 μL of kanamycin and rifampicin in a 50 ml tube with the colony and incubate it 48 hours at 28ºC with shaking must be necessary.

We have decided to repeat the performance of PCR to bind linker RSIAT+TEV with luciferase. We are going to try at 71ºC. Moreover this time, we are going to try it using DMSO at 65 and 67ºC.

REAGENT	VOLUME (μL)
Luciferase pUPD2	1
Buffer HF	10
dNTPs	2
IG16JUL20	2.5
IG16JUN02	2.5
Taq phusion	0.5
DMSO	1.5
H2O milli-Q	30

Ligation using Golden Braid assembly of the next devices:

• promoter 35s: 5’ region: TFL/ Ga20 PCR: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: OK gRNA: psgRNA
• promoter 35s: 5’ region: TFL/ Ga20 cons: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: OK gRNA: psgRNA
• promoter 35s: 5’ region: TFL/ Ga20 PCR: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: XT1 gRNA: psgRNA
• promoter 35s: 5’ region: TFL/ Ga20 KO: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: XT1 gRNA: psgRNA

Pick a single Agrobacterium C58 (promoter35s: NiaTEV protease: Tnos and promoter35s: Renilla: Luc) colony from the plate that has been incubated overnight. Inoculate a starter culture of 5 ml of LB medium with 5 μL of kanamycin and rifampin in a 50 ml tube with the colony and incubate it overnight at 28ºC with shaking.

Run electrophoresis gel of PCR products using 71ºC on the one hand and DMSO at 65 and 67ºC. There is not amplification.

We have decided to repeat again the performance of PCR to bind linker RSIAT+TEV with luciferase. We are going to try at 72ºC. Moreover this time, we are going to try it using DMSO at 68 and 70ºC. In the last case, we use the same quantities that can be observed in the upper table.

Run electrophoresis gel of PCR products using 72ºC on the one hand and DMSO at 68 and 70ºC. There is not amplification.