iGEM 2016 — Team Valencia_UPV

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# Team:Valencia UPV/Notebook

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* PROJECT
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* HUMAN PRACTICES
  + [OVERVIEW](https://2016.igem.org/Team%3AValencia_UPV/Human_Practices)
  + [SOCIAL ENGAGEMENT](https://2016.igem.org/Team%3AValencia_UPV/HP/Silver)
  + [SOLVING REAL PROBLEMS](https://2016.igem.org/Team%3AValencia_UPV/HP/Gold)
  + [IMPACT ASSESSMENT](https://2016.igem.org/Team%3AValencia_UPV/Integrated_Practices)
  + [SAFETY](https://2016.igem.org/Team%3AValencia_UPV/Safety)
* PARTS
  + [BEST PART COLLECTION](https://2016.igem.org/Team%3AValencia_UPV/Part_Collection)
* MODEL
  + [OVERVIEW](https://2016.igem.org/Team%3AValencia_UPV/Model)
  + [Cas9:gRNA Complex Formation](https://2016.igem.org/Team%3AValencia_UPV/CP)
  + [R-Loop Formation](https://2016.igem.org/Team%3AValencia_UPV/Loop)
  + [Cleavage and Reading Frame Shift](https://2016.igem.org/Team%3AValencia_UPV/ORF)
  + [Conclusions](https://2016.igem.org/Team%3AValencia_UPV/Conclusions)
* [ACHIEVEMENTS](https://2016.igem.org/Team%3AValencia_UPV/Achievements)

# 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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/Protocol)

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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/Protocol)
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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/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

![](https://static.igem.org/mediawiki/2016/7/78/T--Valencia_UPV--16704gDNA_clem.jpg)

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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/Protocol)

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.
![](https://static.igem.org/mediawiki/2016/c/c2/T--Valencia_UPV--16705gDNA_rice-ok.jpg)

Repeat: Rice DNA Genome Extraction [Protocol](https://2016.igem.org/Team%3AValencia_UPV/Notebook/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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/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.
![](https://static.igem.org/mediawiki/2016/f/f9/T--Valencia_UPV--16708PCR_gDNA.jpg)

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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/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.
![](https://static.igem.org/mediawiki/2016/b/b6/T--Valencia_UPV--160712dig_35s_5prima-ok.jpg)

* 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

![](https://static.igem.org/mediawiki/2016/f/f8/T--Valencia_UPV--160713dig_targets_pUPD2-ok.jpg)

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.

![](https://static.igem.org/mediawiki/2016/f/f8/T--Valencia_UPV--160713dig_targets_pUPD2-ok.jpg)
*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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/Protocol).

Run electrophoresis gel of the digestion products.

![](https://static.igem.org/mediawiki/2016/6/67/T--Valencia_UPV--160716dig_cons_knock_gRNA-ok.jpg)

Run electrophoresis gel of digested targets in pUPD2.
![](https://static.igem.org/mediawiki/2016/5/50/T--Valencia_UPV--160717dig_comprobacion_puta_mierda-ok.jpg)

* 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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/Protocol).

Run an electrophoresis gel of the digestion products.
![](https://static.igem.org/mediawiki/2016/5/50/T--Valencia_UPV--160717dig_comprobacion_puta_mierda-ok.jpg)

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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/Protocol).

Run an electrophoresis gel of the digestion products. We
will keep the minipreps with “1”.

![](https://static.igem.org/mediawiki/2016/b/bb/T--Valencia_UPV--160720dig_gRNA_cons_KO_TFL_y_Ga20ox.jpg)

* 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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/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.

![](https://static.igem.org/mediawiki/2016/4/44/T--Valencia_UPV--160723dig_Cas9-gRNA_TFL_y_Ga20.jpg)

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.

![](https://static.igem.org/mediawiki/2016/1/1f/T--Valencia_UPV--160725dig_agro.jpg)

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.

![](https://static.igem.org/mediawiki/2016/b/b2/T--Valencia_UPV--160726dig_targets_agro.jpg)

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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/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.

![](https://static.igem.org/mediawiki/2016/8/84/T--Valencia_UPV--160803dig_PCRtargetsGa20yTFL.jpg)

![](https://static.igem.org/mediawiki/2016/0/0f/T--Valencia_UPV--160803_dig_gTS_TFL.Ga20_PCR.jpg)

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.

![](https://static.igem.org/mediawiki/2016/c/c6/T--Valencia_UPV--160805dig_targetsKOyCons_Renilla.jpg)

Prepare the Agroinfiltration with the correct protocol.[Protocol](https://2016.igem.org/Team%3AValencia_UPV/Notebook/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.

![](https://static.igem.org/mediawiki/2016/6/6f/T--Valencia_UPV--160809_dig_gTS_TFL.Ga20_PCR_Ren.jpg)

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.

![](https://static.igem.org/mediawiki/2016/1/19/T--Valencia_UPV--160810_dig_TFL.Ga20_PCR_pUPD2.jpg)

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.

![](https://static.igem.org/mediawiki/2016/e/e5/T--Valencia_UPV--160811_dig_gTS_TFL.Ga20_cons_Renilla_Cas9_XT1gRNA_y_Cas9_XT1gRNA_y_TEV.jpg)

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.

![](https://static.igem.org/mediawiki/2016/b/bd/T--Valencia_UPV--160812_PCR_linkers_AEAAAK_RSIAT_RSIAT-TEV_Luc.jpeg)

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.

![](https://static.igem.org/mediawiki/2016/4/46/T--Valencia_UPV--160814_dig_gTS_Ren_Cas9_XT1gRNA_1.jpg)

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

Run electrophoresis gel of these new Miniprep products.

![](https://static.igem.org/mediawiki/2016/e/ea/T--Valencia_UPV--160814_dig_gTS_Ren_Cas9_XT1gRNA_2.jpg)

Run electrophoresis gel of the PCR which was made on 13-08-2016. The fragment do not amplified with any selected temperature.
![](https://static.igem.org/mediawiki/2016/1/10/T--Valencia_UPV--160814_PCR_RSIAT-TEV_68_69_70_oC.jpg)

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.

![](https://static.igem.org/mediawiki/2016/5/55/T--Valencia_UPV--160815_dig_gTS_TFL-Ga20_cons-PCR-KO_Ren_Cas9_TFL-Ga20-XT1gRNA.jpg)

|  |  |
| --- | --- |
| 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.
![](https://static.igem.org/mediawiki/2016/e/e2/T--Valencia_UPV--160815_dig_agro_Ren_y_TEV.jpg)
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.

![](https://static.igem.org/mediawiki/2016/d/d2/T--Valencia_UPV--160816_dig_RSIAT_Luc_y_AEK_Luc.jpeg)

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.

![](https://static.igem.org/mediawiki/2016/6/63/T--Valencia_UPV--160817_dig_RSIAT_y_Luc_y_dig_agro_Ren_y_TEV.jpg)

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.

![](https://static.igem.org/mediawiki/2016/6/6c/T--Valencia_UPV--160817_dig_agro_Ren.jpg)

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:

![](https://static.igem.org/mediawiki/2016/5/5b/T--Valencia_UPV--160818_dig_gTS_TFL-Ga20_PCR-cons-KO_Ren_Cas9_OKgRNA-XT1gRNA.jpg)

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.

![](https://static.igem.org/mediawiki/2016/2/21/T--Valencia_UPV--160822_dig_gTS_Ren_Cas9_gRNA_52samples.jpg)

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.

![](https://static.igem.org/mediawiki/2016/7/73/T--Valencia_UPV--160822_Cas9_y_RSIAT_Luc.jpg)

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.

![](https://static.igem.org/mediawiki/2016/f/fa/T--Valencia_UPV--160824_dig_gTS_SF.jpg)

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.
![](https://static.igem.org/mediawiki/2016/1/12/T--Valencia_UPV--1608-26_diggTS%2BSF_Cas9%2BgRNA.jpg)

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.

![](https://static.igem.org/mediawiki/2016/e/e0/T--Valencia_UPV--160901dig_gTSGa20-SF-XT1%2BOK.jpg)

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.
Prepare[*Agrobacterium* infiltration](https://2016.igem.org/Team%3AValencia_UPV/Notebook/Protocol%3Ci%3EAgrobacterium%3C/i%3Einfiltration_id) 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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/ProtocolLuciferaseassay_id) 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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/ProtocolLuciferaseassay_id) 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](https://2016.igem.org/Team%3AValencia_UPV/Notebook/ProtocolLuciferaseassay_id)

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