Experiments

Duration

30 minutes sterilization, 30 minutes put the seeds on plates, 1 h transformation, 1,5-2 h examination

Introduction

As a model organism Arabidopsis thaliana Col-0 is and has been well-known and researched and is therefore great to use for a quick estimation of the possible transformation efficiency. We used this organism to create a baseline working with Agrobacterium rhizogenes, to try out our constructs as well as to quickly quantify our transformation efficiency. While we first used dsRed as a marker, we adapted to Ruby, with which we were able to omit the use of a fluorescence microscope and switch to using a light microscope and/or observing the transformation efficiency in our plants by the naked eye.

Material

Sterilization

• 100 Arabidopsis thaliana Col-0 seeds
• 1 1,5 mL reaction tube
• 70% EtOH
• Prepared sodium hypochlorite
• Sterile ddH₂O

Put seeds on plates

• 2 0,5 MS ventilated petri dishes
• small pipette
• sterile small pipette tips

Transformation day

• 4 0,5 MS ventilated petri dishes
• 4 sterile round bleached coffee filters (autoclaved in an aluminum foil bag)
• scalpel
• forceps
• 70% EtOH
• Bunsen burner
• empty sterile petri dish
• 20 mL A. rhizogenes (we used ARqua1 or K599) TY overnight culture with an OD₆₀₀ ~ 1 (with the appropriate antibiotic for selection by antibiotic resistance)

Moving day

• sterile slides
• sterile coverslips
• sterile ddH₂O
• forceps
• 70% EtOH
• bunsen burner
• 5 0,5 MS ventilated petri dishes containing 300 mg/L cefotaxime

Protocol

Sterilization

• To sterilize the seeds they are transferred to a 1.5 mL reaction tube.
• Add 700 µl of 70% ethanol and put on the rotator for 10 minutes.
• Let the seeds sink and remove the ethanol.
• Add 700 µl sodium hypochlorite solution, vortex it and put on the rotator for 5 minutes.
• Remove sodium hypochlorite under the sterile bench and immediately add 1 ml of sterile ddH₂O.
• Remove water and repeat the washing step 3 times, don't remove the water after the last wash.
• Put tubes with sterile seeds in ddH₂O in the fridge at 4 °C for 3-10 days (lasts up to 10 days)

Put seeds on plates after stratification phase (minimum 3 days)

• Place 50 seeds each with a small pipette on 0,5 MS plates containing 1% (w/v) sucrose (25 seeds on one plate) under sterile bench
• Place plates upside down at 21 °C in darkness for 5 days

Transformation day

• Prepare the sterile bench: flame 1 - 2 forceps and a scalpel with the Bunsen burner and EtOH 70% and place them in a sterile petri dish. The lid of the Petri dish serves as a cutting base for the seedlings. Pour the bacteria culture into the empty petri dish as a bath.
• Use the forceps to take the first seedling out of the petri dish and place it on the cutting surface. Cut it at the edge between hypocotyl and root with the scalpel. Repeat this quickly with a few seedlings. Do not leave the cut hypocotyls on the work surface for too long. They dry out very quickly under the sterile bench. It is best to divide a large number of plants into several incubation rounds to reduce stress on the plants.
• Put the already cut seedlings without the roots into the bacteria bath and incubate them there for ~ 7 minutes.
• Place a coffee filter on the 0.5 MS plate with the forceps.
• Carefully remove the plants from the bacteria bath with forceps and place the plants on the 0.5 MS petri dish with a coffee filter.
• Continue in this way until all the plants have been bathed.
• Put the plates in the 22 °C/20°C long day growth chamber for 3 days.

Moving day and first examination

• Prepare the sterile bench: flame 1 - 2 forceps and a scalpel with the Bunsen burner and EtOH 70%.
• Use the forceps to put the seedling on a slide. Drop ddH₂O onto the slide to cover the seedling. Put the coverslip over it.
• Microscope the plants on 100x magnification and evaluate the first transformation results 3 days after transformation.
• Remove the cover glass with the forceps under the sterile bench. Put the seedling gently on a plate with 0,5 MS containing 300 mg/L cefotaxime.
• Let the seedlings grow in the 22 °C/20°C long day growth chamber for 7 days.

Second examination 10 days after transformation

• 10 days after transformation the plants are big enough to evaluate them under a binocular or the bare eye.
• Disinfect a binocular with EtOH 70% under the sterile bench. Flame 2 forceps on the bunsen burner with 70% EtOH. Prepare the plates with the seedlings and new 0.5 MS + cefotaxime plates.
• Place the first petri dish under the bino, open the lid. Examine the seedlings.

Duration

~3 h transformation time

Introduction

With this protocol we started to transform larger agricultural plants, such as Fragaria x ananassa, Taraxacum officinale, and Vigna subterrenea. We aimed to create a Agrobacterium rhizogenes based protocol that makes plant transformation more accessible to workgroups worldwide since it doesn't require a sterile environment but can be performed under non-sterile conditions.

Material

• 20 Plants that were pre-grown in a non-sterile environment. Our plants were grown in the S1 greenhouse, where they also cultivate after transformation, in order to keep the stress of a change of environment as low as possible.
• 400 mL A. rhizogenes (we used ARqua1 or K599) TY overnight culture with an OD₆₀₀ ~ 1 (with the appropriate antibiotic for selection by antibiotic resistance)
• 5 centrifuge tubes
• 100 mM Vanillin stock solution in EtOH
• 20 clean non-sterile flower pots
• 1 tray for the flower pots
• Vermiculite
• Tap water from a watering can
• Non sterile scalpel
• Containers into which the plants fit well during the incubation period, e.g. suitable beakers
• Transparent hood that fits over the pots, e.g. a plexiglas hood or a homemade small greenhouse made of metal sticks and autoclave bags

Protocol

• Transfer a part of the overnight culture to 50 mL centrifuge tubes. Centrifuge the tubes at 4000 xg for 4 minutes until the bacteria have been pelleted. The supernatant is discarded. Fill the tubes again with further overnight culture and resuspend it.
• To this culture 500 uM vanillin is added. The bacteria are incubated by room temperature in vanillin for at least one hour.
• Spill the finished bacteria culture in the beaker.
• Carefully take a plant out of its pot. Remove the root at the "earth edge" between the shoot and the root base with a scalpel. Clean the upper half of the plant with water from possible soil residues.
• Do this swiftly with the other plants.
• Place the rootless plants into the bacterial culture. Allow the plants to incubate in the bacterial culture for 30 minutes.
• Fill the flower pots in the tray with vermiculite. Carefully water the vermiculite with the watering can until the material is evenly soaked.
• After the incubation period, the stump of the plant is carefully put into the vermiculite.
• The hood is put over the tray with the vermiculite pots. The protection remains on the plants for 2 days.

• Depending on the type of plant, the first new roots may appear after about 2 weeks. Before new roots are established, you need to be especially careful watering. Vermiculite retains water very well, and without new roots the plants can quickly rot.
• To examine the cutting sides for new transgenic roots, plants are carefully pulled from the vermiculite after 2 and then after 4 weeks and evaluated.

Duration

~3 h Transformation Day, ~2 h Moving Day

Introduction

We got the lucky opportunity to work with the oaks of the DFG research project "PhytOakmeter". Since the oak clones DF159 are propagated in a sterile environment, we had to design a sterile protocol with which we could transform the oaks via root induction by Agrobacterium rhizogenes. The aim of the protocol is to generate transgenic roots.

Materials

Transformation Day

• 50 circa 8 week old oak cuttings in sterile jars on 0.5 MS medium (no roots, only callus).
• 50 sterile 5 mL reaction tubes
• rack for reaction tubes
• Scalpel
• 2 large forceps
• a sterile base for cutting, e.g. a square petri dish
• 400 mL A. rhizogenes (we used ARqua1 or K599) TY overnight culture with an OD(600) ~ 1 (with the appropriate antibiotic for selection by antibiotic resistance)
• 100 mM Vanillin stock solution in EtOH
• 5 mL pipette
• Electronic pipette controller
• 5 sterile centrifuge tubes
• Bunsen burner
• 70% EtOH
• 10 sterile jars filled with 2 cm 0,5 MS

Moving Day

• large sterile test tubes with dimensions 30x200mm with 30 mL 0,5 MS medium containing 2% charcoal and 500 mg/L cefotaxime
• 2 large forceps
• Bunsen burner
• 70% EtOH
• 2 sterile petri dishes
• Sterile cefotaxime stock solution 150 mg/mL
• Sterile ddH2O

Protocol

Transformation Day

• Treat the sterile bench with UV light and 70% EtOH.
• Transfer a part of the overnight culture to 50 mL centrifuge tubes. Centrifuge the tubes at 4000 xg for 4 minutes until the bacteria have been pelleted. The supernatant is discarded. Fill the tubes again with further overnight culture and resuspend it.
• To this culture 500 uM vanillin is added. The bacteria are incubated in vanillin for at least one hour.
• Burn the forceps and the scalpel with the bunsen burner after washing with 70% EtOH. Place them on the sterile cutting base.
• Put the rack with the 50 reaction tubes under the sterile bench.
• Pipette 4 mL of the prepared bacteria culture in each reaction tube.
• Remove the first oak from the jar with the sterile forceps and place it on the sterile base. The callus is removed with the scalpel. The shoot remains. The oak is placed in a reaction tube with the cutting side down. Proceed quickly in this manner with all further oaks.
• Once all the oaks are in the bacterial medium, turn off the sterile bench and close the window of the sterile bench to reduce the transpiration stress of the oaks.
• The oaks are incubated in the bacteria for 30 minutes.
• The sterile bench is then turned back on and the oaks are placed one at a time in the new sterile jars containing 0,5 MS.
• Put the closed jars back into the growth chamber. The growth conditions are: 25°C, long-day (16 hours (day-)light), ~70µmol light intensity

After 2 days moving day

• Treat the sterile bench with UV light and 70% EtOH.
• Burn the forceps and the scalpel with the bunsen burner after washing with 70% EtOH.
• Fill one petri dish with sterile ddH20. Pour in the second petri dish a cefotaxime bath with 500 mg/L cefotaxime.
• Remove the first oak from the jar with the sterile forceps. Bath the cutting side at first in sterile water. After that, put the cutting side in the cefotaxime bath.
• Place each of the bathed oaks in a large test tube containing 0,5 MS with cefotaxime and charcoal. If possible, insert the shoot into the test tube at an angle so that the cutting side can be seen from the outside. This way the root growth can be observed without taking the oak out of its sterile container.
• Put the closed test tubes into the growth chamber. The growth conditions are: 25°C, long-day (16 hours (day-)light), ~70µmol light intensity.
• Depending on the length of the regeneration phase, growth of transgenic roots is possible after about 3 weeks.

Duration

~3h Transformation time

Introduction

During our work with the non-sterile cut dip bud protocol, we came across an experiment by Sebastian S. Cocioba via social media using rock wool to co-culture bacteria in medium with the plants to be transformed. We picked up this idea and implemented it with our agricultural plants. The protocol is non-sterile and aims to transform plants using Agrobacterium rhizogenes to cultivate new transgenic roots. We tested this protocol with Vigna subterrenea, Fragaria x ananassa, and Taraxacum officinale.

Materials

• 20 Plants that were pre-grown in a non-sterile environment. Our plants were grown in the S1 greenhouse, where they also cultivate after transformation, in order to keep the stress of a change of environment as low as possible.
• 20 non-sterile 50 mL centrifuge tubes with a flat bottom
• 10 normal centrifuge tubes
• Rack for 20 50 mL centrifuge tubes
• Transparent hood that fits over the rack, e.g. a plexiglas hood or a homemade small greenhouse made of metal sticks and autoclave bags
• 20 pre-cut rock wool blocks with dimensions 5x5x5 cm
• New dishcloths
• 1500 mL A. rhizogenes (we used ARqua1 or K599) LB overnight culture with an OD₆₀₀ ~ 1 (with the appropriate antibiotic for selection by antibiotic resistance)
• 2 petri dishes
• 750 mL 0,5 MS Medium
• 100 mM Vanillin stock solution in EtOH
• Scalpel
• Easily disinfectable, clean base (e.g. a planting tray)

Protocol

• Transfer a part of the overnight culture to 50 mL centrifuge tubes. Centrifuge the tubes at 4000 xg for 4 minutes until the bacteria have been pelleted. The supernatant is discarded. Fill the falcons with further overnight culture and centrifuge it again. This procedure is repeated until the bacteria of the 1500 mL overnight culture have been completely harvested.
• Resuspend the bacteria in 750 mL of 0.5 MS medium.
• To this culture 500 uM vanillin is added. The bacteria are incubated by room temperature in vanillin for at least one hour, during which time the falcons can be prepared for transformation.
• Cut the 20 pre-cut rock wool blocks crosswise almost completely. Cut a notch into which you can later insert the stem of the plant.

• The dishcloths are cut into wick-like strips that extend from the center of the rockwool to the bottom in the centrifuge tubes.
• Take off the lids of 20 falcon tubes and fill them with tap water up to about 35 mL.

• Carefully take a plant out of its pot. Remove the root at the "earth edge" between the shoot and the root base with a scalpel. Clean the upper half of the plant with water from possible soil residues.
• Place the open petri dishes on your work base and fill them with the prepared bacterial medium.
• Take a rock wool block and dip it into the Petri dish with bacterial culture until the rock wool is completely soaked with medium. Take a strip of the dishcloth and clamp it into one side of the notch. At the same time, put the shoot of the plant into the other side of the notch. Then carefully put the construct into a centrifuge tube previously filled with water.

• Proceed in this way with the remaining 19 plants until all plants with bacterial medium are in the centrifuge tubes.

• After the plants are transferred to the centrifuge tubes, the tubes are placed in the rack and a hood is put over the rack. The protection remains on the plants for 2 days.
• Depending on the type of plant, the first new roots may appear after about 2 weeks. The falcons must be regularly refilled with fresh tap water.

Duration

1,5 h preparing seeds, 2 h transformation day, 1 h moving day

Introduction

Due to the high relevance in agriculture, we also decided to represent monocots in our project. With the aim to quickly conclude on the transformation efficiency of constructs, we have developed this protocol. With this protocol Setaria sp. can be transformed by Agrobacterium rhizogenes to generate new transgenic roots.

Materials

Preparing Seeds

• 60 Setaria sp. seeds
• 1,5 mL reaction tube
• 70% EtOH
• Prepared sodium hypochlorite
• Sterile ddH₂O
• Pestle and mortar
• Piece of paper
• freezed 1,5 mL reaction tubes with 3,09 mM GA₃ working solution
• KNO₃ stock solution (500 mM KNO₃ in ddH₂O)
• 3 ventilated petri dishes containing 0,5 MS
• Forceps
• Bunsen burner

Transformation day

• 4 ventilated petri dishes with 0,5 MS containing 500 mM vanillin
• Scalpel
• Forceps
• 70% EtOH
• Bunsen burner
• 15 mL A. rhizogenes (we used ARqua1 or K599) TY overnight culture with an OD₆₀₀ ~ 1 (with the appropriate antibiotic for selection by antibiotic resistance)
• 100 mM Vanillin stock solution in EtOH
• 6 sterile 1,5 mL reaction tubes
• 1 empty sterile petri dish
• air-permeable tape to close the petri dishes

Moving day

• 70% EtOH
• Bunsen burner
• 4 ventilated petri dishes with 0,5 MS containing 300 mg/L cefotaxime
• Forceps

Protocol

Preparing Seeds

• To remove the husks of the seeds, place the seeds in the mortar and gently rub them with the pestle for several minutes. Over time, the husks will separate from the actual seed.
• After that, the seeds can be placed in a bowl with a flat surface, or on a sheet of paper. By carefully blowing diagonally across the seeds, the lighter husk residues will detach and be blown away, while the desired seeds will remain on the paper.
• For sterilization of the seeds, they are transferred to a 1.5 mL reaction tube.
• Add 700 µl of 70% ethanol and put on the rotator for 10 min.
• Let the seeds sink and remove the ethanol.
• Add 700 µl sodium hypochlorite solution, vortex it and put on the rotator for 20 min.
• Remove sodium hypochlorite under the sterile bench and immediately add 1 ml of sterile ddH₂O.
• Remove water and repeat the washing step 5 times.

We performed the next two steps for germination induction exclusively on Setaria viridis. In the Setaria species unknown to us, which we subsequently tried with this protocol, germination started significantly faster without gibberellic acid treatment. Thus, it depends on the species whether a gibberellic acid treatment is necessary or not.

• Add 1,41 mL 3,09 mM gibberellic acid solution and 90 uL 500 mM KNO₃ solution to the reaction tube to get a working solution with 2,9 mM gibberellic acid and 30 mM KNO₃
• Put the seeds for 24 h in a 28°C incubator.

If the seeding is not treated with gibberellic acid, the seeds can be plated out immediately after sterilization.

• Sterilize the forceps with the bunsen burner: Dip the tip in EtOH and carefully flame it.
• Use forceps to spread 20 seeds on a 0.5 MS petri dish. The petri dishes are sealed with air-permeable tape and incubated at 26°C/18°C long day in the growth cabinet.

Transformation Day

Germination time varies within Setaria species. The seedlings are transformable after 1-2 weeks, when they are about 2 cm tall and the root can be easily removed with a scalpel.

• Pipette 1.5 mL of overnight bacteria culture into each of the 6 1.5 mL reaction tubes. Centrifuge the bacteria down until a pellet separates. Discard the supernatant. Then resuspend the pellet in 1.5 mL of the remaining bacterial culture.
• Add 500 uM vanillin from the -20 °C freezed vanillin stock solution and let the bacteria incubate at room temperature for 1 hour.
• Prepare the sterile bench: flame 1 - 2 forceps and a scalpel with the bunsen burner and EtOH 70% and place them in a sterile petri dish. The lid of the Petri dish serves as a cutting base for the seedlings.
• Use the forceps to take the first seedling out of the petri dish and place it on the cutting surface. Cut it at the edge between shoot and root with the scalpel. Repeat this quickly with the remaining seedlings.
• Put 10 of the already cut seedlings into each bacteria-filled reaction tube so that the cutting side of the shoot is well covered with culture.
• Let the plants incubate for 15 minutes.
• After incubation, distribute the seedlings on 0.5 MS petri dishes with vanillin.
• Put the plates in the 26°C/18°C long day growth chamber for 3 days.

Moving Day

• Disinfect a binocular with EtOH 70% under the sterile bench. Flame 2 forceps on the bunsen burner with 70% EtOH. Prepare the plates with the seedlings and new 0.5 MS + cefotaxime plates.
• Place the first petri dish under the bino, open the lid. Examine the seedlings.
• Place the plants on the new petri dishes with forceps. Put the plates in the 26°C/18°C long day growth chamber for another week.

A second examination of the potentially transgenic tissue can be performed after another week.

Duration

36 hours

Purpose

Analysis of natural resistance of Agrobacterium to commonly used antibiotics.

Material

• Agrobacterium strains of interest
• LB medium
• Antibiotics of interest
• 96-well plate (clear)
• Plate reader

Protocol

• Prepare precultures of Agrobacterium strains of interest and let them grow overnight in LB without antibiotics.
• Prepare 96-well plate with LB media containing different antibiotic concentrations of antibiotics of interest. Fill wells with plain LB medium for negative and positive controls.
• Add Agrobacterium culture to wells to create an OD₆₀₀ of 0.1. Do not add any Agrobacterium culture to half of the wells filled with plain LB medium to obtain negative controls.
• Incubate plate for 24 h shaking at 28°C.
• Measure OD_{600 of the plate in a plate reader to receive results.}

Duration

3 days

Purpose

Measurement of the strength of Anderson promoters in Agrobacterium.

Construct Design

All constructs contained a pABCa backbone and a lux operon which lays under control of different Anderson promoters. Each construct contained the same ribosome binding site.

Material

• MOPS-1C minimal medium
• 3 96-well plates (clear)
• 96-well plate (white or black with clear bottom)
• LB medium
• Plate reader
• Required antibiotics

Protocol

• Fill each well of a 96-well plate (clear) with 200 µl LB medium and required antibiotics.
• Inoculate wells with your bacterial strains containing the test construct of interest. We used different Anderson promoters that regulated the expression of luminescence. Do at least biological triplicates for each sample and provide proper controls. As controls we provided three blank wells just containing LB medium and a construct containing a dummy promoter.
• Seal plate and incubate plate overnight at 28°C shaking. Cells should be able to grow to the stationary phase.
• Load each well of a new 96-well plate (clear) with 195 µl of MOPS-1C medium and required antibiotics.
• Inoculate the 96-well plate containing MOPS-1C medium with 5 µl of the overnight culture
• Seal plate and incubate at 28°C overnight shaking.
• Measure OD₆₀₀ in a plate reader and dilute all cultures to an OD600 of 0.05 in fresh MOPS-1C medium. The dilution is done in a black or white 96-well plate with a clear bottom.
• Start luminescence measurement in a plate reader for 24 h to receive results.

Duration

3 days

Purpose

Measurement of the strength of inducible promoters in Agrobacterium.

Construct Design

All constructs contained a pABCa backbone and a lux operon which lays under control of different inducible promoters. Each construct contained the same ribosome binding site.

Material

• MOPS-1C minimal medium
• 3 96-well plates (clear)
• 96-well plate (white or black with clear bottom)
• LB medium
• Plate reader
• Required antibiotics
• Required inducers

Protocol

• Fill each well of a 96-well plate (clear) with 200 µl LB medium and required antibiotics.
• Inoculate wells with your bacterial strains containing the test construct of interest. We used different inducible promoters that regulated the expression of luminescence. Do at least biological triplicates for each sample and provide proper controls. As controls we provided three blank wells just containing LB medium and a construct containing a dummy promoter.
• Seal plate and incubate plate overnight at 28°C shaking. Cells should be able to grow to the stationary phase.
• Load each well of a new 96-well plate (clear) with 195 µl of MOPS-1C medium and required antibiotics.
• Inoculate the 96-well plate containing MOPS-1C medium with 5 µl of the overnight culture
• Seal plate and incubate at 28 °C overnight shaking.
• Measure OD₆₀₀ in a plate reader and dilute all cultures to an OD600 of 0.05 in fresh MOPS-1C medium. The dilution is done in a black or white 96-well plate with a clear bottom.
• Start luminescence measurement in a plate reader.
• After 4 hours add appropriate inducers to the samples. To determine crosstalk use inducers of interest to induce samples.
• Continue luminescence measurement in a plate reader for 20 h to receive results.

Duration

3 days

Purpose

Measurement of the strength of ribosome binding sites in Agrobacterium.

Construct Design

All constructs contained a pSRKa backbone and staygold as fluorescence reporter which lays under control of the constitutive Anderson promoter P_J23109. Each construct contained the TB0015 terminator from the Marburg Collection.

Material

• MOPS-1C minimal medium
• 3x 96-well plates (clear)
• 96-well plate (white or black with clear bottom)
• LB medium
• Plate reader
• Required antibiotics

Protocol

• Fill each well of a 96-well plate (clear) with 200 µl LB medium and required antibiotics.
• Inoculate wells with your bacterial strains containing the test construct of interest. We used different ribosome bindings sites that regulated the expression of fluorescence via staygold. Do at least biological triplicates for each sample and provide proper controls. As controls we provided three blank wells just containing LB medium and a construct containing a dummy ribosome binding site.
• Seal plate and incubate plate overnight at 28°C shaking. Cells should be able to grow to the stationary phase.
• Load each well of a new 96-well plate (clear) with 195 µl of MOPS-1C medium and required antibiotics.
• Inoculate the 96-well plate containing MOPS-1C medium with 5 µl of the overnight culture
• Seal plate and incubate at 28 °C overnight shaking.
• Measure OD₆₀₀ in a plate reader and dilute all cultures to an OD₆₀₀ of 0.05 in fresh MOPS-1C medium. The dilution is done in a black or white 96-well plate with a clear bottom.
• Start fluorescent measurement in a plate reader for 24 h to receive results.

Duration

3 days

Purpose

Measurement of the reporters in Agrobacterium.

Construct Design

All constructs contain a pSRKa backbone and the Anderson promoter PJ23102. Each construct contained the B0015 terminator from the Marburg Collection and the same ribosome binding site. Constructs diverged in the used fluorescence or luminescence reporter to test.

Material

• MOPS-1C minimal medium
• 3 96-well plates (clear)
• 96-well plate (white or black with clear bottom)
• LB medium
• Plate reader
• Required antibiotics

Protocol

• Fill each well of a 96-well plate (clear) with 200 µl LB medium and required antibiotics.
• Inoculate wells with your bacterial strains containing the test construct of interest. We used different reporters that are equally regulated and expressed. Do at least biological triplicates for each sample and provide proper controls. As controls we provided three blank wells just containing LB medium and a construct containing a dummy ribosome binding site.
• Seal plate and incubate plate overnight at 28°C shaking. Cells should be able to grow to the stationary phase.
• Load each well of a new 96-well plate (clear) with 195 µl of MOPS-1C medium and required antibiotics.
• Inoculate the 96-well plate containing MOPS-1C medium with 5 µl of the ON culture
• Seal plate and incubate at 28°C overnight shaking.
• Measure OD₆₀₀ in a plate reader. The measurement is done in a black or white 96-well plate with a clear bottom.
• Start fluorescent measurement in a plate reader as a point measurement to receive results.
• Add 50 µl of substrate for the luminescent Reporters to 50 µl of culture.
• Wait 3 minutes after the add-on of the substrate.
• Start luminescent measurement in a plate reader as a point measurement to receive results.

Purpose

Production of competent Agrobacterium cells to receive the created plasmid constructs to test their effects on plant transformation efficiency.

Material

• 1 day old LB liquid culture grown at 28°C of chosen Agrobacterium strain
• LB agar plates containing required antibiotics
• 10% (v/v) glycerol
• 2 ml centrifuge tubes
• liquid nitrogen

Protocol

• Spread freshly growing Agrobacterium cells (1-2 days old) on LB agar plates with required antibiotics.
• Incubate plates overnight at 28°C. The bacterial lawn should cover the whole surface of the plate.
• Wash bacterial cells off the plate using 4 ml ice cold 10% glycerol. Use an inoculation loop to scrape cells from agar and suspend in glycerol solution.
• Transfer solution in two 2 ml centrifuge tubes and centrifuge at 14 000 rpm for 1 min at 4°C.
• Discard supernatant and resuspend each pellet in 1 ml ice cold 10% glycerol.
• Centrifuge tubes at 14 000 rpm for 1 min at 4°C and discard supernatant.
• Resuspend each pellet in 200 µl ice cold 10% glycerol and combine both solutions in one tube.
• Aliquot cells to 80 µl and flash freeze in liquid nitrogen.
• Store cells at - 80°C.

Purpose

Production of competent Agrobacterium cells to receive the created plasmid constructs to test their effects on plant transformation efficiency. This protocol was used to save time, since it requires shorter incubation of Agrobacterium.

Material

• 10 ml of 1 day old LB liquid culture grown at 28°C of chosen Agrobacterium strain
• ddH₂O
• 10% (v/v) glycerol
• liquid nitrogen

Protocol

• Set up fresh culture with required antibiotics from 10 ml preculture to an OD₆₀₀=0.1.
• Grow cells until an OD₆₀₀=0.5-1.
• Incubate cells on ice for 15-30 min.
• Divide culture into two falcon tubes and centrifuge at 4 000 g for 15 min at 4°C.
• Discard supernatant and resuspend each pellet in 45 ml ice cold water.
• Centrifuge tubes at 4 000 g for 15 min at 4°C and discard supernatant.
• Resuspend each pellet in 20 ml ice cold water and centrifuge at 4 000 g for 15 min at 4°C.
• Discard supernatant and resuspend each pellet in 10 ml ice cold 10% glycerol.
• Combine both solutions into one falcon tube and centrifuge at 4 000 g for 15 min at 4°C.
• Discard supernatant and resuspend pellet in 2 ml ice cold 10% glycerol.
• Aliquot cells to 80 µl and flash freeze in liquid nitrogen.
• Store cells at - 80°C.

Purpose

Transform Agrobacterium with a plasmid of choice.

Material

• LB plates with antibiotics
• LB liquid medium
• Electroporation cuvettes
• Electroporator

Protocol

• Use 30-50 µl of ice cold suspension of electrocompetent bacterial cells.
• Mix with 1-4 µl plasmid DNA (1-100 ng) in a sterile centrifuge tube.
• Load mixture into chilled electroporation cuvette (gap=2mm).
• Set electroporation parameters to 2.5 kV, 25 μF capacitance, and 400 Ohm resistance.
• Place electroporation cuvette into cuvette holder and electroporate.
• Add immediately 1 ml LB medium to electroporated cell suspension.
• Transfer suspension to 1.5 ml centrifuge tube.
• Incubate tube at 28°C with rotating (1 h - 3 h).
• Plate 60 µl of each suspension onto LB plates with required antibiotics.
• Incubate for two days at 28°C.
• Verify colonies by colony PCR.

Purpose

Preparation of chemically competent E. coli cells, to introduce plasmids via heat shock transformation.

Material

• Mg2+/Ca2+
• 3.25 g MgCl₂ * 6 H₂O
• 0.6 g CaCl₂ * 2 H₂O
• 200 ml ddH₂O
• Autoclave
• CaCl₂ (100 mM)
• 2.95 g CaCl₂ * H₂O
• 200 ml ddH₂O
• Autoclave
• 80% Glycerol
• LB medium

Protocol

• Day 1: Grow E. coli DH5a overnight in 5 ml LB at 37°C.
• Day 2: Inoculate 100 ml LB with 1 ml of saturated overnight culture of E. coli DH5a cells.
• Incubate at 37°C and 150 rpm until an OD₆₀₀ 0.4- 0.6 is reached (usually 2-3h).
• Incubate on ice for 5 min, keep cells chilled for the rest of the protocol.
• Divide culture into 2 tubes with ~ 40 ml each.
• Centrifuge the cultures at 4°C and 3000 g for 10 min.
• Gently resuspend each pellet with 15 ml of ice cold Mg²⁺/Ca²⁺ solution (Do not vortex!).
• Incubate on ice for 30 min.
• Centrifuge the culture at 4°C and 3000 g for 10 min.
• Resuspend each pellet in 1.6 ml of ice cold 100 mM CaCl₂ solution.
• Incubate on ice for 20 min.
• Combine cells to one tube.
• Add 0.5 ml cold 80% glycerol and swirl to mix.
• Flash freeze in liquid nitrogen a 100 µl aliquots.
• Store at -80°C.

Purpose

Transform E. coli with a plasmid of choice.

Material

• LB medium
• LB agar plates with antibiotics
• Heat block

Protocol

• Defrost stocks of competent cells (100 µl in 1.5 ml reaction tube) on ice.
• Add DNA (2-6 µl, ~ 100 ng) and incubate the suspension for 30 min on ice.
• Heat shock is done by incubating the cells for 45 seconds at 42°C.
• Put samples back on ice for 2 min.
• Add 1 ml of LB medium and incubate for 1 hour at 37°C shaking in order to obtain antibiotic resistance.
• Spread out 100 µl on agar plates with required antibiotics.
• It might be useful to spin down cells at 5000 rpm for 5 minutes.
• Resuspend pellet in 100 µl LB.
• Spread out cells on agar plate with required antibiotic resistance.

Purpose

For efficient Golden Gate assembly of lvl0 and lvl1/2 constructs.

Material

• T4 DNA Ligase (NEB®)
• T4 DNA Ligase Buffer (NEB®)
• BsmbI-v2 (NEB®)
• BsaI (NEB®)

Protocol

Lvl 0 cloning

Reagent	Volume
DNA insert	variable (100 ng)
Entry vector (25 ng/μL)	1 μl
T4 DNA Ligase Buffer	1 μl
T4 DNA Ligase	1 μl
BsmBI	0.5 μl
ddH2O (nuclease free)	ad to 10 μl

Lvl 1/2 cloning

Reagent	Volume
Lvl 0/1 parts (20 fmol)	0.5 µl
T4 DNA Ligase Buffer	1 μl
T4 DNA Ligase	1 μl
BsaI	0,5 µl
BsmBI	0.5 μl
ddH2O (nuclease free)	ad to 10 μl

BsmBI is used for Lvl 0 and Lvl 2 reactions. BsaI used for Lvl 1 reactions.

Thermocycler conditions

Temperature	Time	Cycles
37°C	2 min	50x
16°C	5 min
50°C	10 min	1x
80°C	10 min	1x
10°C	Hold	∞

Purpose

Plasmid assembly.

Material

• Isothermal reaction buffer (5x)
• 25% PEG - 8000
• 500 mM Tris-HCl ph7.5
• 50 mM MgCl₂
• 50mM DTT
• 1 mM each of the 4 dNTPs
• 5 mM NAD

Protocol

Preparation of 5x isothermal buffer

Reagent	Volume
1 M Tris-HCl H 7.5	3 mL
1 M MgCl2	300 µl
100 mM dATP	60 µl
100 mM dCTP	60 µl
100 mM dGTP	60 µl
100 mM dTTP	60 µl
1 M Tris-HCl H 7.5	3 mL
1 M DTT	300 µl
PEG 8000	1.5 g
100 mM NAD	300 µl
dH2O	fill up to 6 mL

Prepare the Master mix as follows

• Thaw one 400 µl aliquot of 5 x isothermal reaction buffer on ice.
• Add 874 µl of ddH₂O.
• Add 1 µl T5 Exonuclease (NEB M0363S).
• Add 25 µl Q5 Polymerase (NEB M0491L).
• Add 200 µl Taq Ligase (NEB M0208L).
• Mix carefully and throughout by pipetting up and down.
• Briefly vortex.
• Spin down.
• Generate 1.5 µl aliquots into 8-fold PCR strips.
• Store in the communal -20°C freezer.
• Perform Quality control of one reaction and document this in the batch control page.

Gibson assembly and transformation

• Thaw one 15 µl aliquot per reaction and include a negative/positive control if necessary.
• Add up to 5 µl DNA and fill up to total volume with dH₂O if necessary.
• Note: DNA should be in equimolar amounts, and a maximum combined DNA amount of 1 pmol is suggested.
• Incubate at 50°C for 1 h in a thermocycler.

Purpose

Amplification of DNA fragments, restriction site removal.

Material

• Thermocycler
• repliQa HiFi ToughMix (QuantaBio)

Protocol

Reaction assembly

Component	Reaction volume for 50 µl	Final concentration
repliQa HiFi ToughMix (2x)	25 µl	1x
Forward Primer	variable	300 nM
Reverse Primer	variable	300 nM
Template	2 - 5 µl	variable
ddH2O (nuclease free)	add to 50 µl

Thermocycler Program

Temperature	Time	Cycles
98°C	10 s	24 - 45
(TM) -5°C	5 s	24 - 45
68°C	5s/kb	24 - 45

Purpose

Verification of presents of desired plasmid.

Material

• Thermocycler
• OneTaq Mastermix (NEB®)

Protocol

• Pick and resuspend a single colony in 20μl ddH₂O and boil it for 10 minutes at 99°C.
• Spin down the solution at 11.000 rpm for 2 min.
• Prepare the reaction mix as follows using the OneTaq MM protocol.
• PCR Master Mix (2X) and 1μl of supernatant as template.
• Thaw and briefly vortex the Taq Master Mix (2X).
• Add reagents on ice.

Component	Reaction volume for 25 µl	Final concentration
One Taq Mastermix	12.5 µl	1x
Forward Primer	0.5 µl	10 µM
Reverse Primer	0.5 µl	10 µM
Template	1 µl	variable
ddH2O (nuclease free)	add to 25 µl

Thermocycler Program

Temperature	Time	Cycles
95°C	30 s	30x
(TM) -5°C	15-60 s	30x
68°C	1 min/kb

Purpose

Verification of the presence of a desired DNA fragment after restriction enzyme digestion or PCR amplification. This method was also used to free PCR samples from template DNA.

Material

• Agarose
• 1% TAE buffer
• Midori Green DNA stain
• DNA ladder
• Loading Dye

Protocol

• Weigh out agarose and solve in 1% TAE buffer by heating up until the solution is completely clear. We prepared 1% agarose gels by dissolving 1 g of agarose in 100 ml TAE buffer.
• After the solution has cooled down to approximately 60 °C, add 4 μl of Midori Green DNA stain when preparing a volume of 100 ml. Pour solution in gel chamber and add combs with desired size to the gel and let gel dry. Pour TAE buffer in gel chamber until the whole gel is covered sufficiently and remove gel combs.
• Add DNA ladder as well as the samples to the gel pockets. If not included yet, add loading dye to samples before loading the gel.
• Connect the gel chamber to power and run gel for 40 - 60 min at 100 V Evaluate the gel in a gel imager.
• If necessary, excise desired DNA bands under UV light.

Purpose

Long-term storing of bacterial strains. Those strains may carry plasmid constructs of interest.

Material

• Bacterial culture of interest
• 80% (v/v) glycerol
• Cryostock tubes
• Liquid nitrogen

Protocol

• Prepare overnight culture of bacterial strain of interest.
• Gently mix 500 µl 80% glycerol and 500 µl of the freshly grown overnight culture in a cryostock tube.
• Flash freeze stock culture in liquid nitrogen.

Material

• Yeast extract
• NaCl
• Tryptone
• Agar-agar, if LB agar is prepared

Protocol

• Dissolve
• 5 g Yeast extract,
• 5 g NaCl and
• 10 g Tryptone in 1 l ddH₂O. To prepare LB agar add 15 g of agar-agar to 1 l of LB medium.
• Autoclave the medium.

Material

• Yeast extract
• Tryptone
• CaCl₂ x H₂O

Protocol

• Dissolve
• 3 g Yeast extract,
• 5 g Tryptone and
• 0.7 g CaCl₂ x H₂O in 1 l ddH₂O.
• Adjust the pH-value to 6.6The pH-value. To prepare TY agar add 15 g of agar-agar to 1 l of TY medium.
• Autoclave the medium.

Material

• 0.05% (w/v) MES
• 1% (w/v) Saccharose
• 0.22% (w/v) Murashige & Skoog medium (MS)
• 0.8% (w/v) plant agar
• KOH
• required antibiotics or inducer

Protocol

• Dissolve
• 0.2 g MES,
• 2 g Saccharose,
• 0.88 g MS and
• 3.2 g plant agar in 400 ml ddH₂O.
• Adjust pH to 5.6 using KOH.
• Autoclave medium.

Material

• MOPS
• Mannitol
• (NH₄)₂SO₄
• MgSO₄
• KOH
• CaCl₂ x 2 H₂O
• FeCl₃ x 6 H₂O
• Biotin
• Thiamin-HCl
• Pantothenate
• H₃BO₃
• MnSO₄ x 4 H₂O
• ZnSO₄ x 7 H₂O
• CuSO₄ x 5 H₂O
• CoCl₂ x 6 H₂O
• NaMoO₄ x 2 H₂O
• K₂HPO₄ x 3 H₂O

Protocol

• Prepare 1 l basic solution by dissolving
• 10 g MOPS,
• 10 g Mannitol,
• 1.05 g (NH₄)₂SO4 and
• 0.12 g MgSO₄ in 1 l ddH₂O.
• Adjust the pH-value of the basic solution to 7.2 and autoclave.
• Prepare vitamin mix by dissolving
• 0.1 mg Biotin,
• 0.1 mg Thiamin-HCl and
• 0.1 mg Pantothenate in 1 ml ddH₂O.
• Sterilize vitamin mix by filtration (diameter ≤ 0.45 µm).
• Prepare 50x oligo element solution by dissolving
• 3.75 g H₃BO₃,
• 2.788g MnSO₄ x 4 H₂O,
• 0.359 g ZnSO₄ x 7 H₂O,
• 0.1563 g CuSO₄ x 5 H₂O,
• 0.0813 g CoCl₂ x 6 H₂O and
• 0.15 g NaMoO₄ x 2 H₂O in 250 ml ddH₂O.
• Sterilize 50x oligo element solution by filtration (diameter ≤ 0.45 µg)
• Add
• 1 ml CaCl₂ x 2 H₂O,
• 1 ml FeCl₃ x 6 H₂O,
• 1 ml vitamin mix,
• 0.2 ml 50x oligo element solution and
• 2 ml K₂HPO₄ x 3 H₂O to the autoclaved basic solution.
• Store all solutions at 4 °C.

Material

• ddH₂O
• Sodium hypochlorite
• Triton X-100

Protocol

Dissolve 2.85 ml Sodium hypochlorite and 0.15 ml Triton X-100 in 12 ml ddH₂O.

Material

• ddH₂O
• 95% ethanol
• Ampicillin
• Spectinomycin
• Streptomycin
• Gentamicin
• Chloramphenicol
• Kanamycin
• Hygromycin
• Tetracycline

Protocol

• Dissolve the stated amount of antibiotic in the specified solution:

Antibiotic	Stock concentration	Solution
Ampicillin	100 mg/ml	ddH2O
Spectinomycin	50 mg/ml	ddH2O
Streptomycin	300 mg/ml	ddH2O
Gentamicin	10 mg/ml	ddH2O
Chloramphenicol	40 mg/ml	95% ethanol
Kanamycin	100 mg/ml	ddH2O
Hygromycin	50 mg/ml	ddH2O
Tetracycline	15 mg/ml	1:1 ddH2O 95% ethanol
Cefotaxim	150 mg/ml	ddH2O

• Sterilize antibiotics that have been dissolved in ddH₂O or partly in ddH₂O by filtration (diameter = 0.22 µm).
• Aliquot antibiotics to 1 ml and store at - 20°C.

Material

• IPTG
• ddH₂O

Protocol

• Dissolve 0.595 g IPTG in 25 ml ddH₂O to receive a stock concentration of 250 mM.
• Sterilize solution by filtration (diameter = 0.22 µm).
• Aliquot solution if desired and store at - 20°C.

Material

• Vanillin
• Taurin
• IPTG

Protocol

• Dissolve the stated amount of antibiotic in the specified solution:

Inducer	Stock concentration	Solution
Vanilin	100 mM	Ethanol
Taurine	null	ddH2O
IPTG	null	ddH2O
Naringenin	250 mM	DSMO
Cuminic acid	0.1 M	Ethanol
Sodium salicylate	100 mM	ddH2O
Anhydrotetracycline	100 mM	1:1 ddH2O ethanol
DHBA	1 mM	Ethanol
Choline Chloride	100 mM	ddH2O
Arabinose	100 mM	ddH2O

• Sterilize antibiotics that have been dissolved in ddH₂O or partly in ddH₂O by filtration (diameter = 0.22 µm).
• Aliquot antibiotics and inducers to 1 ml and store at - 20°C.