The field of aerospace has developed rapidly in recent years. With the instinct curiosity and advancing
technologies, mankind is heading towards the deep space and may carry out interstellar transportation in
the near future. During interstellar expeditions, the storage of information is indispensable.
Even if we draw our attention back to earth, it is estimated in the next 10 years, the Earth's silicon
resources can no longer meet the needs of information storage due to the information explosion. In this
year's iGEM, we thus try to find a new way to store information, especially in the context of space
travel. Among all information storage methods, the DNA data storage, as a comparatively new one, is
promising for this application scenario because of the high information density, long duration, low cost
and so on. Therefore, our team hopes to use DNA to store information for interstellar transportation,
and synthetic biology plays as a perfect bridge between them.
We want to use DNA as a carrier for information storage, which requires determining a proper algorithm to
establish the conversion between the binary content to be stored and the DNA sequences to be served as
carriers. There're many constrains amid such translation between 0101 and ATCG, for instance, DNA needs
to be synthesized in a way that meets the stability requirements and biological capability.
Therefore,
we went through publications and manage to find that Yin-Yang Codec system, primarily developed by
scientist from Shenzhen, China, is able to satisfy the need of transforming files to DNA sequences, as
well as storing information with high density and generating sequences that are easy to synthesize and
structural stable.
Therefore, we selected Yin-Yang Code as our DNA coding method. Meanwhile, considering that we need to
deposit the informative sequence into the genomic DNA of Bacillus subtilis, we need to add primers,
enzyme cleavage sites and aptamer sequences when designing the informative sequences for our project.
There are various storage methods with unique advantages and disadvantages. Of course, DNA itself is relatively stable in nature and there're many methods for scientists today to store DNAs. Among them, storing DNA information sequences in vivo is not only the most natural idea, but also can be amplified accurately at low cost with the replication mechanism while staying stable for an amazingly long time. Therefore, in this project, we intend to store DNA information in vivo.
Since the cosmic environment is different from the earth environment and our spores smay encounter unfavorable factors such as cosmic radiations, extreme temperatures, microgravity, etc., we wish to find a more resistant biological storage carrier, and this is why we think of B.subtilis.
Here're the reasons why we believe that B.subtilis is the microorganism which fits our project the most:
1
B.subtilis is a model organism in the field of synthetic biology. It is well-studied and safe.
2
B.subtilis is the only bacterium-based host able to clone giant DNA above 1000 kbp, which guarantees the insertion of large information content into its genome as we manage to carry about large pieces of information encoded in DNA along the journey in space. The method of how to insert large pieces of exogeneous DNA into bacteria genome is a field with many sophisticated techniques and more methods are under developing.
3
Spores from B.subtilis themselves are blessed with strong tolerance to harsh environments and the very capability of protecting their genome.
4
Spores are studied in the food industry to be modified with the Spore Display System with which we can efficiently anchor target proteins, such as enzymes, to synthesize melanin on the outer surface of the spore in order to give it extra protection.
With these idea in mind, we choose to store DNA information in the genomic DNA of B.subtilis first, and then induce the spore-forming fungi to produce endospores that carry the DNA information about along the journey.
Considering that the environment in the universe is much harsher than that on Earth, we decided to add extra protective strategies to the spores. And a method to realize that is the spore display system. Spore display system employs coat proteins to serve as the anchor for fixing exogeneous proteins intended to be display on the spore surface. This technology has rapidly developed especially in the food industry where scientists manage to display enzymes on the spore surface for increase the enzyme’s stability.
We use this system on our project by choosing cotE and cotB proteins as the anchor. On the one hand, in order to cope with the DNA damage caused by the high intensity radiations in the universe, we manage to build a thick spacesuit on spore surface, that is, to package a layer of melanin. Melanin can absorb various radiations and thus effectively reduce the damages, such as DNA double strands breaks caused by cosmic radiations. We use cot proteins to anchor the tyrosinase and a melanin-binding-peptide named 4B4 on the spore surface, so that the melanin can be generated and sequentially fixed on the spore surface.
Melanin can be classified into different subsets, but all of them are synthesized by various tyrosinases from tyrosine or L-DOPA as the substrates. If we simply want to wrap the spores up with a coat of melanin, we can synthesize the melanin somewhere else but it is definitely more convenient to just anchor the enzyme to the spore surface.
Apart from melanin, Dsup protein is also introduced to our system. Dsup protein is a unique damage suppressor protein found in Tardigrade. It can bind to DNA strands and protect them, allowing Tardigrade to survive even at a high intensity of radiations. We engineer the spore B.suntilis with dsup in order to complement the melanin on the outer surface.
We believe that with these two levels of protection and the instinct resilience of spores themselves, the DNA information sequences we deposited can be preserved very well!
Functional
Verification Experiments
After basic experiments such as WB, SDS-PAGE and FCM for verifying the proteins expressions, we further performed three series of experiments to characterize the functions of exogeneous proteins.
Below are the specific experiments we design to verify:
- Whether Dsup protein can still protect DNA in the procaryotic organism B.subtilis?
- Whether the activity of tyrosinase change in a new environment that is the spore surface?
- Whether melanin-binding-peptide can link melanin successfully since the melanin polymers themselves is just a little bit smaller than a spore.
- Finally, to prove that our project is feasible in design, we shall produce spores, simulating space environment and extract the DNA out testing the mutation or damage rate.
Comet assay experiment
The presence of a large number of cosmic rays and high-energy particles in space poses a serious threat to the storage of our DNA information. In our lab, we can use the UV crosslinker to test easily.
Under UV irradiation conditions, short wavelengths of the UV light carry high energy and cause double strands breaks or damages.
The weight of the broken DNA fragments will be much smaller than the weight of the intact sequence, so the special gel electrophoresis will show a shiny comet head and a faint comet tail. Thus, the more serious DNA is after exposure to UV light, the longer the comet tail shall be. Based on this principle, we used the comet assay to verify the damage of Bacillus subtilis DNA by UV irradiation with or without Dsup protein.
Survival rate after exposed to UV light is also measured.
Tyrosinase Activity and Synthesis of Melanin
The synthesis of melanin requires tyrosinase to serve as catalyst to change the substrates (tyrosine or L-DOPA) into products. In our project, we choose to use the tyrosinase identified from B. megaterium because it does not need caddy proteins, leading it much more convenient to be an exogeneous protein.
The successful expression of tyrosinase can be judged by whether melanin is synthesized after the addition of the substrate; the change curve of melanin content with time can be used to infer the initial expression of tyrosinase and the size of tyrosinase activity by methods of modelling. Also, although the optimal conditions for this enzyme have been clarified the first time it is discovered, we are expressing it in a totally different environment thus considering the new optimal environment such as temperature and pH are relatively unknown.
We therefore design a series of temperature gradient and pH gradient experiments to fully characterize the tyrosinase's activity and kinetics.
Sequencing
The ultimate goal of our project is to establish extra protection measures by constructing a series of components to realize DNA information protection in bacteria spores. Therefore, sequencing, decoding and retrieval of the deposited sequences are necessary after a series of experiments simulating space environments.
We sought help from prof Rao’s lab in China Agricultural University in Beijing to lyse the thick spores with information stored. Genome of lysed spores were then extracted, used for sequencing target fragments and decoding the nucleotide with Yin-Yang codec system. By comparing the obtained results with the information deposited at origin, we can therefore deduce the mutation rate and information retrieval rate, and consequently prove our design.
