Team:Marburg/Cell-Free/In Vivo Diary
Tobacco Chloroplast Transformation - a diary
Developing cell-free systems from chloroplast, we were aware that it would be ideal to show a comparison between our in vitro systems and data obtained in vivo. Therefore, we set out to perform chloroplast transformation in tobacco leaf chloroplast. The goal was to transform a set of constructs that can later on be tested and compared to measurements of the same constructs in our cell-free systems.
Sadly, due to time constraints, several hurdles and the difficult nature of such transformations, we were not able to obtain a fully homoplasmic transformed plant. Nonetheless, we are on our way to get there and with a bit more time might have been able to show interesting results. Follow our journey in this intimate diary and discover what it takes to transform the chloroplast of plants!
06.04.2021
Dear diary,
After spending the last couple of weeks organising the reagents we need for our upcoming experiments, today, we could finally start!
We prepared some simple MS medium with agar to grow our sterile tobacco plants on. The seeds were sterilized and put on the agar plates for germination. I am afraid that the sterilization with bleach and ethanol might have been too much for the seeds. Let’s hope it goes well!
14.04.2021
Dear diary,
the tobacco plants started to germinate! Apparently the sterilization with bleach and ethanol was not too much and they still retained their ability to sprout, nice! They are still very small, just some roots started to come out of the seeds, digging their way through the agar.
I am really excited to see how they develop!
20.04.2021
Dear diary,
today was a sad day… Although most of our little plants have grown quite a bit during the last week, we noticed a contamination on one of the plates… Luckily it is just on that one plate and there are not many seeds on it anyway. This means that we might have a few less plants - good thing we germinated more than we would have needed anyway!
I guess we have to handle our stuff more carefully in the coming weeks!
21.04.2021
Dear diary,
today we prepared the MS medium we need to grow our tobacco plants! The small plantlets can soon be transferred from the plates to bigger jars - I'm so excited to see how well they grow then!
23.04.2021
Dear diary,
today was super exciting! We moved our little tobacco plants from the agar plate to the mason jars we prepared earlier with our growth medium. It was very fun for us to do this for the first time and I think we did a good job on the handling of the small, delicate plants. Let’s hope they grow well! It was a bit tricky to make sure not to hurt their small root system and keep everything intact.
Also, placing them on the agar was also not that easy! The agar is relatively soft, but you still can’t just push the plants inside - at least we didn’t, we were too afraid to hurt them.
We just made a small cut into the agar and placed them there, mostly they were still just on top of the agar with their roots. We were told that this should still be fine,and that they will grow their roots downward into the medium anyway!
Some of the plants were still very small, so let’s see if they survived this procedure.. Most of them look great though!
28.04.2021
Dear diary,
it has been 5 days now since we moved our sterile tobacco plants to the bigger jars. They are growing nicely so far! You can already really see the difference in size!
I hope it will stay this way in the coming weeks until we can finally do our chloroplast transformation… It’s really quite some time we need to grow them now, I hope they won’t get contaminated in the meantime and hold up well under the conditions in the phyto chamber.
05.05.2021
Dear diary,
Another week has passed and boy do the plants look good! They have grown quite a bit since last week, I think they are doing quite well!
There are two plants that are still super small though.. I fear we might have hurt their root system when transferring them from the plate to the mason jar … That’s sad, but I think the remaining plants do really well so far - I’m sure we will still have enough material in the end to perform our chloroplast transformations!
26.05.2021
Dear diary,
A few weeks have passed but when I looked into the phyto chamber , I could not believe my eyes. Our plants mostly look very old and yellowed. They must not have been getting enough nutrients. I went straight to our PI and informed him. According to him, we should start over and germinate new seeds. I plan to do that now too!
Even though it's sad that we can't use these plants, of course I'm not giving up and trying again. I put the old plants into the greenhouse and hope that we could maybe use them for a chloroplast isolation. But right now they look really sad. I'm sure they will look better once they adapted to the greenhouse environment.
02.06.2021
Dear diary,
After our setback, we directly started a new medium for our plants today and sterilized the seeds again with ethanol and bleach. We also sterilized our medium in a large bottle in the autoclave. And in case any contamination would happen, we prepared more seeds than we actually need because as you know, time is very precious in this transformation.
Even though it was a lot of work to prepare everything again,I think it’s worth it! I'm excited for our next try.
12.06.2021
Dear diary,
I looked at our plates again today. The germination of seeds was successful just like last time. It is also very very nice to see that there was no contamination. If our little plants continue to grow like this, we can start soon with the actual transformation! I'm really looking forward to that.
22.06.2021
Dear diary,
today was devastating. Out of all of our plates, 15 were contaminated. I could not believe my eyes. How could this happen? We were so careful. But probably not careful enough. 5 plates are not contaminated. However, the plantlets that are not contaminated look great. Despite the current situation, I would like to continue with the transformation. It's worth a try.
26.06.2021
Dear diary,
Well, the plants are ready for the transfer and we want to do that for sure right next week. For this we have to prepare the media again and autoclave everything. Fortunately, I have already done this before so it should be easy.
28.06.2021
Dear diary,
Today our little seedlings were ready to be transferred into the big jars. Just like two months ago, we sterilized the laminar flow cabinet and all of the tools we needed. We really wanted to make sure that no other contamination would take place, so the tweezers were sterilized with ethanol and a Bunsen burner between handling each little plantlet. We have chosen to transfer 15 small plantlets from plates on which there is contamination. However, these plantlets have not had direct contact with the contamination. And of course, the 5 plantlets from the plates without contamination. We again made an incision in the agar of the jars and lightly pressed the plants into these incisions. Now everyone has to keep their fingers crossed that everything will be fine.
31.06.2021
Dear diary,
The fingers crossed were unfortunately in vain. Everything came as you could have expected. Almost all of our plates were contaminated and I threw most of them away today. The few ones that were not contaminated, I kept. I was very sad about it, but it's not time to give up yet! Here's to a new one!
02.07.2021
Dear diary,
We really do not want to waste any more time, so I immediately started to prepare new medium and new seeds. Hopefully, I can then transfer the plants to the jars already in the next two weeks.
12.07.2021
Dear diary,
I'm so happy, the plates look good and they grew pretty uniformly. Yet there is no contamination on any plate, all seeds germinated as far as i could tell. There is hope.
I'm currently finishing preparing the jars and will transfer the seedlings tomorrow.
13.07.2021
Dear diary,
I transferred the seedlings today. No contamination, Everything is very sterile! Same procedure as every month. If this won't work out, I’m afraid I will lose my nerves. We are running out of time!
26.07.2021
Dear diary,
Do you see the bigger plants in the back? Those were the leftover ones I transferred exactly 4 weeks ago. The ones in the front are from two weeks ago. For the transformation, I would say maybe they could be a bit bigger than the ones we see in the back now. That would mean still two weeks to reach that size and if we want them a bit bigger, I'd say three weeks. If we leave them longer they will probably turn bad again. So I am planning the transformation in around three weeks.
05.08.2021
Dear diary,
The day of transformation is getting closer and closer. I am looking forward to it so much. Today I took another look at the plants and don't they look great? I am thrilled. Everything is planned and I am ready for next week.
12.08.2021
Dear diary,
We have decided to prepare most of the transformation today so that there will be some time tomorrow in case of complications or if we forget something today.
So first thing in the morning we started to prepare the RMOP medium plates. We prepared plates with antibiotics on the one hand and plates without antibiotics on the other. Again, we worked very sterile and autoclaved all media and plates. We also autoclaved all tools, such as tweezers, razor blades and scalpels, which are needed in two days. After that we put the flying disks, the microcarriers, the rupture disks and the stopping screens in ethanol to sterilize them until the day after tomorrow. It took a very long time to autoclave our plates. When the time was up, we transported them in sterile sealed boxes to the laboratory where the gold particle gun is located.
The laminar flow cabinet that we will use for the transformation was also thoroughly cleaned and prepared for the transformation. I am very much looking forward to the day after tomorrow, but I am very afraid that we may have made a mistake. Well, we'll see tomorrow.
13.08.2021
Dear diary,
I was taking another look at the plates that we prepared yesterday and I had to find out that they are unfortunately way too wet. I am pretty sure that we should not use them like that, so I laid them out on a sterile surface to dry until tomorrow. I am aware that this increases the risk of contamination a lot, but I don't know what else to do. I tried my best now to save them. Let's see how they look tomorrow.
14.08.2021
Dear diary,
Yeah you guessed right, the day has finally come! Despite all the setbacks, we have managed to get to this point. We met early in the morning to set everything up and transport the big jars into the lab where we wanted to perform the biolistic bombardment. To make sure that the plants stay sterile, the jars were wrapped with parafilm before transporting them to the laminar flow cabinet that we used. We started off preparing the non-antibiotic plates by putting two sterile Whatman filter papers onto each plate. Then afterwards, we chose the best looking leaves from our beautiful tobacco plants, cut them off with a scalpel and tweezers and put them carefully on top of the filter papers.
As you already know, we have had problems with contamination before, so we have been really careful to work in a sterile way. We left the plates in the laminar flow cabinet and set out to prepare the gold particles to bombard on our selected leaves. The gold particles were washed several times with ice-cold ethanol. We then aliquoted the gold particles and added our DNA. After that, we already had to wash the gold again and resuspend it many times.
After everything possible was prepared, we set to work on the biolistic bombardment. For this, the flying disks were placed in their holders and 10 microliters of the DNA-covered gold particles were pipetted centrally onto each disk. After drying, a rupture disk was placed in the retaining cap. The stopping screen and the flying disk were also assembled in the gold particle gun and a plate with the leaves to be bombarded was placed in each of the corresponding positions. Our instructor showed us how to use the gold particle gun properly and we first practiced the firing sequence. Since it was my first time, it was quite complicated for me at the beginning. But once you've practiced it a bit, it goes very smoothly. For the firing sequence, we first created a vacuum in the particle gun using a helium pump. Through the subsequent firing, we could even hear the rupture disk rupture. The sound was our signal to release the vacuum and remove the ruptured disks from the particle gun. We repeated this with all our constructs and all our plates until all our leaf samples were shelled.
Of course, we also made individual errors during the bombardment, but we documented them carefully. For example, I once placed two rupture disks in the fixture instead of one.
When all leaf samples were bombarded, the plates were sealed with micropore tape and transported back to the phyto chamber, where they will now remain for 2 days until we transfer them to antibiotic-containing plates.
We were in the lab all day today, because the transformation took a lot of time. That was very exhausting. But it was definitely worth it, it's always exciting to get to know new techniques. Now we all need a good night's sleep, but I'm looking forward to seeing what happens next.
16.08.2021
Dear diary,
It's been two days now since we did the tobacco transformation. So now it’s time to transfer the leaves to the plates containing spectinomycin for selection. On these plates, we expect various calli to grow that contain our transgene. Afterwards we will still have to check the calli for spontaneous mutations in the 16SrRNA that could lead to a spontaneous resistance against spectinomycin. We will then do it by expanding the selection and transferring the chosen calli onto plates containing not only spectinomycin but also streptomycin. But this will take some time, probably around 8 weeks. I will tell you about that then.
Today we first cut the leaves into small leaf sections of 1cm^2. We had to be careful not to select central leaf sections directly, as they might be too damaged after the bombing. We focused on small sections around the center and cut them out in a rectangular shape with a scalpel. Using forceps, we carefully transferred each section to the spectinomycin-containing plates, which we had previously labeled.
After all small leaf samples were transferred, we resealed the plates with micropore tape and placed them in the phyto chamber again. Now we wait to see if small green calli form on our plates. I keep my fingers crossed.
24.08.2021
Dear diary,
As you know it was the first time for me doing a plastid transformation in tobacco. Luckily we had a lot of help from our instructor, who explained all the important steps to us and guided us through the whole procedure. Nevertheless some questions were still open about the handling after the transformation. I was insecure about the light intensity that the leaf samples need after transfering them onto antibiotic plates. But there were many more points that were not clear for us. We decided to hold an interview with Dr.Stephanie Ruf and Dr. Daniel Karcher from the Bock department of the Max Planck Institute of Molecular Plant Physiology in Potsdam who are experts in this field. They agreed to the interview and we received a lot of very useful information from them.
It was an amazing experience to be able to talk to experts and I am taking a lot from their advice.
15.09.2021
Dear diary,
today I sadly found another contamination. When I saw it, I removed it directly. We don't want any contamination spreading inside the phyto chamber. Let's hope that this was the only one. The other leaf samples still looked yellowish, but I was able to see some brownish/greenish calli. They will probably take a few more weeks.
29.09.2021
Dear diary,
Today I was taking a look at our leaf samples together with our PI. Since we only have three weeks until the wiki freeze of the iGEM competition, he advised us to transfer a portion of the existing calli to the streptomycin-containing plates soon. This will give us some time to perform experiments on the Spectino- and Streptomycin-resistant calli. I had doubts at first, thinking that the calli would need a little more time, but due to the time pressure we decided to listen to his advice and start the transfer already now. I will start the preparations in two days.
01.10.2021
Dear diary,
Today we prepared the RMOP medium again for another transfer, which we want to start as soon as possible. We sterilized the required antibiotics through a filter and added them to the medium after autoclaving it. We filled half of the plates with medium containing only spectinomycin and the other half now contains spectinomycin and streptomycin. At first, it was inconclusive to me why we should also make plates with only spectinomycin in addition to the plates containing streptomycin, since the leaf samples had already been placed on spectinomycin plates before anyway. In the interview with Dr. Stephanie Ruf and Dr. Daniel Karcher, I asked them this question. They explained to me that it is important to compare the ratio of really transformed plants with the number of plants resistant only by spontaneous mutation. First thing tomorrow I want to do the transfer.
02.10.2021
Dear diary,
Today I actually wanted to transfer the leaf samples. However, when I came to the lab this morning, I found that it was flooded. You can't expect that! I was one of the first people to notice the damage, which is why the water was still relatively high. Then it was clear to me: It's not going to work today! Due to a pipe burst, which had supplied the floors of whole 3 floors with water, we had to postpone our project until tomorrow. Nevertheless, it was an exciting experience!
03.10.2021
Dear diary,
After a delay of one day, today is the day. The calli are put to the test! Will they also survive on the streptomycin media? I am curious.
A total of 14 calli were selected, not all of them were green. As always, the laminar flow was sterilized, as were all the tools used. I mean, the procedure is already known by now.As always, the plates were handled carefully and the tweezers and scalpels were sterilized over a Bunsen burner between each step.
I grabbed each selected calli with the tweezers and used a scalpel to cut them into two equal halves. One half was transferred to a spectinomycin and streptomycin plate respectively and the other to a spectinomycin plate for comparison. To my surprise, the calli were not soft, but in a way very hard and even crispy when cut. For the comparison, I carefully marked the plates beforehand. After transfer, the plates were again sealed with micropore tape and placed in the phyto chamber.
Now I am kind of scared that all of our suspected calli will not survive. But we still have the leaf samples that were not transferred yet. Even though we will not have enough time to get results from all the transformed cells until the wiki freeze, I am excited to continue this experiment afterwards, to see how successful we were.
11.10.2021
Dear diary,
I was taking another look at our calli today. I have to say, except for one calli, I don't see any big differences in the plates. Perhaps it was still too early to transfer the sheets. However, this one calli looks very good! I am curious if we still manage to do a PCR before the wikifreeze to see if our construct was integrated. Sooner or later we will definitely do that!
19.10.2021
Dear diary,
Today was another very happy day at the Voll lab. The green callus that I transferred to the new selection medium, at the beginning of October, grew really big! It was so much material that I could take some for finally performing some assays. I cut off around 1 g for the PCR and fluorescence microscopy. The rest was transferred to a sterilized glass jar with RMOP medium and spectinomycin for selection. Moreover, I realised that more green calli have formed in the selection plates prepared back in August. They have been growing for 9 weeks now. I transferred the callus pieces to new selection plates, dividing them between the streptomycin-spectinomycin plates and only spectinomycin.
20.10.2021
Dear diary,
I have great news! The PCR confirmed the successful integration of transgenes into the plasmid! It is still a long way ahead to achieve transplastomic plants but this result gives me hope I’m getting there soon in the future.