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.

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

Agroinfiltration in Nicotiana benthamiana of C58 with dsRED.

• Take from the glycerinates of Goldenbraid Collection:

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.

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

Orange Clemenules DNA Genome Extraction protocol

• Take from the glycerinates of GoldenBraid Collection:

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.

• 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.

Perform a PCR to bind linker SAGTI with luciferase:

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.

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.

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

Take glycerinated cultures from Goldenbraid Collection:

• 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.

Ligate reaction of 35s:5’ region in pUPD2. Following ligation protocol, BsmbI enzyme is used in this reaction.

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).

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.

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)

Targets ligations in pUPD2 (Orange TFL and Rice Ga20ox). Following ligation protocol, BsmbI enzyme is used in this reaction.

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.

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.

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

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

• 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

• 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)

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 path:https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol].

Run electrophoresis gel of the digestion products.

• 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.

• 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)

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.

• 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.

• 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 path:https://2016.igem.org/Team:Valencia_UPV/Notebook/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

• 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.

Sequencing reaction:

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.

• #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
  

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

• 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
• • inipreps of Agrobacterium with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
  • • 5s: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

• inipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
• • 5s: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

• equencing products have arrived:
• • 6-26:Ga20ox gRNA:psgRNA in α1 - correct
  • U6-26:TFL gRNA:psgRNA in α1 - correct

• • inipreps with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
  • • 5s: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

  
  

  28Jul

  
  

  29Jul

  
  

  01Aug

  
  

  02Aug

  
  

  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

  
  

  04Aug

  
  

  05Aug

  
  

  08Aug

  
  

  09Aug

  
  

  10Aug

  
  

  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

  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.

  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^ 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

  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

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

  We store at -80^:

  Promoter35s	pNos
  TFLKO	Ga20KO
  TFLPCRXT1	Ga20PCRXT1
  TFLPCROK	Ga20PCROK
  TFLconsOK	Ga20consOK

  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

  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

  nfiltration cultures:
  5s: 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
  Transplant agroinfiltrated plants (Nicotiana benthamiana)
  Pick up 6 disks per each plant in order to carry out the luciferase assay on 09/08
  Ligate reaction of:

  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

  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^ 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.
  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^ with shaking.
  iniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
  5s: 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.

  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

  Ligate reactions of TFL PCR and Ga20ox PCR with Renilla

  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

  iniprep with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
  5s: 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.
  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.
  onclusions luciferase assay:
  liminate 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.
  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^ with shaking.
  Targets ligations with Renilla reporters.
  H5α E. coli transformation with the devices:
  5S : 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^
  inipreps with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of:
  5s: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^ 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^ the devices of gTS PCR

  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

  Received the necessary primers to sequence: TFL consensus, TFL knockout, Ga20ox consensus and Ga20ox knockout.
  repare 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^ with shaking. The devices are:
  5S : 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.
  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.

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