It all began at a meeting long-long ago, when one of our PIs, Anke Becker, mentioned that the Opgenoorth AG at our university has been working with a curious oak clone. There, it is multiplied from cuttings to use in plant adaptation research. Furthermore she told us about transformation research. We decided to give it a try and establish a conversation with the Opgenoorth workgroup. Aidana, a member of the plant team, was writing her thesis in that exact workgroup, so she began the correspondence, and the “Oaks for iGEM" project started.
The Editor
Several months of planning and meetings later, Lars Opgenoorth has agreed to give us 50 DF159 oak cuttings for our transformation trial round, and we have set to work.
Dear diary,
Today we are starting the preparation for an oak transformation, how exciting is that? We borrowed 10 big jars from the old iGEM team, thanks to a short recommendation from one of our advisors, rinsed them, and carried them to the autoclave. Then they will be ready to be filled with the medium and become the oaks’ next home.
Karlotta and Dascha
Dear diary,
Marco made us 1.5 L MMMM without activated carbon ( we called it Marco’s Magic Mystery Medium). We picked up the medium, and Julia, our instructor, autoclaved it for us late at night (thanks Julia!).
Aidana
Note from the editor: Since we want to adapt the medium recipe to the work of the propagation protocol, Marco Göttig an associate of Lars Opgenoorth, has kindly prepared the appropriate medium for us. (thanks Marco!)
The Editor
The 10 sterile wake-up jars were filled with MMMM late at night. Unfortunately, we forgot to add a stir fish to our medium before putting it inside the autoclave. As a result, there was a lot of per-hand shaking and even more air bubbling in the medium. Oops! After that, it took only half an eternity until the agar was solid and we could go to bed.
Amanta and Dascha
Little throwback here: if you go to our "Experiments" page, you can find several protocols we have adapted and altered to fit our goals better. Even though the oaks are sterile propagated, which is why we could not adapt them to our non-sterile protocols, we still wanted to take the chance and test a woody plant in a self-developed transformation protocol. Yet, we decided to give our root-inducing Agrobacterium a shot, and rely on natural ability of the bacteria for root induction to generate new roots.
The Editor
Dear diary,
We picked up mason jars from Marco and transferred them to our growth chamber. Then the big 20cm test tubes had to be autoclaved, as it was the following step after the oak transformation and we wanted to be prepared for the next step.
Team
Dear diary,
After a long preparation and organization (thanks, Amanta!) time, we could finally start to transform the oak. The hardest part of the whole protocol was keeping everything as sterile as possible, not the work itself.
Aidana worked on the sterile bench. The small oaks were a bit too dry after 30 minutes of incubation in the bacterium suspension, but the cuttings could moisturize again in mason jars. We have put up a sterilized hood over the oaks, which was a bit of improvisation from our side because we have thought long and hard about how we can protect oaks from drought outside of the jars they were growing in. We decided on covering them with a plastic hood used to contain moisture on plant trays, but as we did not want anything to touch the oaks, we had to put the hood so high, that there was still a lot of air coming in from the back of the sterile bench. That of course resulted in some damage to the plants.
Plants were much more rigid than we thought at first, yet still very fragile and were easily hurt by harsh conditions (due to the absence of cuticula). We hope our little oak fighters make it to the next step!
Aidana, Amanta, Ilayda and Anastasia
Dear diary,
On this day we were getting another batch of MMMM (with charcoal) from Marco, so we needed to pour 50 big test tubes with the medium. Pouring it into test tubes for rooting was a bit tricky, as the carbon tends to settle down and precipitate inside of the tubes, so it had to be done in an ice bath, so that agar solidifies faster and captures the carbon molecules.
Team
Dear diary,
The 53 oaks were transferred today from the jars (5 per jar, MS medium) to the test tube containing charcoal and 500 mg cefotaxime. With the antibiotic, we wanted to reduce the number of bacteria so that the oaks would no longer be exposed to biotic stress. Most of the oaks had dark green crisp leaves, with some lower leaves being yellowish/light green. Unfortunately, black accumulations had formed in the jars around the oak stumps. We suspect it is a fungus. None of the oaks were dead 2 days after transformation. The oaks were each removed individually from the jars with long (sterile, deflamed) forceps, then dunked in a water bath to wash off the large bits of Agrobacterium, and then bathed in cefotaxime bath (500mg/L) for about 1 minute. The oaks were then placed individually in test tubes. Since we had only 50 test tubes prepared with charcoal, we placed 6 very small oaks two at a time in each test tube.
Aidana, Dascha, Amanta
In the course of the next three months we observed the oaks regularly, keeping track of everything we could notice. As a short spoiler: a lot of oaks got contaminated.
The Editor
Dear diary,
Today was the first day we looked into the oaks since we transferred them to the large reagent tubes and put them into a growth chamber. Sadly, there was some mold contamination discovered in oaks 1c, 5e, 6d, 7a, 7f and 9e. Some oaks fell over and stem cut sites were exposed to the air. Most of the oak buds looked good, which left us hoping for the best, although they lost a lot of leaves.
Dascha
Dear diary,
Some oaks seem to be getting a callus tissue on the ends, but it’s hard to tell yet. Apical buds are dying, but there are new lateral buds coming into play, so nothing is lost yet. There is yet more contamination in the oaks, some new tubes are getting moldy. We are hoping for the best, even when our PI is suggesting we try to microscope some of them.
Dascha
Dear diary,
Our oaks have roots!!!
It was reported during an evaluation we are doing once or twice a week, that two of the oaks have actually built roots, isn't it
amazing? They are just white and not hairy, as we hoped, but they are roots! And that's a big first step!
Karlotta
Dear diary,
At this point 4 of the oaks have roots, but there are also many oaks that we had to throw away and even more freshly contaminated ones. We suspect we could not work as sterile as we wanted and there must have been some contamination traveling from tube to tube due to the oaks being closed only with aluminum folie. We are trying to keep the contaminated oaks away from the uncontaminated ones, but we are not sure if it is helping that much, as all of them are in one chamber.
Dascha
Dear diary,
With a heavy heart we have decided to undergo the last counting of round one oaks and take the surviving ones from the tubes for an autopsy - we are going to microscope them and see if there is any red (RUBY) tissue hidden beneath the black medium. The development of hairy roots and/or red roots would be evidence of transformation events.
As a result: there were 4 oaks with roots, half of them with secondary roots. What's even more amazing, the secondary rooted oaks have also turned out to be owners of hairy roots!
We were almost too happy to see a shimmer of red in some plants, but a fluorescence test disappointed us and showed that it was only phenols.
Aidana, Ilayda, Karlotta, Dascha
Dear diary,
Today we again got the MMMM medium from Marco, but this time with the charcoal and poured It into 52 large test tubes. This time we had test tube capes which, we hoped, will minimise contamination from oak to oak in a growth chamber.
Amanta
Dear diary,
Today we are finally able to transform the oaks! There were some issues with the ON culture, the amount we could actually use for a transformation was less than we expected, but it was still enough to centrifuge the culture down and add some vanillin, as those two improvements showed a higher transformation rate with other protocols. Centrifuge tubes were prefilled with 1ml culture, and we were ready to transform!
This time when transforming we did not use any type of hood to cover up the oaks, instead, we just closed the sterile bench and turned the air stream off, while the oaks were still there. This way we made sure the oaks were staying sterile without over-drying. Transformation went well and much faster than last time, as we had some experience now. There were a few fewer jars than needed because of a contamination, but we just combined smaller oaks in one jar and therefore came to exactly 50 transformed plants.
Aidana, Dascha
Dear diary,
After the oaks stayed in the growth chamber for 2 days, they looked much better than last time. The trick with the
sterile bench seemed to have successfully prevented the drying of the oaks and they were looking to bring green and
happy in their new jars, even after treatment with the bacterium.
This Monday is the transfer day when oaks are washed with cefotaxime and then put into new tubes with a charcoal medium
where they stay until further notice (e.g. autopsy).
Transfer went smooth, oaks were bathed in cefotaxime for one minute, after shortly being washed in water to get as much
agar away as possible. After the antibiotic bath, they were put inside the ready-to-use tubes, so that the ends of the
stems where oaks were cut were still visible on the walls of the tube.
Some of the oaks divided themselves during this process, as sometimes there were double plants with a weak link,
therefore we could fill all 52 tubes with an oak in each.
Aidana, Dascha
Dear diary,
This day marks the last oak round 2 evaluation so far. More will follow, but this is the most up-to-date information we
own:
There are 7 contaminated oaks 19 days after transformation, which is way less than last time (it was 10 contaminated
oaks 17 days after transformation). Our adjustments seem to be working, especially considering there's way less leaf
loss in the oaks this time, they weren't crispy and dark green after transformation and keep producing new buds.
All of the oaks are green and 49 have more than 5 alive leaves.
21 oaks have now calluses and three seem to be building roots! This is awesome!
Team
Back to topWe are still counting and evaluating the progress, moreover, an autopsy will probably follow, so stay tuned for curios results!
The Editor