iGEM 2023 — Team marburg

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{% block title %}Notebook{% endblock %}
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{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## Plant Lab

Plant Lab Book 1 2023-02-21 - 2023-05-03
Plant Lab Book 2 2023-05-03 - 2023-08-23
Plant Lab Book 3 2023-08-20 - 2023-10-31
*Arabidopsis* Transformation
*Setaria sp.* Transformation
Oak Transformation

## Engineering

Engineering Lab Book
*Agrobacterium* antibiotic test in liquid media
*Agrobacterium* Hannover 36h flask test
Colony PCR DsRedRoot
Cultivation Tests
PRKS and pABCa stability
Reporter
RUBY recloning protocol
super vir G expression
virB and virG
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{% block title %}Experiments{% endblock %}
{% block hero\_image\_url %}https://static.igem.wiki/teams/4729/wiki/experiments-protocols/bg-hero-experiments.jpg{% endblock %}
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## Plant Protocols

Sterile *Agrobacterium rhizogenes* mediated *Arabidopsis thaliana* protocol

![graphical abstract of the protocol.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/sterile-agrobac-protocol.png)

#### 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.

![You can see half of an open petri dish with bleached round filter paper on the medium. On the plate are 10 0,5 - 1 cm large plants with 2 small green leafs. One hypocotyl has a red color seeable with the bare eye.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/positive-plant-on-plate.jpg)

##### 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.

Unsterile adapted cut-dip-budding protocol via *Agrobacterium rhizogenes*

![graphical abstract of the protocol.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/cdb-protocol-neu.png)

#### 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.
![It's a picture of a big table in the greenhouse. There are 6 trays full of plants on the table and three selfmade table greenhouses made with autoclave bags.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/cdb/le8.jpg)* 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.
![Bird's eye view of a table on which lies a sign with the inscription: If the duck floats, then it is too wet for the plants. Please water carefully! Next to the sign is a plant tray in which sits a small yellow squeaking duck.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/cdb/le7.jpg)

Sterile *Agrobacterium rhizogenes* mediated *Quercus robur* protocol

![graphical abstract of the protocol.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/oak-protocol.png)

#### 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

![small jar with 6 six oaks sitting on white medium.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/quercus/le6.jpg)

#### 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

![Bird's eye view of the sterile workstation on the sterile bench. On the left, you can see a glass jar with oak trees, and in the middle, open petri dishes that are used as a sterile cutting surface. On the far right is a rack with many 5 mL reaction tubes and already some oaks sitting in the tubes.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/quercus/le5.jpg)

##### 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.

![view of the sterile work station for moving the oaks. At first you can see a big jar with oaks. After that are two petri dishes filled first with sterile water and than with cefotaxime solution. Behind that are large test tubes containing dark medium.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/quercus/le7.jpg)

Unsterile *Agrobacterium rhizogenes* mediated protocol with tube + rock wool system

![graphical abstract of the protocol.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/stonewool-protocol.png)

#### 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.
![In the foreground of the picture you can see almost 30 stone wool blocks. The blocks are cut crosswise and lie on a cloth. In the background you can see a Bambara Groundnut plant in a pot.  ](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/tube-rock-wool-system/le1.jpg)* 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.
![Workplace for the transformation: Approximately 30 rock wool blocks that have already been cut into, to the right of the picture is a jar with cut dishcloths. On the left are centrifuge tubes filled with water and some pots with plants in them.  ](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/tube-rock-wool-system/le2.jpg)* 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.
![there is a plant tray in the centre of the picture. You can see two hands in gloves which put the plant rock wool bacteria construct into the tube.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/tube-rock-wool-system/le3.jpg)* Proceed in this way with the remaining 19 plants until all plants with bacterial medium are in the centrifuge tubes.
![in the foreground of the picture is a bambara groundnut ready in the tube construct with green stone wool and a pink dish cloth in it. In the background is a whole rack full of tube constructs with bambara groundnuts.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/tube-rock-wool-system/le4.jpg)* 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.

Sterile *Agrobacterium rhizogenes* mediated *Setaria sp.* Protocol

#### 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.

## Characterization Protocols

Antibiotic resistances in *Agrobacterium*

#### 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 OD600 of the plate in a plate reader to receive results.

Characterization of constitutive promoters

#### 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

![The illustration shows the workflow of constitutive promoter characterisation.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/characterisation-workflow-figure-wiederhergestellt-constitutive.png)

#### 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.

Characterization of inducible promoters

#### 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

![The illustration shows the workflow of inducible promoter characterisation.](https://static.igem.wiki/teams/4729/wiki/experiments-protocols/characterisation-workflow-figure-wiederhergestellt-inducible.png)

#### 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.

Characterization of ribosome binding sites

#### 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.

Characterization of reporters

#### 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.

## Engineering Protocols

Electrocompetent *Agrobacterium* Cells - Volume 1 (after Tóth et al., 2018)

#### 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.

Electrocompetent *Agrobacterium* Cells - Volume 2

#### 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.

*Agrobacterium* Electroporation

#### 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.

Chemically competent *Escherichia coli* DH5a cells

#### 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.

Heat shock transformation of *Escherichia coli*

#### 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.

Golden Gate reaction protocol

#### 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 | ∞ |

Gibson Assembly

#### 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.

PCR

#### 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 |

Colony PCR for *Escherichia coli* and *Agrobacterium rhizogenes*

#### 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 |  |

Agarose gel electrophoresis

#### 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.

Cryostock preparation

#### 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.

## Media and Buffer Protocols

LB (Luria Bertani) Medium

#### 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.

TY (Tryptone + Yeast extract) 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.

MS 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.

MOPS- 1C Minimal 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)2SO4and
+ 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.

Sodium hypochlorite

#### 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.

Antibiotic Stock Solutions

#### 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.

IPTG

#### 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.

Inducer Stock Solutions

#### 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.

{% endblock %}

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{% block title %}Results{% endblock %}
{% block hero\_image\_url %}https://static.igem.wiki/teams/4729/wiki/ruby-and-hairy-ugly-root-1.jpg{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## Antibiotic sensitivity assay

Engineering work normally relies on selection for a desired genotype by antibiotic resistance. *Agrobacterium* has been found to show a quite high natural resistance to some antibiotics (Khafagi et al., 2012), however detailed information for specific strains are often unclear. Therefore we tested the *Agrobacterium* strains used in this project to their natural sensitivity to various antibiotics in the absence of antibiotic resistance genes.

### Results

The antibiotic sensitivity assay performed with *A. rhizogenes* ARqua1 and *A. rhizogenes* K599 showed that both strains show natural tolerance to some antibiotics.

![Antibiotic concentration test in A.rhizogenes, displayed as a lolipop chart](https://static.igem.wiki/teams/4729/wiki/results/antibiotics-sensitivity-assay.png)

**Figure 1**: Wild type *Agrobacterium* strains used in this project were tested for their natural sensitivity to various antibiotics.

Our results show sensitivity of both strains to ampicillin, chloramphenicol and tetracycline, with *A. rhizogenes* K599 being more sensitive to those antibiotics. *A. rhizogenes* ARqua1 has been reported as a streptomycin-resistant derivative of the R1000 strain (Thompson et al., 2020), which also fits our observations. Surprisingly, the K599 strain was not resistant to streptomycin as reported by Alzohairy et al., 2013, but tolerant to spectinomycin. Whereas ARqua1 strain showed to be resistant to high concentrations of streptomycin but not to spectinomycin. When culturing *Agrobacterium* in the lab, we therefore used streptomycin to select for the native ARqua1 strain and spectinomycin to select for the native K599 strain. During our project, we introduced the 35S:RUBY plasmid into both strains with the purpose to identify successfully transformed plants. This plasmid carries a bacterial antibiotic marker that allows selection for this plasmid using streptomycin or spectinomycin. In view of the natural sensitivity of the used *A. rhizogenes* strains, we used the respective other antibiotic. Further tested antibiotics not listed in figure 1 were found to be usable for selection of a desired plasmid carrying the respective antibiotic resistance gene. These antibiotics include gentamicin, hygromycin and kanamycin. Whenever we introduced a second plasmid into either *Agrobacterium* strain, we used plasmids with a pABCa or pSRK backbone, that hold a gentamicin resistance and therefore allowed us a reliable selection for the introduced plasmid.

## Part characterization

One of the main challenges when creating SynBio tools outside the classic prokaryotic model organisms is the lack of thorough characterization of basic parts. This leads many researchers to rely on *E. coli* part characterization data, despite the fact that they do not always constitute the optimal tool for the task at hand. In order to avoid this problem, our team started from the beginning of our project to work on characterizing basic parts that could be used in our target organism *Agrobacterium*.

The characterization construct expressing the LuxABCDE reporter used for our experiments is harbored in a pABCa plasmid backbone. This vector was originally created for expression in *Sinorhizobium meliloti*, and incorporates a single copy repABC-type origin of replication (Döhlemann et al., 2017). The version used by our team was further adapted by one of our PI’s lab to be compatible with Golden Gate assembly (Meier et al., 2023). The assembled plasmids for the promoter characterization were used by one of our PI’s lab in different experiments and kindly provided to us (Seiler, 2023).

### Anderson promoters

#### Purpose and design

The Anderson promoter collection has been thoroughly characterized in the chassis most prevalent in iGEM, such as *E. coli*. For this reason, the collection is extensively used as a standard for constitutive expression in prokaryotes. However, little to no data is available when it comes to non-model prokaryotic organisms. Therefore, our team set out to characterizing their expression strength in *Agrobacterium rhizogenes* strain ARqua1, which was used for most of our plant transformations.

#### Results

Based on the measurements and comparisons to the data originally produced in *E. coli* by iGEM Berkeley 2006, it was observed that the promoters display distinct expression levels in the two organisms. Most of the promoters tested induced only weak expression in *Agrobacterium*, including ones that promote quite high expression in *E. coli*, like J23104.

![Characterization of the Anderson promoter library, tested in A.rhizogenes and E.coli.](https://static.igem.wiki/teams/4729/wiki/results/charac-anderson-library-reformat.png)

**Figure 2**: Characterization of the Anderson Promoter library in *Agrobacterium rhizogenes* ARqua1.

Surprisingly, the strongest promoter of the collection was not J23100, as it is the case for *E. coli*, but J23102, with more than twice the expression levels observed in J23100. These results serve to further drive the argument for the necessity of characterizing basic parts when establishing an organism as a synthetic biology platform.

### Inducible promoters

In addition to the characterization of commonly used constitutive promoters as a proof-of-concept, our team also wanted to employ inducible systems in the project, which were characterized next. Inducible systems are essential for biotechnological applications and for studying gene regulation, since they allow the creation and control of more complex pathways as well as programming desired responses to environmental cues (d’Oelsnitz et al., 2022). Similarly to the constitutive promoters described above, inducible systems that work efficiently in *E. coli* are not necessarily suitable for use in alpha proteobacteria (Kretz et al., 2023). Therefore, we selected 11 inducible promoter systems for characterization, with the goal of identifying a suitable promoter for *VirG* overexpression (see below).

There are several factors that need to be taken into consideration when characterizing an inducible system:

**Input/output** - Each inducible system consists of a promoter that is repressed by a regulator which, in turn, can be relieved by binding to low molecular weight metabolites.

**Leakiness** - Unlike electrical systems, biological circuits do not work in a strict ON/OFF mode. Promoters, even when not activated by an input, still have a basal level of expression. Promoters with a high expression level in their OFF state are considered “leaky".

**Dynamic range** - The expression level of an inducible system can span orders of magnitude from their OFF state to the maximum output strength.

**Cross-talk** - While a single inducible system covers the needs of many projects, several inputs may be necessary to control more complex gene networks. Therefore, it is essential that the different small-molecule inducers are only active on their specific inducible system.

**Cytotoxicity** - In addition to their interference with other inducible systems, the molecules used as inducers may also interfere with essential cellular processes.

In our effort to establish an inducible system that takes into consideration all the above-mentioned factors, we selected 11 inducible systems that are widely used to drive gene expression and/or that had previously been shown to work well in closely related bacteria.

#### Screening of 11 inducible systems in *A. rhizogenes* ARqua1

In this experiment, we aimed to screen as many inducible systems as possible, as we were aware that some may not work in *Agrobacterium*. We selected 9 promoters from the “Marionette Collection", which contains a number of inducible systems highly optimized for high dynamic range and low leakyness in *E. coli* (Meyer et al., 2019). Additionally, Ptac and Ptau were also included (Mostafavi et al., 2014; Stukenberg et al., 2021). Another consideration made when selecting the promoter systems to characterize was to include ones that use non-phenolic compounds as inducers (Ptau, IPTG, Pbetl, and Pbad), in the hope of minimizing cross talk with the native VirA/VirG two component system that responds to phenolic substances like acetosyingone, vanillin, or others (Bolton et al., 1986; Cha et al., 2011).

Based on this data, we decided to further characterize 4 promoters that seemed the most promising: Ptac, Pvan, Pnahr, and Ptau.

![Measuring inducible systems in A.rhizogenes ARqua1 via luminescence.](https://static.igem.wiki/teams/4729/wiki/results/comparing-all-inducible-promoters.png)

**Figure 3**: Characterization of inducible promoters in *A. rhizogenes* ARqua1 via relative luminescence.

**Table 1**: Overview of inducers, their maximal induction concentration and solvent.

| **Inducer** | **Solvent** | **Maximun induction** |
| --- | --- | --- |
| Naringenin | DMSO | 1 mM |
| Vanilin | EtOH | 100 µM |
| Cuminic acid | EtOH | 100 µM |
| IPTG | H2O | 1 mM |
| Sodium salicylate | H2O | 100 µM |
| Anhydrotetracycline | 1:1 H2O EtOH | 200 nM |
| DHBA | EtOH | 1 mM |
| Choline Chloride | H2O | 10 mM |
| Taurine | H2O | 10 mM |
| IPTG | H2O | 1 mM |
| Arabinose | H2O | 4 mM |

The results presented in figure 4 show the luminescence produced by induced vs not induced cultures in the mid-log phase. Out of the eleven promoters tested, only 4 displayed a significant difference in expression level between induced and not induced cultures: Ptac, Pvan, Pnahr and Ptau. Pbad and Pcym also had some response to the induction, albeit extremely faint. From those four promoters, we observe that Ptac and Pvan had the highest overall basal expression, and Ptau the lowest. In addition, Ptau did display the lowest leakiness of all systems tested. Overall, PnahR appeared to have a good middle ground between expression strength and expression tightness. However, upon further investigation, it was found that growth of *A. rhizogenes* ARqua1 cultures in liquid medium was inhibited by sodium salicylate (Figure 4). In *Agrobacterium tumefacies* C58, PttgR, Ptet and Pcym have been shown to have a stronger response than the one observed by our experiment, additionally the toxicity of sodium salicylate was not observed (Qian et al., 2021; Schuster & Reisch, 2021).

![A. rhizogenes cell grwoth during induction](https://static.igem.wiki/teams/4729/wiki/results/cell-growth-during-induction.png)

**Figure 4**: *A. rhizogenes* cell growth during the induction of inducible promoters.

#### Cross-talk assay

The next step was to evaluate the specificity of the selected promoters and their inducers in an independent assay. Overall, we observed a good degree of orthogonality in the promoters tested. The PnahR promoter showed the strongest activation by other inducers, mainly by vanillin and taurine, while Ptau showed some activation by salicylic acid and vanillin (figure 5). While the molecular similarity between sodium salicylate and vanillin could be the cause of the non-specific activation of PnahR by vanillin, the same is not the case for taurine. In *E. coli*, PnahR remained unresponsive to vanillin (Meyer et al., 2019).

![Cross-talk between inducible systems.](https://static.igem.wiki/teams/4729/wiki/results/cross-talk-inducible-systems.png)

**Figure 5**: Evaluation of the specificity of selected promoters and their inducers in an independent assay.

### Reporters in *A. rhizogenes*

In addition to the characterization of the commonly used constitutive Anderson promoters and inducible promoters, we wanted to characterize different reporters, codon optimized for *Agrobacterium*, to facilitate new and characterized parts to the iGEM community. Reporters are essential in molecular biological applications, because they allow to measure promoter activity. Once expressed in cells, the reporter is assayed by either directly measuring the reporter protein itself or assessing its enzymatic activity, correlating the strength of the upstream promoter element with the amount of reporter produced. Reporter genes are used as indicators of transcriptional activity in cells.

#### Purpose and design

Our test constructs for the reporter characterization used the pSRK backbone, the 0-09 RBS3 DM, the 0-11 rrnB, TB0015 terminator, the PMPJ23102 promoter and the respective reporter to characterize. We tested three fluorescent reporters, mNeonGreen, Scarlet and Staygold as well as Nanoluc a luminescent reporter. The advantages of luminescence reporters are higher sensitivity and lower background noise. All reporters were synthesized and codon optimized for *A. rhizogenes*. Designing our reporter test cassette we chose to use the strongest of the previously characterized anderson promoters, PJ23102. Lacking knowledge about the behavior of our reporters in *Agrobacterium*, a strong promoter was the most promising option, in order to get qualitative proof of their performance, even for weak reporters.

For the characterization we transformed our constructs in both of our *Agrobacterium* strains, ARqua1 and K599.

#### Results

Figure 6 displays the results generated for the fluorescent reporters. The fluorescence intensity was measured using an excitation wavelength of 552 nm for red fluorescent proteins and 488 nm for green fluorescent proteins. The figure shows the expression strengths measured in Arqua1 and K599.

![](https://static.igem.wiki/teams/4729/wiki/results/fluorescence-reporters-in-a-rhizogenes.png)

**Figure 6**: Expression strength of selected fluorescent reporter genes in ARqua1 and K599.

Among the three reporters, mNeongreen demonstrated the highest fluorescence intensity, indicating a robust expression under the tested conditions. It surpassed both mScarlet and Staygold in terms of reporter strength. The high expression in strain ARqua1 suggests that mNeongreen could be a preferable choice especially when characterizing weak promoters or RBS. However, mScarlet represents a viable alternative with moderate expression levels. Staygold, while still functional, may be less desirable when high fluorescence intensity is required, demonstrating only a slightly higher expression compared to the Dummy negative control. In K599, mNeongreen is also the strongest Reporter. However, Staygold has a higher fluorescence intensity in K599 than mScarlet. Recognizing a missing negative control for the K599 strain, those findings should be considered with reservations.

ARqua1 is the strain with the highest fluorescence intensity in all measurements. The overall performance was notably superior compared to K599.

![](https://static.igem.wiki/teams/4729/wiki/results/luminescence-reporters-in-a-rhizogenes.png)

**Figure 7**: Comparison between the expression strength of NanoLuc in ARqua1, K599 and a Dummy.

The overall performance was notably superior in ARqua1 compared to K599.

## Plant transformation

### Baseline transformation efficiency of *Agrobacterium rhizogenes* in *Arabidopsis thaliana*

In our RhizoGene project, we aim to increase the efficiency of *Agrobacterium rhizogenes*-mediated transformation of plants, simplify transformation protocols, and expand the range of usable plant chassis. In order to determine the transformation efficiency of individual strains within the shortest possible time, we adapted a transformation protocol published in 2016 using the model organism Arabidopsis thaliana (Mai et al., 2016).

Arabidopsis, a well-established plant model organism, possesses several attributes that made it an ideal candidate for our research. Its compact size allowed us to efficiently study numerous plants within limited growth chamber space, while its short cultivation time facilitated rapid and precise prototyping of transformation protocols.

To assess transformation efficiency, we initially used *Agrobacterium rhizogenes* ARqua1 with the fluorescent reporter gene dsRed. However, scoring the fluorescent reporter in the transgenic plants was very time consuming. Also, the actual transgenic area could only be delimited imprecisely. In addition, a fluorescence microscope was necessary for the evaluation. Our goal was to establish a particularly low-threshold protocol for upcoming iGEM teams. To address this accessibility concern, we transformed the 35S:RUBY plasmid into *Agrobacterium rhizogenes*, offering the advantage of scoring transformation events by naked eye via red coloured betalain, simplifying and expediting the assessment process (He et al., 2020).

![In the picture you can see a plate with several small Arabidopsis plants, with the roots cut off. In three of the plants the stem below the leaves is colored pink.](https://static.igem.wiki/teams/4729/wiki/results/pt/3daysonplate.jpg)

**Figure 8**: Arabidopsis thaliana 3 days after transformation with *Agrobacterium rhizogenes* ARqua1.

To classify the upcoming experiments, we established a baseline using our Arabidopsis thaliana protocol by replacing the bacterial strain with *Agrobacterium rhizogenes* ARqua1 containing the 35S:RUBY plasmid with the RUBY reporter gene and a hygromycin resistance on the T-DNA.

We transformed a total of 179 baseline plants in multiple rounds of experiments, plating about 50 sterilized and stratified seeds for each round. Five days after germination in the dark, the shoots of the seedlings were long enough to be separated from the roots. The wounded shoots were incubated in Agrobacterium suspension for 7 minutes. After 3 days, the Arabidopsis seedlings can be examined for the first time under the microscope for red tissue. New hairy root cells are not formed at this time point. Subsequently, the plants are plated out on petri dishes containing cefotaxime 300 mg/L to kill the Agrobacterium. Seven days later, i.e. 10 days after transformation, red hairy root tissue had formed if transformation was successful. Successful transformation of one rhizodermis cell is required for hairy root cell development. However, the formation of betalains does not occur with every formatting root. This can be explained by different combinations of native T-DNAs and RUBY T-DNA integration into the plant cells.

![Graph with the caption: A. thaliana transformation baseline - 35S:RUBY (HygR) in ARqua1.](https://static.igem.wiki/teams/4729/wiki/results/erbegnisse-tabelle.png)

**Figure 9**: Perentage of RUBY positive plants 3 and 10 days post infection under different infection conditions.

Three days after transformation, 46% of the 179 seedlings developed the red pigment betalain. This shows the expression of the RUBY reporter, corresponding to a transient or stable transformation of the T-DNA. More than 20% of the seedlings died between the first and second evaluation. One explanation for the high lethality could be the heightened plant defense due to the challenge by the bacteria, another explanation could be the particular stress of the plants due to a rough handling by the experimenters. Especially by incautious handling, microscopy under a coverslip can lead to high stress for the plants, from which some do not recover.

![The image was taken through a microscope. On the slide you can see an Arabidopsis plant with two small green leaves and a pink node in the middle of the shoot.   ](https://static.igem.wiki/teams/4729/wiki/results/pt/10dayscallus.jpg)

**Figure 10**: 10 days after transformation with *Agrobacterium rhizogenes* ARqua1 - Arabidopsis thaliana has formed hairy, ruby-red positive callus tissue.

![The image was taken through a microscope. On the slide you can see three large hairy roots which are completely ruby red.   ](https://static.igem.wiki/teams/4729/wiki/results/pt/ruby-and-hairy-ugly-root-1.jpg)

**Figure 11**: 10 days after transformation with *Agrobacterium rhizogenes* ARqua1 - Arabidopsis thaliana has formed hairy and completely ruby red roots.

While working on our baseline, we came to consider whether it might be interesting at a later stage to select the transgenic tissue by the inserted hygromycin resistance. Since hygromycin is expensive, we thought of cloning another antibiotic resistance as a selection marker into the 35S:RUBY plasmid that would be more widely applicable by other labs and iGEM teams. After some literature survey we decided to use kanamycin resistance. Even though we did not use the kanamycin resistance in 35S:RUBY:KanR afterwards, we initially ran a second baseline to check if ARqua1 with the modified plasmid gave the same results as the first baseline.

As you can see in Figure 9, 3 days after transformation, we could observe a transformation event in 47% of the 284 *Arabidopsis thaliana* used. Remarkably, after 10 days, 35% of the plants counted on day 3 died. In fact, since the Hygromycin baseline (Figure 9), several new team members were incorporated in the plant laboratory who were still untrained in appropriate lenient handling. This confirms the hypothesis that a significant proportion of the deceased plants were caused by improper handling and cannot be explained by the virulence of the bacterium alone.

### Test for enhancement of natural virulence of *Agrobacterium rhizogenes* ARqua1 in *Arabidopsis thaliana* by treatment with vanillin

While part of our RhizoGene team focused on targeting *Agrobacterium* virulence genes using inducible promoters, we looked in parallel for other ways to increase the transformation efficiency in the newly developed protocols for different plant species. During our research, we came across a publication in which different molecules were used to increase transformation efficiency (Cha et al., 2011).
For *Agrobacterium*-mediated gene transfer into plant hosts, the substance acetosyringone is often favored, which is naturally secreted by wounded dicotyledonous plants and induces *Agrobacterium* virulence genes. However, according to this paper, as an alternative to acetosyringone, other structurally similar phenolic compounds can also induce the virulence genes. For this reason, we decided to test vanillin in *Agrobacterium* culture to investigate an increase in transformation efficiency. This also offers the advantage that vanillin is significantly cheaper than acetosyringone. To achieve a higher probability of transformation events, we also centrifuged half of the bacterial culture with an OD(600) of approximately 1 and resuspended the pellet in the remaining culture. We added 500 µM vanillin to the bacterial culture with increased titer one hour before transformation.

The results 3 days after transformation were outstandingly good: 87% of the plants showed RUBY expression whereas without vanillin only 47% of the plants were RUBY positive. In this transformation, there were no dead plants after transformation. This proves that higher transformation efficiency with careful treatment does not lead to increased plant lethality at all. These overwhelmingly good results gave hope that more plant species could be successfully transformed by this adaptation of the protocol. The amazing results with Bambara groundnut confirmed this assumption (see results below).

### Test of enhancement of the transformation efficiency by using the strain K599 in *Arabidopsis thaliana*

In addition to increasing the virulence of Agrobacterium strain ARqua1, we also searched for bacterial strains that might perform better in the plant species we transformed. In a comparison paper between Agrobacterium strains ARqua1, K599 and R1000, K599 showed superior results in transformation experiments compared to ARqua1 with a higher virulence (Foti & Pavli, 2020). Therefore, we tested Agrobacterium rhizogenes K599 in Arabidopsis thaliana to observe the transformation efficiency with this strain. In K599, we transformed our 35S:RUBY:KanR plasmid.

Similar to the increase in virulence by vanillin, 3 days after transformation by K599, there is a increase in RUBY positive plants with 72% compared to baseline with ARqua1 with 47% (Figure 9). A low death rate of treated plants also suggests that the higher virulence of K599 did not have a negative impact on the plants.

### Test of enhancement of the transformation efficiency by using our composite parts

Our team also focussed on the construction of composite parts which should enhance *Agrobacterium rhizogenes*-mediated plant transformation. Each version of our composite part consists of an inducible promoter, a version of VirG and one of 2 different backbones. The general idea was to fine-tune the expression of the transcriptional activator VirG as the master switch of all other virulence genes. Therefore, we were looking for an inducible promoter with a high dynamic range, low leakiness and no cross reactivity. The promoters of our choice were Ptac and Ptau which can be induced by IPTG and taurine (see above). The two versions TiBo542 and super80 of VirG differ in the independence of the sensory kinase VirA of the latter. Whereas the pABCa has a single copy for Agrobacterium. The pSRK entry vector (Khan et al 2008) carries the pBBR1 (broad host range) ori and was initially selected for our VirG overexpression constructs, due to its medium copy number in Alphaproteobacteria and compatibility with *E. coli* (Blázquez et al., 2023;Antoine & Locht, 1992). Additionally, we wanted to use the pABCa backbone with its single copy ori. We tested different constructs containing various combinations of the previously mentioned parts in plants.

*A. thaliana* transformation via ARqua1 containing Ptac\_TiBo542[VirG]\_pSRK was the first construct tested (Figure9). After 3 days we saw a total amount of 32% of plants which expressed RUBY. Seven days later, the number of RUBY-positive plants had increased to 57%. Thus, transformation efficiency after 3 days is 15% lower compared to the baseline with ARqua1 35S:RUBY:KanR. After10 days the efficiency increased 25%, making the transformation 11% more efficient compared to the baseline.

The next construct we tested was the improved version Ptac\_super80[VirG]\_pSRK. When analyzing the transformation efficiency after 3 days we saw a drop of RUBY positive plants to 29% compared to the baseline (Figure 9). Both results were against our previous expectations, however after several rounds of trouble shooting we came up with the idea that Ptac still shows relatively high leaky expression despite having a big difference in expression levels of induced to uninduced. To evade leakiness we chose the lower expressed Ptau.

When testing Ptau\_super80[VirG]\_pSRK we observed a slight improvement to 37% RUBY positive plants. Nonetheless, it is important to note that the total percentage was still lower than during the RUBY baseline experiments without ARqua1 containing any additional constructs. Upon evaluating our most recent results 10 days post-transformation with constructs containing the pSRK backbone, we were surprised to witness an unexpected increase in the number of RUBY-positive plants compared to the 3-day post-transformation results. **We were especially surprised by the Ptac\_TiBo542\_pSRK transformation results which showed a transformation efficiency gain of 11% in comparison to the 35S:RUBY:KanR baseline**. The current efficiency levels now seem to be on par with the outcomes from the baseline experiments. With the assumption that pABCa behaves similarly, we anticipate obtaining comparable results ten days after transformation, and we anticipate these results to be available within the next week.

#### Plasmid stability assay

Plasmid incompatibility occurs when two plasmids cannot stably coexist in the same bacterial cell line over multiple generations. Typically, closely related plasmids are incompatible, while distantly related ones are compatible. Incompatibility often arises from shared replicons with identical Rep protein specificity or controlling elements. Competition can also stem from similar partitioning systems. This incompatibility may be reciprocal, leading to equal chances of loss, or unidirectional, favoring the plasmid with additional advantages, such as a second replicon. Although classification systems exist for certain hosts, many plasmids defy categorization due to the absence of systematic incompatibility tests on all known plasmids.

Many of the most widely used origins of replication from *E. coli*, such as ColE1 do not work in Alphaproteobacteria. Hence, some of the plasmid backbones used by our team contained 2 origins of replication: one for viability in *E. coli* during cloning steps and one for replication in *Agrobacterium*, or alternatively, a broad host range ori. Additionally, our iterations of the best composite part were designed to co-exist in *Agrobacterium* with the 35S:RUBY:KanR plasmid carrying the plant selection marker and the pVS1 ori. Therefore, the stability of plasmids carrying different origins of replication was tested in strains also carrying the 35S:RUBY:KanR plasmid.

##### pSRK backbone & 35S:RUBY:KanR plasmid

The pSRK entry vector carries the pBBR1 (broad host range) ori and was initially selected for our VirG overexpression constructs, due to its medium copy number in Alphaproteobacteria and compatibility with *E. coli* (Antoine & Locht, 1992; Blázquez et al., 2023). However, after the observations from plant transformations, we noticed that strains carrying both plasmids displayed lower transformation efficiency when compared to strains solely carrying the 35S:RUBY plasmid. This led to the suspicion that the two plasmids might be unstable when co-existing in *Agrobacterium*, negatively affecting cell health and thus decreasing overall transformation efficiency.

In order to verify this hypothesis, we conducted a stability assay in *A. rhizogenes* ARqua1. *A. rhizogenes* ARqua1 competent cells carrying the 35S:RUBY:KanR plasmid were transformed with the pM4\_1\_002 VirG overexpression plasmid and plated on selection media (Gentamicin + Spectinomycin). Colonies were picked and screened via colony-PCR to verify the presence of both plasmids. A cryo stock from one colony was used to inoculate liquid LB medium without antibiotics, gentamicin + streptomycin, and gentamicin + spectinomycin, which were then incubated overnight. On the following day, a 20 µL sample was taken from the overnight culture and frozen away, 1 µL of the overnight culture was used to inoculate fresh medium for a new overnight culture. This process was repeated until samples from 5 days were collected.

![pSRK amplification via cPCR (517 bp amplicon)](https://static.igem.wiki/teams/4729/wiki/composite-part/gelbild-composite.png)

**Figure 10**: Stability assay results via cPCR of pSRK and 35S:RUBY:KanR with and without added antibiotics. Samples were taken throughout 5 consecutive days.

The gel images in figure 10 show that the combination of pSRK (pBBR1 ori) and 35S:RUBY (pVS1 ori) is not stable in *A. rhizogenes* ARqua1. After 4 days, both plasmids were lost in plain LB and LB(gen+strep) cultures. In LB (gen+spec), 35S:RUBY:KanR was still detected on the 4th day and then lost on the 5th. All cultures were visibly thin on day 4 and 5.

Based on these results, we opted to use the pABCa backbone for our VirG expression constructs.

As a final test we therefore decided to use the single copy ori in the pABCa backbone to express our VirG construct. And evaluate the transformation efficiency in Arabidopsis.

![](https://static.igem.wiki/teams/4729/wiki/composite-part/pabca.png)

**Figure 11**: Distribution of different phenotypes after transformation 3 days post infections (dpi) with ARqua1 containing Ptac\_super80\_pABCa (A) and Ptac\_TiBo\_pABCa (B).

When testing Ptac\_TiBo542[VirG]\_pABCa construct we observed a transformation rate of around 36% contradicting our prior belief of pABCa being compatible with the 35S:RUBY:KanR ori. We experienced similar results when testing Ptac\_super80[VirG]\_pABCa only seeing around 48% RUBY-positive plants.

### The transformation of crops: The cut-dip-budding protocol

In our ambitious desire to improve transformation methods for non-model organisms, we decided to test the recently published cut-dip-budding protocol (Cao et al., 2023). The plants we selected for our work were Bambara groundnut *Vigna subterrenea*, the garden strawberry *Fragaria x. ananassa* and the regional dandelion *Taraxacum officinale*.

In the first experimental run, we cut the roots of the plants grown in a non-sterile environment, incubated the shoots of the plants in *Agrobacterium rhizogenes* ARqua1 for 15 minutes, and then placed the experimental plants in vermiculite. We evaluated the results after 2 and 4 weeks by removing the plants from the vermiculite and examining the cutting site with the naked eye.
Of the 14 Bambara groundnuts used, no plant showed root growth after 2 or 4 weeks. 19 of the 31 dandelion plants showed root growth after 2 weeks, as did 7 of the 8 strawberry plants. Unfortunately, none of the plants developed hairy roots or RUBY expression. Accordingly, a transformation event probably did not occur in the first experiment.

In the second experimental run, we increased the incubation time in *Agrobacterium rhizogenes* ARqua1 culture. We used 9 strawberry plants, 18 dandelions and 12 Bambara groundnuts. In this experiment, spontaneous root formation also occurred in strawberries and dandelions. None of the plants showed signs of positive transformation events or transgenic tissue in the phenotype.

In accordance with the promising results in the study with our workhorse *A. thaliana*, we increased the titer of actively dividing *Agrobacterium rhizogenes* ARqua1 in the medium in experiment number 3. We also added 500 µM vanillin to the culture one hour before incubation of the plants to induce virulence of the bacteria. Fortunately, between experiments 2 and 3, we were able to begin work in another greenhouse that had stable temperature and humidity. Since we were also able to transform in this greenhouse, we were able to minimize environmental stress to the plants.

Experiment number 3 was conducted with 9 strawberry plants, 19 Bambara groundnuts and 6 dandelion plants. Surprisingly, after two weeks, 3 Bambara groundnut plants showed root growth for the first time, with the roots also showing hairy patches. This result was highly exciting, as this is phenotypic evidence of transformation events. Four weeks after transformation, as many as 8 Bambara groundnuts had developed roots. However, no RUBY marker was visible. After moving to the new greenhouse, we observed a particularly strong drought stress of the plants after transformation, as the solar radiation was more intense during summertime. Most of the strawberry and dandelion plants died within 2 weeks, so no RUBY expression or hairy roots developed.

![In the picture you can see a root about 3 cm long growing out of a cutting side, the beginning of the root is hairy.   ](https://static.igem.wiki/teams/4729/wiki/results/pt/hairy-root-vs.jpg)

**Figure 12**: Development of a hairy root in bambara groundnut 2 weeks after transformation with *Agrobacterium* ARqua1.

![Two hands with gloves hold a bambara groundnut plant with a handful of root system.](https://static.igem.wiki/teams/4729/wiki/results/pt/vsroot-system.jpg)

**Figure 13**: Strongly developed root system of bambara groundnut 8 weeks after transformation with *Agrobacterium* ARqua1.

After 4 weeks, we reexamined the experimental plants trial no. 3, which by this time had developed a mature root system. To our amazement and excitement, two of the Bambara groundnut plants developed ruby red root sections. This means that the T-DNA with the reporter gene of 35S:RUBY:KanR was successfully transformed into Bambara groundnut which means that our adapted protocol suits to transform this poorly investigated tropical crop plant plant. This is a real breakthrough that makes us proud, especially since Bambara groundnut has not been successfully transformed according to our literature survey at that time.

![Image through binoculars showing a root section with a red color stripe running through it.](https://static.igem.wiki/teams/4729/wiki/results/pt/vsredroot.jpg)

**Figure 14**: Red root section of bambara groundnut 8 weeks after transformation with *Agrobacterium* ARqua1.

![Image through binoculars showing a root section with a red color stripe running through it.](https://static.igem.wiki/teams/4729/wiki/results/pt/ruby-root-2.jpg)

**Figure 15**: Red root section of bambara groundnut 8 weeks after transformation with *Agrobacterium* ARqua1.

### Implementing a new transformation method: The stone wool method

During the implementation of the cut dip budding protocol, we came across a method by Sebastian Cocioba via social media by chance. In fact, while working with the CDB protocol, we had thought about a way to further reduce the stress of the plants, as the roots suffered even when being carefully removed from the vermiculite. In the new method, rockwool is soaked with *Agrobacterium* culture and placed in a centrifuge tube filled with tap water. The plant, stripped of its roots, is placed in this assembly with the cutting side down and co- cultured with the bacteria. If root growth occurs, the roots can be assessed through the transparent tube wall. Expecting to follow up on the positive results with Bambara groundnut, we transformed dandelion, strawberry, and Bambara groundnut with *Agrobacterium rhizogenes* ARqua1 using the rockwool method. This involved adding 500 µM vanillin to the culture as in our previous, very successful CDB run and centrifuging the culture to double titer.

![A plant with many leaves is in the foreground in a centrifuge tube containing green rockwool and a pink dishcloth.](https://static.igem.wiki/teams/4729/wiki/results/pt/stonewoolaufbau.jpg)

**Figure 16**: Bambara groundnut after performing the rockwool method.

After the impressive transformation efficiency of *Agrobacterium* strain K599 in *A. thaliana*, we decided to transform our crop plants with this strain as well. We proceeded as described above. For now we are still waiting for upcoming results.

### Establishing *Agrobacterium rhizogenes*-mediated transformation in *Quercus robur*

In a great collaboration with Lars Opgenoorth from the PhytOakmeter project at the University of Marburg, we got the opportunity to work with the oak clone DF159. Since the clone is axenically propagated, it was necessary to develop a sterile transformation protocol specifically adapted to this propagation procedurel. If you want to read more about our self-developed protocol, you can [click here](https://2023.igem.wiki/marburg/oak-diary) for our "Oak diary".

In the first round of experiments, we used the *Agrobacterium* strain ARqua1 to transform 53 oak plantlets. After the transformation day, we transferred the oaks to a medium containing 500 mg/L of cefotaxime and activated charcoal two days later. We then conducted an evaluation once or twice a week for three months, documenting health status and new, potentially transformed tissue.

In the first week after transformation, almost all oaks lost their leaves due to the enormous transpiration stress the plants had suffered under the sterile bench. Only after about 3 weeks, lateral buds sprouted and new leaves formed. At 4 weeks after transformation, we were pleased to observe root formation in two of the oaks, but no hairy roots. In fact, the culture in activated charcoal medium for evaluation turned out to be very impractical, as the formation of new tissue could only be observed inaccurately. At 12 weeks after transformation, we removed the oaks from the medium, evaluated them by visual inspection, and microscopically examined cross-sections of the roots and shoots in hopes of detecting ruby red tissue. At that time 27 of the 53 plantlets had been lost due to contaminations. Of the 26 plants that were not contaminated, 4 oaks had formed new roots. Two of these oaks had also developed hairy roots. Unfortunately, we were unable to detect RUBY in the oaks, indicating the transfer of one of the endogenous T-DNAs of the ARqua1 strain. Nonetheless, using a completely self-created transformation protocol, we were able to detect transformation events in two Quercus robur, which is tremendous success, since we were not aware of any report on an existing oak transformation protocol. This gave us hope that further improvement of the protocol might provide even better results.

![in the picture is a part of a large test tube with black medium in it. In the medium sits a small oak with a white root visible.](https://static.igem.wiki/teams/4729/wiki/results/pt/quercusroot.jpg)

**Figure 20**: Quercus robur with a visible root 5 weeks after transformation with *Agrobacterium* ARqua1.

![jar with 6 small oak plants which are suffering from drought stress.](https://static.igem.wiki/teams/4729/wiki/results/pt/crispyquercus.jpg)

**Figure 21**: Quercus robur 2 days after transformation suffering from drought stress.

After the promising results of the first experiment, we repeated the transformation again with 51 oak plantlets. This time we made some adaptations to reduce the drought stress during handling under the laminar flow hood. The detailed description of the performed method can be found on the [experiment page](https://2023.igem.wiki/marburg/experiments). As a bacterial strain we used *Agrobacterium* K599 andinduced the virulence according to the promising results in Bambara groundnut with 500 µM vanillin. In addition, the bacterial culture was spun down and subsequently resuspended to increase the bacterial titer of the suspension twofold.
At the time of wiki publication, the transformation was 19 days ago, therefore no statement about the transformation events can be made yet. Nevertheless, it can already be seen that the further developed method has had a positive influence on the oaks. On the one hand, only one oak shed its leaves after the transformation, and new buds also formed within the first week. After 2 weeks 21 oaks have already built calluses. As an innovation, we have the oak's cutting edges on the outer wall of the new test tube.

![More than 21 large test tubes containing dark medium and healthy, green oaks.](https://static.igem.wiki/teams/4729/wiki/results/pt/healthyquercus.jpg)

**Figure 22**: Quercus robur 14 days after transformation with *Agrobacterium rhizogenes* K599.

### Establishing *Agrobacterium rhizogenes*-mediated transformation in *Setaria viridis*

Agriculture relies mainly on a handful of monocotyl crops such as maize, wheat and rice. Despite their immense agricultural and nutritional significance, monocots, which represent the most crucial food crops in terms of both quantity and calories, have been explored by fewer than five iGEM teams throughout the history of the competition. When aiming to facilitate *Agrobacterium*-mediated plant transformation and broaden the spectrum of genetically accessible plants it is also important to think about testing in monocots. Since monocots don't release phenols like acetosyringone after wounding the virulence genes of *Agrobacterium* do not get activated and therefore the monocot does not get transformed (Cao et al., 2023). To circumvent this, phenolic additives like vanillin and acetosyringone are added to the bacterial culture and also the growing medium of the infected plants. In this context decoupling of VirG from VirA might result in an induction of the general virulence without the need of additives. When trying to work with the emerging model-organism for monocots Setaria viridis, commonly known as green millet, we encountered several challenges. At first we had to overcome the hurdle of seed sterilization. For this we had to come up with a way of completely removing the husks around the seeds so that our sterilization solution can reach every part of the seed. After successfully plating out several rounds of seeds which did not contaminate had virtually no seeds germinating so we looked for ways to break seed dormancy. After troubleshooting and literature research we found a way to break seed dormancy by adding a gibberellic acid (Sebastian et al., 2014). After our first trial of transforming the seedlings we surprisingly got 40% RUBY-positive plants after 3 days and even 53% after 10 days, slightly exceeding the baseline results for Arabidopsis. All following germinating trials were unsuccessful which forced us to ultimately change to a close relative of our chosen plant: Setaria italica, commonly known as foxtail millet. This plant is cheap, readily available and germinates even without the need of added gibberellic acid. However, after several trials with multiple constructs and conditions we couldn't detect the RUBY reporter. This could be due to the fact that the 35S:RUBY:KanR promoter is relatively weak in monocots (Jang et al., 2002). Sadly we couldn't explore the possibility to exchange the promoter and optimize RUBY expression in Setaria during the time of our iGEM project.

![](https://static.igem.wiki/teams/4729/wiki/plant-design/pd17.jpg)

**Figure 23**: Distribution of different Setaria viridis phenotypes 3 and 10 days post infection with ARqua1 containing 35S:RUBY:KanR.

![](https://static.igem.wiki/teams/4729/wiki/results/setaria-viridis-transformation.png)

**Figure 24**: MS agar plate containing selection of S. viridis plants some of which show visible RUBY.

## References

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