Notebook
When we thought of developing our project to provide UV protection for plants on Mars, we wanted it to be easy to replicate and we expected our method to be as effective as possible, by modifying large numbers of plants. In order to accomplish this mission, we received the help of the CSIC researchers throughout the project, who guided us on which methods we should use. Thanks to them, we were able to develop our project as they gave us the opportunity to do it in a comfortable way, supplying us with materials, equipment, and the required place.
If you remember, our main objective is to provide UV protection for plants on Mars by boosting the plant's natural photoprotective response. Therefore our strategy is set by 3 different fields of research: to start with, we want to verify if the increase of pigments protects from UV, leading us to characterize Arabidopsis plants overexpressing PAP1 and characterize Arabidopsis plants exposed to volatile compounds. Simultaneously, we used synthetic biology to engineer multi resistant plants to UV: producing Arabidopsis plants overexpressing PAP1 and UVR8 genes, with the construction of a biobrick that contains both genes and a Kanamycin resistant marker.
Laboratory work played a very important role in our project and it lasted about 2 months, from early April to mid June. As we are high school students, some of us were not as familiarized with laboratory practices as we wanted to be. So, we spent a few weeks learning everything we need to be aware of and to know about the laboratory. Our PI, Sarah G. Hualde gave us classes about the procedures we were going to follow inside the lab, about the protocols, proper equipment use and how to work safely there, by learning what are the necessary security measures. As a multidisciplinary team, we did not have a specific job for every team member, whereas we tried to rotate in every laboratory job as we wanted every team member to participate in every labor. We thought that this would be the best way to learn and to enjoy the laboratory phase.
WEEK 1
Our first week inside the laboratory was very practical as we started with easy procedures to get to know the new workplace. We prepared some Murashige and Skoog Mediums (MS) with sucrose and without sucrose. After this, we tackle these mediums. As it is widely known, they are used as a plant tissue culture growth medium. Also, we prepared some LB Agar Mediums and tackled them.
Victor pouring the solution into the test tube.
Solution being shacked.
WEEK 2
During this week we started with the construction of our biobrick, following our third field of research. As we have explained, our biobrick consists of two specific genes: UVR8 and PAP1, also we added the Kanamycin resistance as it is one of the most frequently used selection markers for obtaining transgenic plants.
We started with reaction 1 where we cloned 35S promoter, UVR8 cDNA and NOS terminator. This reaction contained the enzyme BsaI and T4 Ligase and was placed in a thermocycler with the following program: 25 cycles of 37ºC 2', 16ºC 5'.
After this we electroporate this reaction into E. coli bacteria. Our first reaction inside the laboratory… What was going to happen?
Well, the next day we came back to the laboratory and saw the results. Oh no! There were only blue colonies! If the plasmid was correctly constructed the colonies must be white. We were very disappointed as we did not accomplish the assignment. But we did not come apart and remake the reaction and this second time we obtained white colonies as we wanted to.
After that, we took some colonies and put them inside some falcons with LB liquid and left them overnight at 37ªC and shaking.
At the same time, we sowed WT and PAP1 Arabidopsis seeds on MS plates. These seeds that contain the PAP1 gene, were transformed by the previous NBG team.
Andrea preparing reaction 1.
White and Blue bacteria colonies.
WEEK 3
We carried out the plasmid preparation via the miniprep protocol. To verify that the plasmid was OK we digested it with restriction enzymes. We used type II restriction enzymes (in this case: EcoRI and PstI). The expected fragments were:
- 2574 bp.
- 1601 bp.
- 654 bp.
- 622 bp.
An agarose gel was used to separate these fragments.
After an hour, we saw that everything was correct. And the plasmid was correctly built.
At the same time, during this week we had to transfer the WT and PAP1 plantlets from the MS plate to soil.
WEEK 4
Ole! As the first reaction was done successfully, we could proceed with reaction 2. In this case, our objective is to unify in the same construction the two selected genes: the UVR8, produced in reaction 1, and the PAP1 gene selected by the previous NBG team.
To prepare the second reaction, we had to follow the same procedure as with reaction 1. In this reaction, we used BsmBI, T4 ligase and the pDGB1Ω2 plasmid. We set the reaction in a thermocycler with the same conditions that in the reaction 1 (foto 9) . After electroporation we place our plates at 37ºC overnight.
Sergio preparing reaction 2.
Later on, we introduced it inside a thermal cycler as we wanted to unify the mixture and after this, we electroporate reaction 2 as we did it with reaction 1.
We left it inside the fridge, to see better the results, and the next day we came back to the laboratory and saw that we obtained white colonies as we wanted to.
After that, we took some colonies and put them inside some falcons with LB liquid and left them overnight at 37ªC and shaking.
On the other hand, we also had to continue with the cultivation of the plants. This week we air out the oil and we select and leave only one plant per pot.
Selected plants one week after transferred to soil.
WEEK 5
This was a very important week, we start with the characterization of Arabidopsis plants overexpressing PAP1 and characterization of Arabidopsis plants exposed to volatile compounds.
First of all, to finish with reaction 2 we follow the same procedures as with reaction 1 in week three. We made the miniprep protocol, digest the plasmid and finally, the electrophoresis in agarose gels, in order to analyze the size of the fragments. Everything was in order!
To prove if Arabidopsis overexpressing PAP1 and Arabidopsis exposed to volatile compounds had higher protection against UV radiation, we characterized them.
It is known that plants exposed to volatile compounds grow faster and have an increase the amount of photoprotective pigments. We use Alternaria Alternata fungus as the producer of volatile compounds. We put our WT plants with A. alternata fungus inside some plastic boxes and covered them with PVC film.
WT Arabidopsis thaliana plants in contact with Alternaria alternata fungus.
At the same time, we exposed both groups of Arabidopsis, WT and PAP1, to a mix of UV-B and white light for 2 hours and 30 minutes inside a phytotron. Two days after UV-B exposure and before the biochemical characterization of the plants, we carried out a visual exam, comparing the damage produce by UV-B exposure to WT plants and PAP1 plants.
Arabidopsis plants exposed to UV-B light inside the phytotron.
WEEK 6
One month and a half later after the start of the laboratory work, we started with the reaction 3 to produce our biobrick. In this last reaction we introduced the kanamycin resistant gene (Kn).
In order to prepare reaction 3, we had to follow the same procedure as with reaction 1 and reaction 2. In this reaction, we used BsaI, T4 ligase and the pDGB3a1 plasmid. We set the reaction in a thermocycler with the same conditions that in the reaction 1 (foto 9) . After electroporation we place our plates at 37ºC overnight.
Alex and Ana preparing the reaction 3.
At the same time, we acclimated the Arabidopsis WT plants that were being exposed to the volatile compounds.
Leyre showing the Agrobacterium tumefaciens bacteria culture.
Agrobacterium tumefaciens bacteria culture.
WEEK 7
The next day we came back to the laboratory and took some white colonies and put them inside some falcons with LB liquid and left them overnight at 37ºC and shaking. We made minipreps, digestion with restrictions enzymes and electrophoresis in agarose gels. Our final plasmid was finish.
In our last laboratory week, we finished the 3rd reaction, and, exactly as with the previous ones, We carried out the plasmid preparation via the miniprep protocol. We verified that everything inside the plasmid was well displayed, making the digestion of the plasmid using EcoRI and PstI restriction enzymes, and the expected fragments were:
- 6345 bp
- 3176 bp
- 2639 bp
- 654 bp
Finally we have our biobrick finish! Now it is time to transform Agrobacterium tumefaciens with PAP1-UVR8 plasmid. This A. tumefaciens will be used to transform A. thaliana plants.
Why Agrobacterium tumefaciens?
Agrobacterium tumefaciens is a bacteria that lives in the ground and infects the stems of plants that are wounded. Moreover, this bacteria inserts a part of its genetic information into the plant's cells. This DNA has some genes that express specific compounds, opines, which serve as a source of nitrogen for the A. tumefaciens. How do we use this attribute? Through genetic engineering, we replaced this gene with the one we were interested in, inserting it into the plant. Allowing us to introduce the construction we created into the Arabidopsis' DNA by using the Agrobacterium tumefaciens natural method of infection.
The method used to transform Arabidopsis is called the floral dip method. This method consists of dipping Arabidopsis flower buds in a suspension that has modified Agrobacterium cells, with our constructed plasmid. This way, we favor contact between the female gametes and Agrobacterium.
That same week, we did our last UV radiation experiment, irradiating the WT plants that had been exposed to A. alternata volatiles. Two days after the UV treatment we carried out a visual exam of the plants and we harvested the irradiated plants and the control plants (non irradiated with UV)
Do you remember the other plants we irradiated 2 weeks ago? It was time to do the pigments measurement. This data will give us information about if our work had been successful and our plasmid worked. To do that, we used the anthocyanins and carotenoids measurement protocol, which consist of different steps, anthocyanins having a more long process.
Anthocyanins and carotenoids measurement protocols.
Carotenoids measurement protocol:
Lichtenthaler, H. K., & Buschmann, C. (2001). Chlorophylls and carotenoids: Measurement and characterization by UV‐VIS spectroscopy. Current protocols in food analytical chemistry, 1(1), F4-3.
Anthocyanins:
Teow, C. C., Truong, V. D., McFeeters, R. F., Thompson, R. L., Pecota, K. V., & Yencho, G. C. (2007). Antioxidant activities, phenolic and β-carotene contents of sweet potato genotypes with varying flesh colours. Food chemistry, 103(3), 829-838.