Aim: to form a level 1 construct (full transcriptional unit) from level 0 modules in a in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.

Add relevant DNA to a PCR tube at a 2:1 ratio of insert to acceptor (dilutions may be required depending on starting DNA concentration).

Add enzymes and reaction buffers according to the template shown below.

Make up the reaction mix to 15 µL using distilled water.

Reaction component	Volume (µL)
Level 1 acceptor	Ratio 2:1 (Insert: acceptor)
Insert 1	Ratio 2:1 (Insert: acceptor)
Insert 2	Ratio 2:1 (Insert: acceptor)
Insert 3	Ratio 2:1 (Insert: acceptor)
T4 reaction buffer	1.5
BSA buffer	1.5
BsaI	0.5
T4 DNA Ligase	0.5
Distilled water	Make up to 15 µL

Cycle the reaction mix in a PCR machine according to the program given below.

Step	Temperature (°C)	Time (minutes: seconds)
1	37	0:20
2*	37	3:00
3*	16	4:00
4	50	5:00
5	80	5:00
6	16	∞

*Steps 2-3 cycled x26

Aim: to form a level 2, multigene construct from level 1 modules in a in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction..

Add relevant DNA to a PCR tube at a 2:1 ratio of insert to acceptor (dilutions may be required depending on starting DNA concentration).

Add enzymes and reaction buffers according to the template shown below.

Make up the reaction mix to 15 µL using distilled water.

Reaction component	Volume (µL)
Level 1 acceptor	Ratio 2:1 (Insert: acceptor)
Insert 1	Ratio 2:1 (Insert: acceptor)
Insert 2	Ratio 2:1 (Insert: acceptor)
Insert 3	Ratio 2:1 (Insert: acceptor)
T4 reaction buffer	1.5
BSA buffer	1.5
Bpi1	0.5
T4 DNA Ligase	0.5
Distilled water	Make up to 15 µL

Cycle the reaction mix in a PCR machine according to the program given below.

Step	Temperature (°C)	Time (minutes: seconds)
1	37	0:20
2*	37	10:00
3*	16	10:00
4	37	10:00
5	65	20:00
6	16	∞

*Steps 2-3 cycled x3

Aim: to make LB Agar plates for growing bacteria

Add the following components to a reaction mix:

10% w/v Tryptone

10% w/v NaCl

5% Yeast extract

Make up with distilled water, ensuring all solid is mixed thoroughly. Pour into appropriately sized Erlenmeyer flasks with 1.5% w/v agarose in each flask, place sponge stopper on top and autoclave.

Aim: to make LB growth media for culturing bacterial colonies.

Add the following components to the reaction mix:

10% w/v Tryptone

10% w/v NaCl

5% Yeast extract

Make up with distilled water, ensuring all solid is mixed thoroughly. Pour into appropriately sized Erlenmeyer flasks, place sponge stopper on top and autoclave.

Aim: to get DNA expression in E. coli.

• Remove chemically competent E. coli (DH5-alpha) cells from the -80°C freezer and thaw on ice.
• Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.
• Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.
• Preheat water bath to 42°C.
• Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).
• Transfer back onto ice for 5 mins.
• Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.
• Spread plate 100 µL onto plates containing the relevant antibiotics* and incubate over night at 37°C.

Aim: to get DNA expression in E.coli.

Remove 50 µL of electrocompetent E.coli cells from the -80°C freezer and thaw on ice.

Add 5 µL of DNA and transfer to a clean 1m elcteroporation cuvette.

Electroporate cells at 1.8kV using a Bio-rad micropulser machine.

Add 500 µL of LB broth to the elctroporated cells and transfer to a clean 1.5 mL Eppendorf tube.

Incubate at 37°C with shaking for 1 hour.

Spread plate 100 µL onto plates containing the relevant antibiotics* and incubate over night at 37°C.

Aim: to get DNA expression in Agrobacterium tumefaciens to allow infiltration into Nicotiana Benthamiana.

Remove 50 µL of electrocompetent Agrobacterium GV3101 cells from the -80°C freezer and thaw on ice.

Add 5 µL of DNA and transfer to a clean 1m electroporation cuvette.

Electroporate cells at 1.8kV using a Bio-rad micropulser machine.

Add 500 µL of LB broth to the elctroporated cells and transfer to a clean 1.5 mL Eppendorf tube.

Incubate at 28°C with shaking for 1 hour.

Spread plate 100 µL onto plates containing the relevant antibiotics* and incubate over night at 28°C.

Aim: to select colonies in which the lacZ has dropped out of the acceptor, indicating it has been replaced by the desired construct.

Spread plate 40 µL of XGAL (40 mg/ml)and 100 µL of IPTG (0.5mM) onto LB agar plates containing the relevant antibiotic*.

Leave to dry before plating 100 µL of E.coli containing the construct of interest.

Incubate at 37°C overnight

Pick white colonies- these should have taken up the plasmid of interest which replaces lacZ

Aim: to determine the size (bp) of the DNA plasmid expressed by the colony picked. This helps us to check that the construct is what we expected.

Pick a single colony and add to a PCR tube containing the following components:

Component	Volume (µL)
dNTPs	2.5
Primer 1 (Sense)	1.0 (10mM stock)
Primer 2 (Antisense)	1.0 (10mM stock)
10x NH4 Reaction Buffer	2.0
MgCl2 Solution (50mM)	2.0
BIOTAQ DNA Polymerase	0.5
Distilled water	6.0

Cycle the PCR mix according to the program detailed below.

Step	Temperature (°C)	Time (minutes: seconds)
1	96	2:00
2*	96	0.30
3*	55	0.30
4*	72	0.30
5	16	∞

*Steps 2-4 cycled x34

Run 15 µL of the PCR product with 2 µL of loading dye on a 1% agarose gel, alongside a 1Kb ladder to determine the size of the DNA within the construct.

Aim

The following protocols were used with QIAGEN QIAprep miniprep kits.

• Create an overnight culture- Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with shaking.
• Centrifuge 1-5ml of overnight culture at >8000 for 3 minutes in an Eppendorf tube to form a bacterial pellet; discard the supernatant.
• Re-suspend bacterial pellet in 250µl of P1 Buffer (kept at <5oC).
• Add 250µl of P2 Buffer and invert 4-6 times to mix thoroughly. This reaction is left for no longer than 5 minutes before completing the next step.
• Add 350µl of N3 Buffer and invert 4-6 times to mix thoroughly.
• Centrifuge for 10 minutes at 13,000rpm
• Decant supernatant into a spin column, centrifuge for 30-60 seconds, and discard the flow-through.
• Add 750µl of PE Buffer to the spin column and centrifuge for 30-60 seconds to wash the DNA. Discard the flow-through and centrifuge for a further 1 minute to remove any remaining buffer.
• Remove the top section of the spin column and place in a clean 1.5ml Eppendorf tube. Add 50µl of EB Buffer to the column; let it stand for 1 minute before centrifuging for a further 1 minute to elute the DNA.

Aim: to sequence our DNA in order to check content of our constructs

• Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes. All DNA concentrations were previously determined using a nanodrop.
• Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl)
• Send off both 10µl samples for sequencing.

Aim: To get expression of our constructs in Nicotiana benthamiana leaves, allowing our system to be tested.

From a single agrobacterium colony or glycerol stock inoculate 5 mL LB media containing the appropriate antibiotic(s)*. Grow overnight at 28 °C, 250-300 rpm.

Pellet by centrifugation at RT (19-23 °C), 4000 rpm for 15 minutes. Resuspend in 2% sucrose, 1% Murashige and Skoog (MS) salt, 150 μM acetosyringone. Incubate at RT (19-23 °C) for at least 3 hours.

Infiltrations are made by injecting the culture to the underside of a leaf with a blunt syringe. Plants are left in a growth room at a temperature around 19-23 °C during co-incubation of the Agro and the plant.

Aim: To analyse the expression of each of our constructs in Nicotiana benthamiana to allow us to see how well our system is working.

We used an epifluorescent microscope to view the results of our infiltrations into Nicotiana benthamiana.

Remove a small section of the leaf from an area that has been infiltrated and place it on a microscope slide with a coverslip over the top.

Place the slide under the epifluorescence microscope, adjusting the focus and light settings accordingly.

Capture relevant images of infiltrated areas of interest.

The following antibiotic concentrations have been used for antibiotic selection throughout the project. Antibiotics have been added to agar plates and overnight cultures in order to select for bacteria carrying the desired plasmid.

Antibiotic	Use	Stock concentration	µL/ mL required
Chloramphenicol	PSB1C3 constructs	33mg/ml	1.5
Kanamycin	Level 0 constructs	30mg/ml	1
Carbenicillin	Level 1 constructs	100mg/ml	1
Ampicillin	Level 1 constructs	100 mg/ml	1
Spectinomycin	Level 2 constructs	100 mg/ml	1
Rifampicin	Agrobacterium tumefaciens	10mg/ml	5
Gentamicin	Agrobacterium tumefaciens	10mg/ml	2