Team:Valencia UPV/Notebook

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Notebook

Valencia UPV team works with standard plant protocols and with the assembly system Goldenbraid, which works for assembly of Phytobricks. The protocols mentioned in the notebook can be found in the page "Protocols". We hope this page can help to follow or repeat our work easily, and that our protocols can help future teams working with plant synthetic biology or with simple bacteria experiments. Enjoy our journey through the work of our team!

Protocols

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



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.

Run electrophoresis gel of digested targets in pUPD2.

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 Agrobacterium C58 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.



Run electrophoresis gel of digested targets in pUPD2.

• 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.

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”.

• 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.

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.

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 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 PCR targets Ga20 and TFL. In the second gel, digested TFL and Ga20 PCR from gTS are run. 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.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.

13Aug

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.

16Aug

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

• AEK linker : Luc in pUPD2
• RSIAT linker : Luc in pUPD2

Digestion of Minipreps with NotI. Incubate 1 hour at 37ºC.

Run electrophoresis gel of digestion products. We run 4 samples of RSIAT and 4 samples of AEK. It can be observed the sample of AEK – 4. We only can keep this one.



DH5α E. coli transformations with the products of ligate reactions that were made on 15 /08/2016.

Ligate reactions of gTS 2.0:
Promoter 35s: TFL/Ga20 (KO/cons/PCR) : AEK linker : Luc : Tnos in 3α1

Devices	Volume (μL)
Tnos	1
Promoter 35s	1
TFL / Ga20 (PCR / cons / KO)	1
AEK linker: Luc	1
3α1 plasmid	1
Bsa 10x	1.2
Buffer ligase	1.2
Bsa I	1
T4 ligase	1
H20 milliQ	2.6

We have decided to repeat again the performance of PCR to bind linker RSIAT+TEV with luciferase. We are going to try with and without DMSO at 60, 61, 62, 64 and 65ºC. Now, we are going to increase the extension phase. Instead of 30 seconds, we are going to try with 2 minutes.

Run electrophoresis gel of PCR products. There is amplification in all temperatures but at 65ºC the amplification is a little worse. It can be checked that DMSO helps the amplification. We keep the PCR which have been amplified at 64 ºC using DMSO.

Luciferase glycerinate is taken out of Golden Braid collection because of the Miniprep is empty.

Ligate reaction of RSIAT+TEV in pUPD2

Reagent	Volume (μL)
RSIAT + TEV: Luc	1
pUPD2	1
BSA10X	1.2
Ligase Buffer	1.2
BsmbI	1
T4 ligase	1
H2O milli-Q	5.6

17Aug

Transform E. coli DH5α with ligation products of gTS 2.0 (AEK linker) with TFL (PCR/consensus/KO) and Ga20 oxidase (PCR/consensus/KO). Incubate 2 hours at 37ºC and plate it.

Pick a single E. coli DH5α (promoter35s: TFL/Ga20ox (PCR/consensus/KO): Tnos - promoter35s: Renilla: Luc – Cas9: Tnos – U6-26 Ga20 gRNA: psgRNA) 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 10 ml tube with the colony and incubate it overnight at 37ºC with shaking.

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

• RSIAT : Luc in pUPD2 in E. coli DH5α
• Luc in pUPD2 in E. coli DH5α
• Promoter 35s: Renilla: Tnos in Agrobacterium C58
• Promoter 35s: NiATEV protease: Tnos in Agrobacterium C58

Digestion of Minipreps with NotI. Incubate 1 hour at 37ºC.

Run electrophoresis gel of digestion products. It has not left any result.



Repeat ligate reactions of RSIAT+TEV: Luc in pUPD2. Transform in DH5α and plate it.

Nanodrop measurement:

• Promoter 35s: Renilla: Tnos in 3α2: 36.6 ng
• Promoter 35s: NiATEV protease: Tnos in 2α2: 12.6 ng

There is DNA but a wide band should be seen. We cannot see anything.

PCR purification of RSIAT:Luc in pUPD2 due to the digestion has not gone well. DNA concentration of the PCR is 55.6 ng/ μL

Ligate reaction of RSIAT:Luc in pUPD2

Nanodrop measurement of promoter 35s: NiaTEV protease: Tnos in 2α2. (We check the glycerinate E. coli Miniprep to guarantee that it is correct. If not, it could be cause that we cannot see any band in Agrobacterium C58.

There is 94.5 ng/ μL so it is correct. We are going to pick up colonies in NiATEV plate and we do again the minipreps in C58 Agrobacterium.

Digestion of promoter 35s: Renilla: Tnos in C58 using the restriction enzyme HindIII. (3α2)

Run electrophoresis gel of digestion products. Unexpected bands appear. We are going to do the digestion of the promoter 35s: Renilla: Tnos in E. coli just to check that it is correct.



Platting the ligation product RSIAT+TEV: Luc in pUPD2. Incubate it at 37ºC overnight.

Transform E. coli DH5 α with the device RSIAT: Luc in pUPD2. Incubate 1 hour at 37ºC and plate it.

18Aug

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

• TFL / Ga20 (PCR and consensus) – Renilla – Cas9 – TFL / Ga20 gRNA
• TFL / Ga20 (KO and PCR) – Renilla – Cas9 – XT1 gRNA

Digestion of Minipreps using EcoRI. Incubate 1 hour at 37ºC.

Run electrophoresis gel of digestion products:

Pick E. coli DH5α (gTS- Renilla + Cas9: gRNA with combinations previously explained) colonies from the plate that has been incubated overnight. inoculate a starter culture of 4 ml of LB medium with 4 μL of kanamycin or Cloramphenicol in a 10 ml tube with the colony and incubate it overnight at 37ºC with shaking.

19Aug

Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of gTS – Renilla – Cas 9- gRNA. There are 60 samples.

Digestion of Miniprep products. 52 samples are in α1 so it is used the restriction enzyme EcoRI. 8 samples are in pUPD2 so the enzyme used is NotI.

21Aug

Ligate reaction of gTS (TFL / Ga20 cons/KO/PCR) – SF in 3Ω2

22Aug

Run electrophoresis gel of digestion products.



Transform ligation products of gTS + SF in E. coli DH5α. Incubate 1 hour at 37ºC. Plate it.

Digestion of Miniprep: promoter 35s: Cas9: pNOS

Run electrophoresis gel of promoter 35s: Cas9: pNOS, Cas9 no digested and RSIAT:Luc of PCR purified.

23Aug

RSIAT:Luc and RSIAT+TEV:Luc

Pick a single E. coli DH5α (gTS + SF) 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 overnight at 37ºC with shaking.

Ligate reaction of RSIAT:Luc and RSIAT+TEV in pUPD2.

24Aug

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

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

Run electrophoresis gel of digestion products.



Pick a single Agrobacterium C58 (promoter35s: Renilla) colony from the plate that has been incubated overnight. Incubate it overnight at 28ºC with shaking.

Transform E. coli DH5α with RSIAT:Luc and RSIAT+TEV::Luc.

Split-Cas9 gBlocks have arrived: C-Cas91 and C-Cas92 in TMV – N-Cas91 and N-Cas92 in PVX.

Ligate reactions of Split-Cas9 in pUPD2.

Ligate reactions of:

• Promoter35s: TFL(PCR/cons):link: Luc: Tnos + SF in 3Ω2 and promoter35s: Cas9: Tnos + U6-26: TFL gRNA: psgRNA
• Promoter35s: TFL(PCR/KO):link: Luc: Tnos + SF in 3Ω2 and promoter35s: Cas9: Tnos + U6-26: XT1:XT2 gRNA: psgRNA
• Promoter35s: Ga20(cons):link: Luc: Tnos + SF in 3Ω2 and promoter35s: Cas9: Tnos + U6-26: TFL gRNA: psgRNA
• Promoter35s: Ga20(KO):link: Luc: Tnos + SF in 3Ω2 and promoter35s: Cas9: Tnos + U6-26: XT1:XT2 gRNA: psgRNA

We do not put Ga20 PCR ligations due to the band in electrophoresis gel is not the expected.

25Aug

Transform E. coli DH5α with the ligations previously made. Both Split-Cas9 and gTS + SF – Cas9 + gRNA. Incubate those 2 hours at 37 º C and plate it.

Pick a single E. coli DH5α (RSIAT and RSIAT+TEV) 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 10 ml tube with the colony and incubate it overnight at 37ºC with shaking.

Pick up a single E. coli DH5α again of promoter35s: Ga20PCR: link: Luc: Tnos + SF

PCR to try our own thermocycler (Nóbelcycler)

REAGENT	VOLUME (μL)	PROGRAM
Luciferase pUPD2	1	TEMPERATURE	TIME
Buffer HF	10	94ºC	5 minutes
dNTPs	2	94ºC	35x	30 seconds
IG16JUN02	2.5	70ºC		30 seconds
IG16JUN18	2.5	72ºC		2 minutes
Taq UVAT	0.5	72ºC	10 minutes
H2O milli-Q	31.5	16ºC	∞

26Aug

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

• RSIAT:Luc in pUPD2
• RSIAT + TEV: Luc in pUPD2
• Promoter 35s: Ga20 PCR: Link: Luc + SF in 3Ω2
• Promoter35s: Renilla: Tnos in C58 Agrobacterium in 2α2
• Promoter 35s: Ga20 PCR/cons/KO: AEK: Luc in C58 Agrobacterium in 3α1
• Promoter 35s: TFL PCR/cons/KO: AEK: Luc in C58 Agrobacterium in 3α1

Digestion of minipreps using NotI (pUPD2), EcoRI (α1), HindIII (α2) and EcoRV (Ω2)

Run electrophoresis gel of digestion products.

Ligation of gTS Ga20 ox + SF in 3Ω2 due to the bands of this device are not the expected ones.

Ligation of gTS + RSIAT: TEV in 3α1.

Refresh C58 Agrobacterium tumefaciens cultures with the aim of infiltrating in N.benthamiana on 27/08. We are going to refresh the following cultures:

• Promoter35s: TFL (PCR/ cons/ KO): AEK: Luc: Tnos
• Promoter35s: Ga20 (PCR/ cons/ KO): AEK: Luc: Tnos
• Promoter35s: Luc: Tnos
• pNOS: luc: Tnos
• Cas9- XT1:XT2
• Cas9 – TFL gRNA and Cas9 – Ga20 gRNA

Take out the glycerinate of promoter35s: NiaTEV: Tnos(GB-0594). Triple groove is made.

Agrobacterium C58 transformations with:

• Promoter35s: Renilla: Tnos

27Aug

Prepare Agroinfiltration

	Ga20 PCR	Ga20 cons	Ga20 KO	TFL PCR	TFL cons	TFL KO	35S	pNOS	XT1 Cas9 1	XT1 Cas92	Cas9 – TFL gRNA	Cas9 Ga20 gRNA
Initial OD	0.49	0.51	0.45	0.21	0.52	0.53	0.45	0.18	0.19	0.17	0.24	0.23
Vi culture (mL)	0.82	0.78	0.89	1.90	0.77	0.75	0.89	2.22	5.26	5.88	1.67	1.74
Vi Agroinfiltration solution (mL)	9.18	9.22	9.11	8.1	9.23	9.25	9.11	7.78	4.74	4.12	8.33	8.26

Culture mix – 10 plants of Nicotiana benthamiana

• gTS KO + gRNA -
• gTS cons + gRNA OK
• gTS PCR + gRNA OK
• gTS PCR + gRNA -
• promoter 35s: Luc + gRNA -
• pNOS: Luc + gRNA -

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

• gTS TFL / Ga20 (PCR/ cons/ KO) + SF – Cas9: gRNA
• Cas 9 N in pUPD2
• Cas9 C in pUPD2

Digestion of minipreps using EcoRI and NotI.

Run electrophoresis gel of digestion products.

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

Digestion with HindIII. Incubate 1 hour at 37ºC.

Run electrophoresis gel of digestion products.

Pick E. coli DH5α (RSIAT and RSIAT+TEV) colonies 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 10 ml tube with the colony and incubate it overnight at 37ºC with shaking.

• gTS Ga20 PCR – SF in 3Ω2
• promoter 35s: Ga20 PCR: RSIAT + TEV: Luc: Tnos in 3α1
• promoter 35s: Ga20 cons: RSIAT + TEV: Luc: Tnos in 3α1
• promoter 35s: Ga20 KO: RSIAT + TEV: Luc: Tnos in 3α1
• promoter 35s: TFL PCR: RSIAT + TEV: Luc: Tnos in 3α1
• promoter 35s: TFL cons: RSIAT + TEV: Luc: Tnos in 3α1
• promoter 35s: TFL KO: RSIAT + TEV: Luc: Tnos in 3α1

29Aug

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

• 3α1
• Promoter35s: TFL (PCR/ cons/ KO): RSIAT + TEV: Luc: Tnos in 3α1
• Promoter35s: Ga20 (PCR/ cons/ KO): RSIAT + TEV: Luc: Tnos in 3α1
• gTS: Ga20 PCR + SF en 3Ω2

Digestion of Miniprep products using EcoRI and EcoRV.

Run electrophoresis gel of digestion products. TFL KO has not gone well.

Ligate reactions of:

• N-split Cas9 in PVX
• C-split Cas9 in TMV
• gTS TFL/Ga20 (PCR/ cons/ KO)

Transform C58 Agrobacterium with the next devices:

• gTS TFL cons – SF – TFL gRNA – Cas9
• gTS TFL PCR – SF – XT1 gRNA – Cas9
• gTS TFL PCR – SF – TFL gRNA – Cas9
• gTS TFL KO – SF – XT1 gRNA – Cas9
• gTS Ga20 cons – SF – Ga20 gRNA – Cas9
• gTS Ga20 KO – SF – XT1 gRNA – Cas9

Ligate reactions of gTS TFL KO RSIAT + TEV in 3α1

Ligate reactions of promoter 35s: Ga20 PCR: linker: Luc: Tnos –SF + Cas9: XT1 and Ga20 gRNA

Transform E. coli DH5α with N-Split Cas9 and C- Split Cas9, gTS RSIAT in 3α1 and TFL KO – RSIAT+TEV in 3α1. Incubate 16 hours at 37ºC and plate it.

30Aug

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

Transform in E. coli DH5α of Ga20 PCR + SF – Cas9: XT1 gRNA and Ga20 PCR + SF – Cas9: Ga20 gRNA. Incubate 2 hours at 37ºC and plate it.

31Aug

Repeat gTS TFL KO RSIAT + TEV: Luc in 3α1

Pick Agrobacterium C58 (gTS + SF – Cas9: gRNA) colonies from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of Kanamycin and 4 μL of Rifampin in a 10 ml tube with the colony and incubate it overnight at 28ºC with shaking.

Pick E. coli DH5α (gTS Ga20 PCR + SF – Cas9: gRNA) colonies 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 overnight at 37ºC with shaking.

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

• Ga20 / TFL (PCR – cons – KO) + RSIAT in 3α1
• N-Cas9 (Kanamycin)
• C-Cas9 (Kanamycin)
• Renilla C58 in 3α2

Digestion of Miniprep products using EcoRI and HindIII.

Run electrophoresis gel. We throw away Ga20 / TFL (PCR – cons- KO) + RSIAT, C-Cas9 with kanamycin.

Transform E. coli DH5α with promoter35S: TFL KO: RSIAT + TEV: Luc: Tnos in 3α1. Incubate 2 hours at 37ºC and plate it.

Pick up a single blue colony from C-Cas9 plate in order to digest it with EcoRI and compare it with the correct colony. In this way, we will be able to compare blue and white colonies.

01Sep

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

• gTS Ga20 PCR – SF – Cas9 – U6: Ga20 gRNA and XT1 gRNA + blue colony of C-Cas9

Digestion of Miniprep products using EcoRI. Incubate 1 hour at 37ºC.

Run electrophoresis gel. After digesting blue colony of C-Cas9 , we know that the resistance is kanamycin and the restriction site is EcoRI.



Refresh C58 Agrobacterium tumefaciens cultures with the aim of infiltrating in N.benthamiana on 27/08. We are going to refresh the following cultures:

• Promoter 35s: TFL (PCR/ cons/ KO): AEK: Luc: Tnos
• Promoter 35s: Ga20 (PCR/ cons/ KO): AEK: Luc: Tnos
• Promoter 35s: Luc: Tnos
• pNOS
• Cas9: XT1/XT2
• Cas9: TFL gRNA
• Cas9: Ga20 gRNA

Transformed plate of TFL KO: RSIAT + TEV in 3α1 has not gone well. We are going to repeat the transformation. Incubate 1 hour at 37ºC and plate it.

Repeat digestion done on 31 /08/2016 using EcoRI.

Run electrophoresis gel again. We throw away the samples: Ga20 PCR – Ga20 cons – Ga20 KO and TFL PCR – TFL cons – TFL KO.

02Sep

Repeat ligate reaction of Ga20/ TFL (PCR – cons – KO)

Samples	Primers
TFL PCR	DNA genomic extraction
TFL cons	IG16 JUL09 + IG16 JUL10
TFL KO	IG16 JUL11 + IG16 JUL12
Ga20 PCR	DNA genomic extraction
Ga20 cons	IG16 JUL13 + IG16 JUL14
Ga20 KO	IG16 JUL15 + IG16 JUL16

Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of gTS + SF – Cas9: gRNA in Agrobacterium C58.

Refresh C58 Agrobacterium tumefaciens cultures with the aim of infiltrating in N.benthamiana on 27/08. We are going to refresh all cultures with AEK linker.

Transform in C58 Agrobacterium:

• Ga20 (PCR/ cons/ KO): RSIAT + TEV: Luc: Tnos in 3α1
• TFL (PCR/ cons/ KO): RSIAT + TEV: Luc: Tnos in 3α1
• U6: XT1 gRNA XT2 gRNA in 3α1
• U6: TFL gRNA gRNA
• U6: Ga20 gRNA

Digestion of gTS + SF – Cas9: gRNA in C58 and 3α1 (to check that it is correct.

Run electrophoresis gel of digestion products. All samples have gone well.

Transform the next ligations in C58 of Agrobacterium: Ga20/TFL (PCR/ cons/ KO): RSIAT: Luc: Tnos

Plate in C58 of Agrobacterium gTS with RSIAT+TEV.

03Sep

Pick E. coli DH5α (Ga20 / TFL (PCR/ cons/ KO) : RSIAT: Luc: Tnos) colonies 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 overnight at 37ºC with shaking.

Agrobacterium infiltration in Nicotiana benthamiana with gTS 2.0 (linker AEK) + promoter35s + pNOS + Cas9:gRNA

	35s: Luc	Pnos: Luc	Ren	TFL KO	Ga20 KO	Ga20 PCR	TFL PCR	Ga20 cons	TFL cons	XT1 Cas91	XT1 Cas92	Cas9 – TFL gRNA	Cas9 Ga20 gRNA
Initial OD	0.53	0.6	0.61	0.62	0.58	0.65	0.60	0.69	0.64	0.67	0.77	0.68	0.67
Vi culture (mL)	1.13	1	0.66	0.97	1.03	0.92	1	0.86	0.94	1.19	1.039	0.88	0.9
Vi Agroinfiltration solution (mL)	13.87	14	14.34	14.03	13.97	14.08	14	14.14	14.06	13.81	13.961	14.12	14.1

04Sep

Ligate reaction of TFL KO: RSIAT + TEV : Luc in pUPD2

Pick Agrobacterium C58 (Ga20 (PCR/ cons/ KO) and TFL (PCR/ cons): RSIAT + TEV: Luc: Tnos and U6: TFL/ Ga20/ XT1:XT2 gRNA) colonies from the plate that has been incubated overnight. 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 overnight at 28ºC with shaking.

05Sep

Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of Ga20 / TFL (PCR/ cons/ KO): RSIAT: Luc in 3α1

Digestion of Miniprep products using EcoRI. Incubate 1 hour at 37ºC.

Run electrophoresis gel of digestion products.

Transform PVX and TMV in Agrobacterium C58 due to there is not growth in their plates. Incubate 2 hours at 28ºC

Transform TFL KO: RSIAT + TEV in DH5α

06Sep

Pick E. coli DH5α (promoter35s: TFL KO: RSIAT+TEV: Luc: Tnos in 3α1) colonies 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 overnight at 37ºC with shaking.

Transform Agrobacterium C58 with gTS RSIAT (except TFL cons)

Ligate reaction of promoter35s: TFL cons: RSIAT: Luc + Tnos in 3α1

Transform E. coli DH5α with promoter35s: TFL KO: RSIAT: Luc: Tnos in 3α1.

Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of gTS RSIAT + TEV of Ga20 (PCR / cons/ KO) + TFL (PCR/ cons) + Ga20 gRNA + TFL gRNA + XT1 gRNA.

Digestion of Miniprep products using EcoRI.

Run electrophoresis gel of digestion products.

07Sep

Minipreps with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of TFL KO: RSIAT + TEV (3α1)

Digestion of miniprep products using EcoRI. Incubate 1 hour at 37ºC

Run electrophoresis gel of digestion products. It has not run in the correct way.

Pick E. coli DH5α (promoter35s: TFL KO: RSIAT+TEV: Luc: Tnos in 3α1 and promoter35s: TFL cons: RSIAT: Luc in 3α1) colonies 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 overnight at 37ºC with shaking.

Refresh C58 Agrobacterium tumefaciens cultures with the aim of infiltrating in N.benthamiana on 08/08. We are going to refresh the following cultures:

• Promoter35s: TFL (PCR/ cons/ KO): AEK: Luc: Tnos
• Promoter35s: Ga20 (PCR/ cons/ KO): AEK: Luc: Tnos
• Promoter35s: Luc: Tnos
• pNOS: luc: Tnos
• Cas9- XT1:XT2
• Cas9 – TFL gRNA and Cas9 – Ga20 gRNA

08Sep

Pick E. coli DH5α (C-Split Cas9, N-Split Cas9, gTS RSIAT Ga20 (PCR/cons/KO) and gTS RSIAT (KO/PCR)) colonies 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 overnight at 37ºC with shaking.

Minipreps with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of TFL KO: RSIAT + TEV (3α1) and TFL cons RSIAT

Digestion of miniprep products using Buffer EcorRI. Incubate 1 hour at 37ºC

Run electrophoresis gel of digestion products. Only sample number 6 of TFL cons RSIAT has run well.
PrepareAgrobacterium infiltration following:

	35s: Luc	Pnos: Luc	Ren	Ga20 PCR	Ga20 cons	Ga20 PCR	Ga20 KO	TFL PCR	TFL cons	TFL KO	XT1 Cas9	Cas9 – TFL gRNA	Cas9 Ga20 gRNA
Initial OD	0.557	0.574	0.745	0.621	0.580	0.65	0.579	0.568	0.690	0.605	0.652	0.608	0.747
Vi culture (mL)	1,077	1,047	0,805	0,966	1,034	1,036	1,056	0,87	0,992	0,987	0,803	0,96	1,077
Vi Agroinfiltration solution (mL)	13,923	13,953	14,195	14,034	13,966	13,964	13,944	14,13	14,008	14,013	14,197	14,04	13,923

10Sep

Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of C58

Pick Agrobacterium C58 (gTS Ga20PCR – SF- Cas9 – Ga20 gRNA + gTS Ga20PCR – SF- Cas9 – XT1 gRNA + gTS TFL cons- RSIAT ) colonies from the plate that has been incubated overnight. 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 overnight at 28ºC with shaking.

Glycerinate all devices in Agrobacterium

11Sep

Digestion of Miniprep products using EcoRI. Incubate 1 hour at 37ºC.

Run electrophoresis of digestion products.

12Sep

Luciferease assay of gTS (TFL and Ga20) with AEK linker

We throw out from Golden Braid collection the glycerinate number GB0584. Incubate over night at 37ºC

13Sep

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

• Ga20 PCR + SF – Cas9: XT1 gRNA
• Ga20 PCR + SF – Cas9: Ga20 gRNA
• 35s: TFL cons: RSIAT: Luc: Tnos
• 35s: Renilla: Tnos (1α2)

Digestion of Miniprep products using EcoRI (3α1) and HindIII (1α2)

Run electrophoresis gel of digestion products. Order in gel: TFL cons RSIAT - XT1 gRNA – Ga20 gRNA – Renilla

Transform in C58 N-Split Cas9 and C-Split Cas9 because of the digestion was wrong. C-Split cas9 is C58Psoup.

Transform promoter 35s: Renilla: Tnos in C58

Plate 35s:Ren:Tnos (C58) and N-Cas9 (C58)

14Sep

We are going to optimize ligate reaction of TFL KO with RSIAT+TEV. With this aim, we are going to measure DNA concentration of:

Device	DNA concentration (ng/μL)
3α1	133.0
Promoter 35s	73.2
RSIAT+TEV:Luc	230.4
pNOS	65.4

After obtaining the optimal value we are going to do the ligate reaction:

Reagent	Volume (μL)
Promoter 35S	1.4
3α1	2
TFL KO	0.5
RSIAT + TEV: Luc	1.12
Tnos	1.2
BSA10X	1.2
Ligase Buffer	1.2
BsmbI	1
T4 ligase	1
H2O milli-Q	1.38

15Sep

Pick a single E. coli DH5α (N-Split Cas9 in PVX) 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 overnight at 37ºC with shaking

Transform in C58 pSoup Agrobacterium promoter C-Split Cas9 (TMV) (3 α2)

Primer IG16SEP01 has arrived

Pick a single E. coli DH5α (gTS TFL KO RSIAT+TEV) 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 overnight at 37ºC with shaking.

16Sep

Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of TFL KO and Nóbel colonies.

Digestion of Miniprep products using EcoRI. Incubate 1 hour at 37ºC

Run electrophoresis gel of digestion products. Nóbel colonies are perfect but TFL KO no.

Transform Renilla in 1α2 with pSoup

Refresh gTS Ga20 AEK in order to prepare the infiltration the following day.

17Sep

Prepare infiltration

	35s: Luc	Pnos: Luc	Ren	Ga20 PCR	Ga20 cons	Ga20 KO	XT1 Cas9	Cas9 Ga20 gRNA
Initial OD	0.643	0.693	0.643	0.710	0.745	0.653	0.669	0.673
Vi culture (mL)	0.93	0.866	0,933	0,845	0.805	0.918	0,9036	0.8915
Vi Agroinfiltration solution (mL)	14.07	14.134	14,067	14,155	14.195	14.082	14,197	14.1085

19Sep

Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of PVX in C58.

Digestion of Miniprep product using EcoRI. Incubate 1 hour at 37ºC

Run electrophoresis gel. Just a band is observed over 10Kb.

20Sep

Ligate reaction

Reagent	Volume (μL)
C-Cas9	1
TMV	1
BSA10X	1.2
Ligase Buffer	1.2
BsmbI	1
T4 ligase	1
H2O milli-Q	5.6

Transform C-Split Cas9 in E. coli DH5 α and plate it.

Digestion N-Cas9 in Agrobacterium using EcoRI. Incubate 1 hour at 37ºC

Run electrophoresis gel. Two bands are observed at 13 and 3/4 Kb.

21Sep

Pick Agrobacterium C58 (35s: Renilla: Tnos) colonies from the plate that has been incubated overnight. Inoculate a starter culture of 4 ml of LB medium with 4 μL of Kanamycin and 4 μL of Rifampin in a 10 ml tube with the colony and incubate it overnight at 28ºC with shaking.

Pick colonies of C-Cas9 in TMV following the method explain before.

Refresh cultures in order to prepare the Agroinfiltration

22Sep

Prepare PCR in order to probe Nóbel thermocycler.

REAGENT	VOLUME (μL)	PROGRAM
Luciferase pUPD	1	TEMPERATURE	TIME
Buffer HF	5	94ºC	5 minutes
dNTPs	2	94ºC	35x	30 seconds
IG16JUN02	2.5	63ºC	30 seconds
IG16JUL18	2.5	72ºC	2 minutes
Taq UVAT	0.5	72ºC	10 minutes
H2O milli-Q	31.5	16ºC	∞

Miniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of C-Cas9 TMV (10 colonies) and Renilla in C58
Digestion of miniprep products using HindIII

Run electrophoresis gel.

Refresh gTS AEK TFL cultures in order to infiltrate.

Prepare Agroinfiltration of gTS AEK Ga20

	35s: Luc	Pnos: Luc	Ren	Ga20 PCR	Ga20 cons	Ga20 KO	XT1 Cas9	Cas9 Ga20 gRNA
Initial OD	0.344	0.355	0.287	0.309	0.358	0.352	0.342	0.383
Vi culture (mL)	1.16	1.126	2.787	1.94	1.67	1.136	2.34	2.088

23Sep

Repeat Digestion of TMV using HindIII. Incubate 1 hour at 37ºC

Digestion Renilla 1α2 (Agrobacterium) using BamI

Run electrophoresis gel. Bands from TMV are right whereas Renilla is not ok.

24Sep

Refresh Agrobacterium cultures in order to infiltrate the following day:

• 35s: Luc:Tnos
• pNOS:Luc:Tnos
• 35s: TFL PCR: AEK: Luc: Tnos
• 35s: TFL cons:AEK:Luc:Tnos
• 35s: TFL KO: AEK: Luc:Tnos
• 35s: Cas9: Tnos – U6: TFL gRNA: psgRNA
• Cas9- XT1:XT2
• 35s: Ren:Tnos

Digestion of Renilla suing PvuI, BanI and BclI. Incubate 1 hour at 37ºC

Run electrophoresis gel. All samples are ok

25Sep

Minipreps with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of Renilla in C58

Pick up samples in order to carry out luciferase assay tomorrow

Digestion of Renilla usin BanI. Incubate 1 hour at 37ºC

Run electrophoresis gel.

Electroporation of 4 Nóbel samples and a control using the electroporator from the laboratory.

Prepare infiltration

	35s: Luc	Pnos: Luc	Ren	TFL PCR	TFL cons	TFL KO	XT1 Cas9	Cas9 TFL gRNA
Initial OD	0.353	0.321	0.338	1.61	1.60	1.06	2.20	0.386
Vi culture (mL)	8.87	8.75	26.4	13.4	13.4	8.94	17.8	17.9

26Sep

Pick up colonies of TMV C-Cas9

Measure DNA concentration using Nanodrop in order to send them to iGEM.

Take from glycerinate SAGTI:Luc in pUPD2

Luciferase assay

Refresh gTS TFL RSIAT in order to prepare infiltration.

27Sep

Prepare a PCR for Nóbel thermocycler.

Prepare infiltration of TFL RSIAT

	35s: Luc	Pnos: Luc	Ren	TFL PCR	TFL cons	TFL KO	XT1 Cas9	Cas9 TFL gRNA
Initial OD	0.345	0.348	0.354	0.348	0.347	0.371	0.346	0.336
Vi culture (mL)	1.16	1.15	1.69	1.72	1.73	1.08	2.89	2.38

28Sep

Minipreps C-Cas9 TMV in C58

Digestion C-Cas9 TMV using HindIII. Incubate 1 hour at 37ºC

Run electrophoresis gel of PCRs (thermocycler). Control is ok but the sample not.

Run electrophoresis gel of digestion. All of them are perfect.

Sequencing reaction of Ga20 cons AEK

Pick up samples of TFL AEK and luciferase assay are done.

Prepare infiltration of Ga20 RSIAT

	35s: Luc	Pnos: Luc	Ren	Ga20 PCR	Ga20 cons	Ga20 KO	XT1 Cas9	Cas9 Ga20 gRNA
Initial OD	0.627	0.728	0.750	0.654	0.699	0.636	0.632	0.597
Vi culture (mL)	1.27	1.098	1.6	1.83	1.7167	1.26	3.16	3.35

29Sep

Prepare a PCR in order to prove our thermocycler.

Glycerinating C-Cas9 in TMV (DH5α), C-Cas9 in TMV (C58) and N-Cas9 in PVX (C58)

Refresh N-Cas9, C-Cas9, Cas9-XT1 and XT1:XT2 + TMV-GFP, TMV-BFP, TMV –dsRED and PVX-dsRED

04Oct

Prepare infiltration

	35s: Luc	Pnos: Luc	Ren	TFL PCR	TFL cons	TFL KO	XT1 Cas9	Cas9 TFL gRNA
Initial OD	0.21	0.51	0.49	0.28	0.22	0.21	0.57	0.50
Vi culture (mL)	1.9	0.78	1.22	0.97	2.73	1.9	2	1.75

05Oct

Prepare infiltration

	35s: Luc	Pnos: Luc	Ren	Ga20 PCR	Ga20 cons	Ga20 KO	XT1 Cas9	Cas9 Ga20 gRNA
Initial OD	0.46	0.48	0.54	0.52	0.54	0.48	0.60	0.60
Vi culture (mL)	0.87	0.83	1.11	1.15	1.11	0.833	1.67	1.67

06Oct

Infiltrate U6 –XT1:XT2 gRNA in Split-Cas9

Luciferase assay of TFL RSIAT

10Oct

Refresh TFL SAGTI + TFL SAGTI:gRNA:Cas9

Luciferase assay of Ga20 RSIAT

Genome extraction of N.benthamiana in order to check if Split-Cas9 works or not.

PCR Split-Cas9

11Oct

Refresh TFL KO with all possible linker in order to infiltrate it

Digestion of Split-Cas9. Incubate it 4 hours at 37ºC. There is a resistant band so we can suspect that the mutation has been produced.

PCR purification from electrophoresis gel