Team:Valencia UPV/Notebook
Notebook
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.
- 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.
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:
| Plasmid | GB Code |
|---|---|
| 35s:Cas9:nopaline synthase terminator (Tnos) | GB0639 |
| Luciferase (Luc) in pUPD2 | GB0096 |
| Tnos in pUPD2 | GB0037 |
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.
| 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 | ∞ | |
Run electrophoresis gel of Clemenules DNA (agarose 1%).
45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE
1X. Voltage used is 100 V.
Gleva Rice DNA Genome Extraction
Run electrophoresis gel of Luciferase PCR product. 45 mL
agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X.
Voltage used is 100 V.
Ligation Reaction of SAGTI:Luciferase into a pUPD2.
Transform E. coli DH5α with it. The method that is
necessary to carry out this procedure is explained in
protocols
Check DNA concentration with NanoDrop.
| SAMPLE | DNA Concentration(ng / μL) |
|---|---|
| Rice Gleva 1 | 22.8 |
| Rice Gleva 2 | 17.3 |
Run electrophoresis gel of Gleva rice DNA. We have
checked that there isn’t DNA.
Repeat: Rice DNA Genome Extraction Protocol
Check DNA concentration with NanoDrop.
| SAMPLE | DNA Concentration(ng / μL) |
|---|---|
| Rice Gleva 1 | 294.9 |
| Rice Gleva 2 | 193.7 |
Run electrophoresis gel of Gleva rice DNA. It observed that
genome extraction is correctly done.
Take glycerinated cultures from Goldenbraid
Collection:
| GB part | Plasmid | Antibiotic | Number GB |
|---|---|---|---|
| psgRNA | pUPD | Ampicillin | 0645 |
| U6-26 | pUPD | Ampicillin | 1001 |
- Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
-
- psgRNA in pUPD2
- U6-26 in pUPD2
Primers IG16JUL01, IG16JUL02, IG16JUL03, IG16JUL04,
IG16JUL05, IG16JUL06, IG16JUL07 and IG16JUL08 arrived.
gBlocks of - 35s:5’ region - have arrived.
We perform a PCR of Clemenules Orange and Gleva Rice Genome
Extraction following the protocol.
We want to obtain a fragment of the gene TFL of the orange
DNA extraction and the gene Ga20ox of the rice DNA
extraction.
| Sample | Initial concentration(ng/μL) | Final concentration(ng/μL) | Initial volume(μL) | Final volume (μL) |
|---|---|---|---|---|
| TFL 1 | 3153.8 | 150 | 4.756 | 100 |
| TFL 2 | 4527.9 | 150 | 3.31 | 100 |
| Ga20ox 1 | 294.9 | 150 | 50.8647 | 100 |
| Ga20ox 2 | 193.7 | 150 | 77.44 | 100 |
| Reagent | Volume(μL) | Program | ||
|---|---|---|---|---|
| TFL DNA | 1 | Temperature | Time | |
| Buffer HF | 10 | 98°C | 5 minutes | |
| dNTPs | 2 | 98°C | 35x | 30 seconds |
| IG16JUL01 (TFL_For) | 2.5 | 64°C | 30 seconds | |
| IG16JUL02 (TFL_Rev) | 2.5 | 72°C | 30 seconds | |
| Taq phusion | 0.5 | 72°C | 10 minutes | |
| H2O milli-Q | 31.5 | 16°C | ∞ | |
| Reagent | Volume(μL) | Program | ||
|---|---|---|---|---|
| Ga20ox DNA | 1 | Temperature | Time | |
| Buffer HF | 10 | 98°C | 5 minutes | |
| dNTPs | 2 | 98°C | 35x | 30 seconds |
| IG16JUL03 (Ga20_For) | 2.5 | 64°C | 30 seconds | |
| IG16JUL04 (Ga20_Rev) | 2.5 | 72°C | 30 seconds | |
| Taq phusion | 0.5 | 72°C | 10 minutes | |
| H2O milli-Q | 31.5 | 16°C | ∞ | |
Ligate reaction of 35s:5’ region in pUPD2. Following
ligation
protocol, BsmbI enzyme is used in this reaction.
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)
| Sample | DNA Concentration(ng / μL) | DNA Concentration(ng) |
|---|---|---|
| 1 | 105.2 | 5035.2 |
| 2 | 104.6 | 5035.2 |
Targets ligations in pUPD2 (Orange TFL and Rice Ga20ox).
Following ligation
protocol, BsmbI enzyme is used in this reaction.
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.
Run electrophoresis gel of digested targets in pUPD2.
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
| 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
- 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 Agrobacterium C58 with:
-
- 35s:5’ region:TFL PCR:Luc:Tnos (3α1)
- 35s:5’ region:Ga20ox PCR:Luc:Tnos (3α1)
Plating the last devices in 2 plates with
Agrobacterium C58. Incubate 2 days at 28°C.
- Minipreps (2 samples for each device) with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
-
- 35s:5’ region:TFL target control positive:Luc:Tnos
- 35s:5’ region:Ga20ox target control positive:Luc:Tnos
- 35s:5’ region:TFL target consensus:Luc:Tnos
- 35s:5’ region:Ga20ox target consensus:Luc:Tnos
- U6-26:sgRNA TFL:psgRNA (scaffold)
- U6-26:sgRNA Ga20ox:psgRNA (scaffold)
- Ligation using Golden Braid assembly of the next devices:
-
- 35s:Cas9:Tnos – U6:TFL sgRNA:psgRNA (scaffold) (Ω1)
- 35s:Cas9:Tnos – U6: Ga20ox sgRNA:psgRNA (scaffold) (Ω1)
| Reagent | Volume (μL) |
|---|---|
| Promotor 35s:Cas9 : Tnos | 1 |
| U6-26:TFL sgRNA:psgRNA / U6-26:Ga20ox sgRNA:psgRNA | 1 |
| 3Ω1 | 1 |
| BSA10X | 1.2 |
| Ligase Buffer | 1.2 |
| BsmbI | 1 |
| T4 ligase | 1 |
| H2O milli-Q | 4.6 |
Digestion of the minipreps with EcoRI which have been done
before. Incubate 1 hour at 37°C. There is a total of twelve
minipreps. It has been followed the Miniprep digestion
protocol.
Run electrophoresis gel of the digestion products.
Run electrophoresis gel of digested targets in pUPD2.
- Pick a single E. coli DH5α colony from the plates that have been incubated overnight. The devices are:
-
- 35s:TFL Knock-out:Luc:Tnos (4 samples)
- U6-26:Ga20ox sgRNA:psgRNA (2 samples)
Inoculate a starter culture of 4 ml of LB medium with 4 μL
of kanamycin in a 50 ml tube with the colony and incubate
it 16 hours at 37°C.
- Transformation in DH5α E. coli with:
-
- 35s:Cas9:Tnos – U6:TFL sgRNA:psgRNA (scaffold) (Ω1)
- 35s:Cas9:Tnos – U6:Ga20ox sgRNA:psgRNA (scaffold) (Ω1)
Plating E. coli transformations in plates with LB +
agar + IPTG + XGal and incubate it overnight at 37°C.
- Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
-
- U6-26:Ga20ox sgRNA:psgRNA
- 35s:5’ region:TFL Knock-out:Luc:Tnos (3α1)
Digestion of the minipreps with EcoRI which have been done
before. Incubate 1 hour at 37°C. There is a total of six
minipreps. It has been followed the Miniprep digestion
protocol.
Run an electrophoresis gel of the digestion products.
| Lanes | Samples | Verification |
|---|---|---|
| 1 | 1 Kb molecular weight marker | - |
| 2 | gRNA Ga20ox 1 | correct |
| 3 | gRNA Ga20ox 2 | correct |
| 4 | gRNA Ga20ox 3 | correct |
| 5 | gRNA Ga20ox 4 | correct |
| 6 | TFL knock-out 1 | correct |
| 7 | TFL knock-out 2 | correct |
| 8 | 1 Kb molecular weight marker | correct |
However, despite the fact that electrophoresis gel have
correctly run, we have decided to repeat the ligation
reactions. We have not get the expected results for a
gRNA.
- Ligation using Golden Braid assembly of the next devices:
-
- 35s:5’ region:TFL target control positive:Luc:Tnos
- 35s:5’ region:Ga20ox target control positive:Luc:Tnos
- 35s:5’ region:TFL target consensus:Luc:Tnos
- 35s:5’ region:Ga20ox target consensus:Luc:Tnos
- U6-26:sgRNA TFL:psgRNA (scaffold)
- U6-26:sgRNA Ga20ox:psgRNA (scaffold)
| Reagent | Volume(μL) | Reagent | Volume(μL) |
|---|---|---|---|
| TFL/Ga20ox gRNA | 1 | 35s:5’region | 1 |
| U6-26 | 1 | TFL/Ga20ox consensus TFL/Ga20ox knock-out | 1 |
| psgRNA | 1 | luciferase | 1 |
| 3α1 | 1 | Tnos | 1 |
| BSA10X | 1.2 | 3α1 | 1 |
| Ligase Buffer | 1.2 | BSA10X | 1.2 |
| BsaI | 1 | Ligase Buffer | 1.2 |
| T4 ligase | 1 | BsmbI | 1 |
| H2O milli-Q | 3.6 | T4 ligase | 1 |
| H2O milli-Q | 2.6 |
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.
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:
| 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.
- 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
- 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.
- 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.
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.
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. GA |
| 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 |
- 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.
Pick up the samples of infiltrated plants. We keep 3
disks per plant in a Eppendorf tube and we take 2 samples
of each plant.
Luciferase assay is made following Protocol
.
After analyzing all the data obtained, it seems that the
system works but we must optimized it to increase the
signal range. In this way, we will be able to distinguish
signal from noise.
- Conclusions luciferase assay:
-
- Eliminate 5’ region of the device
- Change linker sequence
- Add renilla in luciferase assay
- Change reporter to +1
- Use pLess as control
- Use a Wild Type as control
- Insert devices in cis
- Design a consensus target as longer as the amplified.
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 |
- DH5α E. coli transformation with the devices:
-
- 35S : 5’ region : TFL KO : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
- 35S : 5’ region : TFL consensus : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
- 35S : 5’ region : Ga20ox KO : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
- 35S : 5’ region : Ga20ox consensus : Luc : Tnos - promoter35s: Renilla:Tnos in Ω2
E. coli plating in plates with Kanamycin
(antibiotic). Incubate 16 hours at 37°
- Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
-
- 35s:5’region:TFL PCR:Luc:Tnos in α1
- 35s:5’region:Ga20oxPCR: Luc: Tnos in α1
Pick a single E. coli DH5α (TMV CDS) colony from the
plate that has been incubated overnight. Inoculate a
starter culture of 4 ml of LB medium with 4 μL of kanamycin
in a 50 ml tube with the colony and incubate it overnight
at 37°C with shaking.
Digestion of minipreps with EcoRI. Incubate 1 hour at
37°
Run electrophoresis gel of PCR targets Ga20 and TFL. In the second gel, digested TFL and Ga20 PCR from gTS are run. We remain the
samples 1.
Store at -80°C the devices of gTS PCR
| 1 | 35S:5’ | pUPD2 | CAM |
|---|---|---|---|
| 2 | SAGTI:Luc | pUPD2 | CAM |
| 3 | 35s:5’:TFLPCR:Luc:Tnos | 3α1 | KAN |
| 4 | 35s:5’:Ga20PCR:Luc:Tnos | 3α1 | KAN |
Ligate reactions of TFL PCR and Ga20ox PCR with
Renilla
| Reagent | Volume(μL) |
|---|---|
| 35s:5’region: TFL/Ga20oxPCR: Luc: Tnos | 1 |
| Promoter35s: Renilla: Tnos | 1 |
| pUPD2 | 1 |
| BSA10X | 1.2 |
| Ligase Buffer | 1.2 |
| BsmbI | 1 |
| T4 ligase | 1 |
| H2O milli-Q | 4.6 |
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.
Pick a single E. coli DH5α (promoter35s: 5’
region: TFL/Ga20oxPCR: Luc: Tnos - promoter35s: Renilla:
Tnos ) colony from the plate that has been incubated
overnight. Inoculate a starter culture of 4 ml of LB medium
with 4 μL of kanamycin in a 50 ml tube with the colony and
incubate it overnight at 37°C with shaking.
- Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
-
- 35s: 5’ region: TFL PCR: Luc: Tnos - promoter35s: Renilla: Tnos
- 35s: 5’ region: Ga20oxPCR: Luc: Tnos - promoter35s: Renilla: Tnos
- 35s: 5’ region: Ga20oxconsensus: Luc: Tnos - promoter35s: Renilla: Tnos
- 35s: 5’ region: Ga20oxKO: Luc: Tnos - promoter35s: Renilla: Tnos
Digestion of minipreps with EcoRV. Incubate 1 hour at
37°
Run electrophoresis gel of the digestion products: TFLK01,
TFLK02, TFLcons01, TFLcons02, GAK01, GAK02, GAcons01,
GAcons02.
Prepare the Agroinfiltration with the correct protocol.Protocol
Centrifuge the cultures at 3000 rpm during 15 minutes. We
discard the supernatant and it is necessary to resuspend in
5mL of Agroinfiltration solution. Let shaking it a RT
during 2 hours. OD’s measurement.
| device | Volume of culture (mL) | Volume of Agroinfiltration solution (mL) |
|---|---|---|
| Cas9 - TFL gRNA | 0.7 | 9.3 |
| Cas 9 - Ga20ox gRNA | 0.7 | 9.3 |
| Cas 9 - XT1: XT2 | 0.59 | 9.41 |
| TFL KO | 0.67 | 9.33 |
| TFL PCR | 0.73 | 9.27 |
| Ga20ox consensus | 0.71 | 9.28 |
| Pnos | 0.68 | 9.32 |
| 35s : Luc | 0.78 | 9.22 |
| Ga20ox PCR | 0.74 | 9.26 |
| TFL consensus | 0.71 | 9.29 |
| Ga20ox- KO | 0.73 | 9.27 |
- Infiltration cultures:
-
- 35s: Luc: Tnos Laboratory controls
- Pnos: Luc: Tnos Laboratory controls
- gTS TFL KO + Cas9 - XT1:XT2 Positive controls
- gTSGa20oxKO + Cas9 - XT1:XT2 Positive controls
- gTS TFL PCR + Cas9 - XT1:XT2 Negative controls
- gTS Ga20oxPCR + Cas9 - XT1:XT2 Negative controls
- gTS TFL PCR + Cas9 - TFLgRNA Samples
- gTS TFL cons + Cas9 - TFLgRNA Samples
- gTS Ga20oxPCR + Cas9 - Ga20gRNA Samples
- gTS Ga20oxcons + Cas9 - Ga20gRNA Samples
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:
| devices | Volume (μL) |
|---|---|
| 35s : 5’ region: Ga20/TFL KO: Luc: Tnos - 35s: Renilla: Tnos - 35s: hCas9: Tnos: U6: Ga20/TFL gRNA: psgRNA | 1 |
| 35s : 5’ region: Ga20/TFL cons: Luc: Tnos - 35s: Renilla: Tnos - 35s: hCas9: Tnos: U6: Ga20/TFL gRNA: psgRNA | 1 |
| 3 α 1 plasmid | 1.2 |
| Bsa 10X | 1.2 |
| Buffer ligase | 1 |
| Bsa I | 1 |
| T4 ligase | 1 |
| H20 milliQ | 4.6 |
U6:Ga20oxgRNA: psgRNA - 35s: Cas9: Tnos - Miniprep number 2
was empty. We have resuspended it with 40 μL of H20 milliQ
and we have checked the DNA concentration with the
Nanodrop. The results show us that the DNA concentration in
the Eppendorf was 140 ng/ μL so we have used it.
Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless)
Spin of 35s: 5’ region: TFL/Ga20oxPCR: Luc: Tnos – 35s:
Renilla: Tnos
Transform in C58 Agrobacterium 35s: Renilla : Tnos
(3 α2)
Plating promoter35s: Renilla: Tnos (3α2)
Luciferase assay
| Promoter35s | pNos |
|---|---|
| TFLKO | Ga20KO |
| TFLPCRXT1 | Ga20PCRXT1 |
| TFLPCROK | Ga20PCROK |
| TFLconsOK | Ga20consOK |
Transform the products of ligation in DH5 α. Incubate it at
37°C during 2 hours.
We store at -80°:
| 5 | 35s:5’:Ga20cons:Luc:Tnos | 3α1 | KAN |
|---|---|---|---|
| 6 | 35s:5’:Ga20KO:Luc:Tnos | 3α1 | KAN |
| 7 | 35s:5’:TFLcons:Luc:Tnos | 3α1 | KAN |
| 8 | 35s:5’:TFLKO:Luc:Tnos | 3α1 | KAN |
| 9 | 35s:5’:Ga20cons:Luc:Tnos-35s:Ren:Tnos | 3Ω2 | SPEC |
| 10 | 35s:5’:Ga20KO:Luc:Tnos-35s:Ren:Tnos | 3Ω2 | SPEC |
| 11 | 35s:5’:Ga20PCR:Luc:Tnos-35s:Ren:Tnos | 3Ω2 | SPEC |
| 12 | 35s:5’:TFLcons:Luc:Tnos-35s:Ren:Tnos | 3Ω2 | SPEC |
| 13 | 35s:5’:TFLKO:Luc:Tnos-35s:Ren:Tnos | 3Ω2 | SPEC |
| 14 | 35s:5’:TFLPCR:Luc:Tnos-35s:Ren:Tnos | 3Ω2 | SPEC |
| 15 | U6:Ga20sgRNA:psgRNA | 3α1 | KAN |
| 16 | U6:TFLsgRNA:psgRNA | 3α1 | KAN |
| 17 | 35s:Cas9:Tnos-U6:Ga20gRNA:psgRNA | 3Ω1 | SPEC |
| 18 | 35s:Cas9:Tnos-U6:TFLgRNA:psgRNA | 3Ω1 | SPEC |
| 19 | Ga20PCR | pUPD2 | CAM |
| 20 | TFLPCR | pUPD2 | CAM |
Run electrophoresis gel of: 35s: 5’ region: TFL/Ga20oxPCR:
Luc: Tnos – 35s: Renilla: Tnos. We keep the Miniprep number
1.
Refresh the cultures of TFL PCR (pUPD2) and Ga20oxPCR
(pUPD2) because we suspect that these cultures are the
correct ones but we are not sure so we want to check them.
The cultures that we use to refresh were made on
12/07/2016. It is important to remember that we need them
to assemble the gTS with the new linkers.
Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless)
Spin of TFL / Ga20oxPCR in pUPD2
Digestion of minipreps with NotI. Incubate 1 hour at
37°
Run electrophoresis gel of Miniprep products. We have made
a small gel so we have mix 0.45 g of Agarose with 45 mL of
TAE 1X. Voltage used is 100V. Both samples are correct.
Pick a single E. coli DH5α (promoter35s: 5’ region:
TFL/Ga20oxcons: Tnos - promoter35s: Renilla: Tnos - 35s:
Cas9: Tnos - U6: TFL/Ga20oxgRNA: psgRNA) colony from the
plate that has been incubated overnight. Inoculate a
starter culture of 4 ml of LB medium with 4 μL of
chloramphenicol in a 50 ml tube with the colony and
incubate it overnight at 37°C with shaking.
We throw out from Golden Braid collection the glycerinate
number 1107 (Cas9 - XT1gRNA) and 0549 (35s: TEV: Tnos)
Ligation reaction of 35s: 5’ region: TFL/ Ga20: Tnos - 35s:
Renilla: Tnos + 35s: Cas9: Tnos - U6: TFL/Ga20oxgRNA:
psgRNA
| devices | Volume (μL) |
|---|---|
| gTS TFL/ Ga20ox- Renilla | 1 |
| Cas9 - gRNA | 1 |
| 3 α 1 plasmid | 1.2 |
| Bsa 10x | 1.2 |
| Buffer ligase | 1 |
| Bsa I | 1 |
| T4 ligase | 1 |
| H20 milliQ | 4.6 |
Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
- Promoter 35s: 5’region: TFL/Ga20 cons: Luc: Tnos – p35s:Renilla:Tnos – p35s:Cas9:Tnos – U6-26: XT1:XT2 gRNA: psgRNA
- Promoter35s: 5’region: Cas9: Tnos – U6-26: XT1:XT2 gRNA: psgRNA
- Promoter 35s: protease NiaTEV: Tnos
Ligation reaction of:
- promoter 35s: 5’ region: TFL/ Ga20 KO: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: XT1:XT2 gRNA: psgRNA
- promoter 35s: 5’ region: TFL/ Ga20 PCR: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: XT1:XT2 gRNA: psgRNA
Digestion of minipreps TFL/Ga20 cons with EcoRI. Incubate 2 hour at 28ºC.
Run electrophoresis gel of Miniprep products. They have not gone well and maybe, we have picked up blue colonies. This hypothesis explains that the digestion is the same that 3α1 plasmids without any insert. 
Transform in C58 Agrobacterium culture by electroporation using 1440 kV during 5 mS:
- Promoter35s: protease NiaTEV
- Promoter35s:Renilla:Luc (3α2) due to the last time, culture was platting on the wrong plate.
Incubate 2 hours at 28ºC and plating it. Incubate during 48 hours at 28ºC.
Transform in DH5α E. coli culture by electroporation using 1500kV during 5mS:
- promoter 35s: 5’ region: TFL/ Ga20 PCR: Luc: Tnos – promoter 35s: Renilla: Tnos + promoter 35s: hCas9: Tnos – U6-26: OK gRNA: psgRNA
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.
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 | ∞ | |
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 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. Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of: 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. Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of: Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of gTS – Renilla – Cas 9- gRNA. There are 60 samples. Ligate reaction of gTS (TFL / Ga20 cons/KO/PCR) – SF in 3Ω2 Run electrophoresis gel of digestion products. RSIAT:Luc and RSIAT+TEV:Luc Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of gTS + SF 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. Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of: Prepare Agroinfiltration Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of: Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of PVX. Repeat gTS TFL KO RSIAT + TEV: Luc in 3α1 Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of: Repeat ligate reaction of Ga20/ TFL (PCR – cons – KO) 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. Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of Ga20 / TFL (PCR/ cons/ KO): RSIAT: Luc in 3α1 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. Minipreps with E.Z.N.A ®.® Plasmid Mini Kit I, Q(capless) Spin of TFL KO: RSIAT + TEV (3α1) 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.
Run electrophoresis gel of Miniprep products. We cannot interpret the results. We have decided to repeat the digestion again.
Digestion of minipreps with EcoRI. Incubate 1 hour at 37ºC.
Run electrophoresis gel of these new Miniprep products.
Run electrophoresis gel of the PCR which was made on 13-08-2016. The fragment do not amplified with any selected temperature.
Run electrophoresis gel of this last digestion. We have decide to repeat the ligate reaction because of appearing an unexpected band.
Ga20 cons – Ren – Cas9 – Ga20 gRNA x4 Ga20 PCR – Ren – Cas9 – XT1 gRNA x4 Ga20 PCR – Ren – Cas9 – Ga20 gRNA x4 TFL PCR – Ren – Cas9 – XT1 gRNA x4 TFL cons – Ren – Cas9 – TFL gRNA x4 TFL KO – Ren – Cas9 – XT1 gRNA x4 TFL cons – Ren – Cas9 – TFL gRNA x4 Ga20 KO – Ren – Cas9 – XT1 gRNA x4
Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
Digestion of these last Minipreps with HindIII. Incubate 1 hour at 37ºC.
Run electrophoresis gel of digestion products. We cannot distinguish anything in the gel so we have decided to pick up more colonies from the plate.
Inoculate a starter culture of 5 ml of LB medium with 5 μL of kanamycin and rifampicin in a 50 ml tube with the colony and incubate it 48 hours at 28ºC with shaking must be necessary.
We have decided to repeat the performance of PCR to bind linker RSIAT+TEV with luciferase. We are going to try at 71ºC. Moreover this time, we are going to try it using DMSO at 65 and 67ºC. REAGENT VOLUME (μL) Luciferase pUPD2 1 Buffer HF 10 dNTPs 2 IG16JUL20 2.5 IG16JUN02 2.5 Taq phusion 0.5 DMSO 1.5 H2O milli-Q 30
Ligation using Golden Braid assembly of the next devices:
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.
Digestion of Minipreps with NotI. Incubate 1 hour at 37ºC.
Run electrophoresis gel of digestion products. We run 4 samples of RSIAT and 4 samples of AEK. It can be observed the sample of AEK – 4. We only can keep this one.
DH5α E. coli transformations with the products of ligate reactions that were made on 15 /08/2016.
Ligate reactions of gTS 2.0:
Promoter 35s: TFL/Ga20 (KO/cons/PCR) : AEK linker : Luc : Tnos in 3α1Devices 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 pUPD2Reagent 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
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:
Digestion of Minipreps with NotI. Incubate 1 hour at 37ºC.
Run electrophoresis gel of digestion products. It has not left any result.
Repeat ligate reactions of RSIAT+TEV: Luc in pUPD2. Transform in DH5α and plate it.
Nanodrop measurement:
There is DNA but a wide band should be seen. We cannot see anything.
PCR purification of RSIAT:Luc in pUPD2 due to the digestion has not gone well. DNA concentration of the PCR is 55.6 ng/ μL
Ligate reaction of RSIAT:Luc in pUPD2
Nanodrop measurement of promoter 35s: NiaTEV protease: Tnos in 2α2. (We check the glycerinate E. coli Miniprep to guarantee that it is correct. If not, it could be cause that we cannot see any band in Agrobacterium C58.
There is 94.5 ng/ μL so it is correct. We are going to pick up colonies in NiATEV plate and we do again the minipreps in C58 Agrobacterium.
Digestion of promoter 35s: Renilla: Tnos in C58 using the restriction enzyme HindIII. (3α2)
Run electrophoresis gel of digestion products. Unexpected bands appear. We are going to do the digestion of the promoter 35s: Renilla: Tnos in E. coli just to check that it is correct. 
Platting the ligation product RSIAT+TEV: Luc in pUPD2. Incubate it at 37ºC overnight.
Transform E. coli DH5 α with the device RSIAT: Luc in pUPD2. Incubate 1 hour at 37ºC and plate it.
Digestion of Minipreps using EcoRI. Incubate 1 hour at 37ºC.
Run electrophoresis gel of digestion products:
Lanes Samples Verification 1 1 Kb molecular weight marker correct 2 TFL PCR and TFL gRNA 1 correct 3 TFL PCR and TFL gRNA 2 correct 4 TFL cons and TFL gRNA 1 correct 5 TFL cons and TFL gRNA 2 correct 6 TFL KO and XT1 gRNA 1 correct 7 TFL KO and XT1 gRNA 2 correct 8 TFL PCR and XT1 gRNA 1 correct 9 TFL PCR and XT1 gRNA 2 correct 10 Ga20 PCR and Ga20 gRNA 1 correct 11 Ga20 PCR and Ga20 gRNA 2 correct 12 Ga20 cons and Ga20 gRNA 1 correct 13 Ga20 cons and Ga20 gRNA 2 correct 14 Ga20 KO and XT1 gRNA 1 correct 15 Ga20 KO and XT1 gRNA 2 correct 16 Ga20 PCR and XT1 gRNA 1 correct 17 Ga20 PCR and XT1 gRNA 2 correct 18 1 Kb molecular weight marker correct
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.
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.
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.
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.
Digestion of minipreps with EcoRV. Incubate 1 hour at 37ºC.
Run electrophoresis gel of digestion products.
Pick a single Agrobacterium C58 (promoter35s: Renilla) colony from the plate that has been incubated overnight. Incubate it overnight at 28ºC with shaking.
Transform E. coli DH5α with RSIAT:Luc and RSIAT+TEV::Luc.
Split-Cas9 gBlocks have arrived: C-Cas91 and C-Cas92 in TMV – N-Cas91 and N-Cas92 in PVX.
Ligate reactions of Split-Cas9 in pUPD2.
Ligate reactions of:
We do not put Ga20 PCR ligations due to the band in electrophoresis gel is not the expected.
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 ∞
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:
Take out the glycerinate of promoter35s: NiaTEV: Tnos(GB-0594). Triple groove is made.
Agrobacterium C58 transformations with: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
Minipreps with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin of:
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.
Digestion of Miniprep products using EcoRI and EcoRV.
Run electrophoresis gel of digestion products. TFL KO has not gone well.
Ligate reactions of:
Transform C58 Agrobacterium with the next devices:
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.
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.
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:
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.
Digestion of Miniprep products using EcoRI. Incubate 1 hour at 37ºC.
Run electrophoresis gel. After digesting blue colony of C-Cas9 , we know that the resistance is kanamycin and the restriction site is EcoRI.
Refresh C58 Agrobacterium tumefaciens cultures with the aim of infiltrating in N.benthamiana on 27/08. We are going to refresh the following cultures:
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.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:
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.
Agrobacterium infiltration in Nicotiana benthamiana with gTS 2.0 (linker AEK) + promoter35s + pNOS + Cas9:gRNA35s: 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
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.
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
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.
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:
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.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 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 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 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 DNA concentration (ng/μL) 133.0 73.2 230.4 65.4 Volume (μL) 1.4 2 0.5 1.12 1.2 1.2 1.2 1 1 1.38 35s: Luc Pnos: Luc Ren Ga20 PCR Ga20 cons Ga20 KO XT1 Cas9 Cas9 Ga20 gRNA 0.643 0.693 0.643 0.710 0.745 0.653 0.669 0.673 0.93 0.866 0,933 0,845 0.805 0.918 0,9036 0.8915 14.07 14.134 14,067 14,155 14.195 14.082 14,197 14.1085 Volume (μL) 1 1 1.2 1.2 1 1 5.6 VOLUME (μL) PROGRAM 1 TEMPERATURE TIME 5 94ºC 5 minutes 2 94ºC 35x 30 seconds 2.5 63ºC 30 seconds 2.5 72ºC 2 minutes 0.5 72ºC 10 minutes 31.5 16ºC ∞ 35s: Luc Pnos: Luc Ren Ga20 PCR Ga20 cons Ga20 KO XT1 Cas9 Cas9 Ga20 gRNA 0.344 0.355 0.287 0.309 0.358 0.352 0.342 0.383 1.16 1.126 2.787 1.94 1.67 1.136 2.34 2.088 35s: Luc Pnos: Luc Ren TFL PCR TFL cons TFL KO XT1 Cas9 Cas9 TFL gRNA 0.353 0.321 0.338 1.61 1.60 1.06 2.20 0.386 8.87 8.75 26.4 13.4 13.4 8.94 17.8 17.9 35s: Luc Pnos: Luc Ren TFL PCR TFL cons TFL KO XT1 Cas9 Cas9 TFL gRNA 0.345 0.348 0.354 0.348 0.347 0.371 0.346 0.336 1.16 1.15 1.69 1.72 1.73 1.08 2.89 2.38 35s: Luc Pnos: Luc Ren Ga20 PCR Ga20 cons Ga20 KO XT1 Cas9 Cas9 Ga20 gRNA 0.627 0.728 0.750 0.654 0.699 0.636 0.632 0.597 1.27 1.098 1.6 1.83 1.7167 1.26 3.16 3.35 35s: Luc Pnos: Luc Ren TFL PCR TFL cons TFL KO XT1 Cas9 Cas9 TFL gRNA 0.21 0.51 0.49 0.28 0.22 0.21 0.57 0.50 1.9 0.78 1.22 0.97 2.73 1.9 2 1.75 35s: Luc Pnos: Luc Ren Ga20 PCR Ga20 cons Ga20 KO XT1 Cas9 Cas9 Ga20 gRNA 0.46 0.48 0.54 0.52 0.54 0.48 0.60 0.60 0.87 0.83 1.11 1.15 1.11 0.833 1.67 1.67
