Competent Agrobacterium Tumefaciens cells

The creation of competent Agrobacterium Tumefaciens cells is a 5-day procedure. In our case the Agrobacterium Tumefaciens strain we use is C58C1.

Day 1

• Streak the preferred Agrobacterium Tumefaciens strain on solid LB medium with rifamycin (50μg/mL)
• Incubate for 2 days at 28℃ in dark

Day 3

• Pick up a single colony and start a 5mL liquid culture in LB with rifamycin (50μg/mL)
• Incubate (shaking) at 28℃ until saturation (overnight)

Day 4

• Add 100μL of the saturated culture in a 2L flask with 200mL of LB with rifamycin (50μg/mL)
• Incubate (shaking) overnight at 28℃

Day 5

• Measure OD₆₀₀ (should be ~0.8-1)
• Split culture in 50mL Falcon tubes and pellet cells at 3000rpm for 10 min (4℃)
• Discard the supernatant
• Dilute cells in every Falcon in 1mL cold CaCl2 20mM
• Aliquot 100μL of cells in 1.5mL Eppendorf tubes and freeze them immediately in liquid nitrogen
• Store at -80℃

E. coli Transformation

We use E.coli DH10B cells for our project’s needs. This protocol’s steps are the following:

• Rest E.coli DH10Bcells on ice for 3-5 mins
• Add ~100ng of DNA
• Rest on ice for 30 min
• Heat shock at 42℃ for 1 min and thaw on ice for 1 min
• Add up to 1mL liquid LB
• Incubate at 37℃ (shaking) for 1h
• Pellet cells at 2100 rcf for 2 min and discard the supernatant
• Resuspend the pellet in LB leftovers
• Plate in selective solid medium and incubate overnight at 37℃

Agrobacterium tumefaciens Transformation

For the purpose of our project we use Agrobacterium Tumefaciens C58C1 strain. The transformation procedure we use is the following:

• Thaw competent Agrobacterium Tumefaciens C58C1 cells on ice for 30 minutes.
• Add ~5μg of plasmid DNA and cold shock the cells (freeze in liquid nitrogen)
• Incubate at 37℃ for 1 hour
• Add sterile liquid LB up to 1mL and incubate (shaking) at 28℃ for 2-3 hours
• Pellet the cells (3000g for 2min) and discard the supernatant
• Resuspend the pellet in LB leftovers and plate in solid selective medium
• Incubate at 28℃ for 2 days in the dark

Isolation of plasmid DNA (mini-preps)

The process of plasmid isolation can be separated into three main steps: Alkaline Lysis, Ethanol Precipitation and Dilution. For this procedure 3 solutions will be needed: Solution I,II and III.

Alkaline Lysis:

• Pellet cells and discard all of the supernatant (2x)
• Resuspend the pellet at 100μL of Solution I
• Add 200μL of Solution II, mix by turnaround and let sit for a maximum of 5min
• Add 150μL of Solution III and mix by turnaround
• Centrifuge at 13800g for 10min and collect the supernatant in a clean tube

Ethanol Percipitation:

• Add 900μL of 100% Ethanol and rest in ice for 2min
• Centrifuge at 13800g for 20min and discard the supernatant
• Wash pellet with 60μL 70% Ethanol
• Centrifuge at 13800g for 5min, discard the supernatant and remove Ethanol completely
• Air dry in hood for 10min

Dilution:

• Dilute pellet in 30μL dH₂O (sterile)

Agroinfiltration

Agroinfiltration is a procedure in which we inject Agrobacterium Tumefaciens C58C1 containing our prefered gene into plant leaves to make them express this gene’s protein.

The procedure takes 2 days.

Day 1:

• Start an overnight liquid culture of Agrobacterium Tumefaciens C58C1 (28℃ )

Day 2:

• Pellet the cells at 2800 rpm for 10 minutes (4℃ )
• Discard the supernatant and resuspend the pellet in 10mM MgCl₂ solution (~5mL)
• Pellet the cells again, discard the supernatant and resuspend the pellet in 1mL MM solution (10mM MgCl₂ and 10mM MES)
• Measure the concentration of the cells using a photometer and dilute until the absorption at 600nm is 0.5
• Make a small nick using a small needle at the leaf’s underside
• Inject a small amount of the cell solution using a syringe

Note: The plant used in this procedure must be unwatered.

Golden Gate Cloning

We used Golden Gate cloning to connect the parts we needed to be expressed together.

This protocol was adjusted as NEB instructed.

For 20μL reaction:

Thermocycler settings are the following:

Digestion and Ligation for 25 cycles

A-tailing

For a 50μL reaction

Incubate at 72℃ for 30 min

Ligation of A-tailed products

For a 20μL reaction

Incubate at room temperature for 2 hours

Colony PCR

For a 50μL reaction

Thermocycler settings

High Fidelity PCR

We use High Fidelity PCR for a plethora of our experiments and it’s the main method to multiply our parts and constructs.

For a 50μL reaction:

Restriction Digestions

In our case we used the restriction enzymes NdeI and SacI. We used the protocol provided by NEB. For a 50μL reaction:

Incubate for 5-15 min at 37℃