Integrated Human Practice

Our lab has no experience of working on B.subtilis before and so it's a tough decision to make which strain should we use and how do we get the specific strains, commercially or from our institute.

Prof.Liu is from the School of Life Science and Biotechnology and working on B.subtilis. we met him in his lab and he briefly introduced several common-used strains to us. We finally decided to use WB800N, because this strain is a highly proteinase-deficient one, which can guarantee the stability of tyrosinase, a protein produced and anchored on the outer surface of our spores.
Prof.Liu then listened to our design of engineerings on the bacteria and shared with us some documentations on common manipulations such as how to do the electrotransformation. Besides, to our great surprise (literally), he kindly gave us a tube of the strain WB800N he freezed in refrigerator for 10 years!

We decide on our strain of B.subtilis to use and successfully get the WB800N in glycerol stock, which spured the process of our wet-lab work.

During our own research on papers in the field of spore display system, we were surprised to find few papers working on multi-anchoring proteins and this made us wondering whether our prospect of using different proteins, namely cotE and cotB, feasible, or maybe some obstacles existed but we couldn't find clues in research papers. So we contact Prof.Wang from Qilu University of Technology and he is an expert in employing the spore display system in the food industry.

Prof.Wang had a casual conversation with us about our questions on the use of anchoring proteins. He mentioned that different Cot proteins have different weights as distributing on the spore surface. Therefore, we could not only use different Cot proteins to display our target proteins but also we can even to determine the quantity of our total amount! He encouraged us to do so since the amount of enzyme and its spatial structure matters a lot to the enzyme activity. He also pointed out some typical difficulties we may meet in doing experiments with spore display technology, such as the method of sporulation and spore purification.

We learned a lot during the chat with professor Wang, especially about the feasibility of our project. We had confirmed using two or even more Cot protein is somewhat accessible so that we can continue designing our project. Besides, his suggestion of quantifying our enzyme and melanin-binding peptide really inspired us to think about the effect of quantity ratio. This idea leads to our efforts in searching for related studies concerning the distribution of spore surface protein (but we finally give up to do so since that would lead to too large plasmid transferred). Besides, his mentioning of enzyme activity, which maybe slightly or largely differentiate from the original molecule in its pure protein state made us design experiments on enzyme activity and kinetics tests.
What's more, we later figured out that a published paper whose authors also used plasmid pDG1730 (which we plan to use to transfer DNA information) is from Prof.Wang's lab! So we e-mailed for the plasmid and he kindly gave us!

Doctor Yin is from Fan' Lab in the Institute of Translational Medicine. Prof.Fan is a academician of the Chinese Academy of Sciences and his research interest is largely on the nucleic acid materials and DNA data storage technology. We meet them for professional advice on the general idea for our space information traveling with DNA storage technology and store them in the B.subtilis.

After reading our basic plan, Doctor Yin thought our design is practical by combining DNA data storage and B.subtilis.
During the conversation, we discussed the benefits of storing DNA in vivo. We were a little bit confused about this at that time since from reading papers related, we learned that scientists are exploring storing DNA in many materials, for instance, the inorganic materials such as silicon or salts. However, Doctor Yin tell us that storing DNA in vivo may not be the one with most density, compared to storage in solid, but it is now widely used when researchers trying to test the stability and fidelity of their DNA information. This is partly due to the ease of later manipulations, such as sequencing the DNAs, which we plan to do as well. And transferring DNA information in vivo, mostly to E.coli cells though, are convenient in labs.
After listening to our plan of designing a new mode of encoding DNA information, she suggested that many code book developed by scientists worldwide are similar in basic logic but having their own features or application scenario. We could get some inspirations from previous works and make a codebook featuring usage in B.subtilis, which may be specifically restricted by the rules or genome structure of B.subtilis.

Through the conversation, we are more confident on our project design of making the use of microorganisms to store DNA information instead of the inorganic ones, since in vivo storage is a more mature technology with much more convenience for sequential manipulations. Besides, we gain some tips to develop a unique coding method, leading us to search for more related information. We later figuring out that apart from common rules we've known, DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. And this rule became a constrain in our modeling.

Lin is a doctor candidate studying in the lab of Prof. Wang from Qilu University of Technology and interested in the techniques of spore display system. During the process of our project, we encountered many problems because few of our team members nor the labs in our university has abundent experience or materials in working with B.subtilis.

We got in contact luckily with Lin and had several discussions online. When we handled WB800N and our plamids pDG1730 (gifted by their lab) for the first time, Lin was closely stay with us and provide timely suggestins on the process.

The help from Lin definitely guarantees the fundamental success of our experiments, she gave us lots of advice on the culture and sporulation of B.subtilis. For example, from her kind illustration on the principle, we finally decided to DIY a sporulation medium since purchasing the specific Difco medium from abroad would be quite challenging under the pandemic. Besides, When we got in stuck in the failure of selecting the correct strain which is supposed to be transferred with our Amp-resistant plasmid, she saved us by suggesting changing the common usage of Amp to Cm for the plasmid may be dual-resistant and showing antibiotic-preferrence in different species.

At the beginning of our design, we were enamoured in the idea of astrobiology which is part of our core value and so much correlates with the fantasies and imaginations and many interesting problems arised from the concerns regarding what's beyond our knowledge today. However, we were gradually confused by many theories and terminologies within the large and vague range of space synthetic biology. Therefore, we managed to get in contact with a scholar in this field and Doctor Shen kindly shared with us his understandings.

Doctor Shen is from The Institute of Geology,Chinese Academy of Geological Sciences and work in the lab of Prof. Wei Lin. Prof.Lin is interested in the astrobiology, and their lab even designed a ballon-like experimental platform to study microbial metabolisms in the near space, where the conditions is very much stimulating Mars so that scientists can use it to perform experiments conveniently!
Doctor Shen firstly discuss with us differences between the those various terms. Tough difficult, but these an still be telling part. Astrobilogy, cosmic biology, axmen... are definitely supposed to use under different circumstances. In this term, our project, depending on its application scenario, can also be devided in to dffererent catagories.
We also asked questions regarding the environments of outer space to deal with the problem of radiations. Doctor Shen recognized our emphasis on radiation especially when protecting DNAs, he gave us some information on what scientists have found about the possible mechanisms contributing to the damage on DNAs caused by the fatal radiations everywhere in the vacuum space. Last but not least, we consulted Doctor Shen about the popularization of astrobiology in the public. Prof. Lin once wrote a significant review regarding the astrobiology development in China and appeal for popularizing astrobiology among the public to attract more people, especially the youth to devote themselves in the amazing field and promote the development of astrobiology both home and worldwide.

Through the conversation, we are more confident on our project design of making the use of microorganisms to store DNA information instead of the inorganic ones, since in vivo storage is a more mature technology with much more convenience for sequential manipulations. Besides, we gained some tips to develop a unique coding method, leading us to search for more related information. We later figured out that apart from common rules we've known, DNA previously handled by this host was limited to that with GC content similar to or lower than that of the B. subtilis genome. And this rule became a constrain in our modeling.

After we confirmed the primary topic of interstellar trip, we were eager to gain more knowledge about the conditions human need to live in space in long term. Coincidentally ,We noticed an online lecture about how humans could live on Mars for more than a year by Prof. Wei ,leading us to contact him for advice and information.

During our online contact, Prof. Wei shared his thoughts on how humans could live on Mars for more than a year, and then recommended Professor Lin Wei, an expert in Geology, and Professor Zhu Xian, an expert in synthetic biology to us. He highly acknowledged our idea of space applications.

It was Prof.Wei's generous help that gave us an opportunity to meet Prof.Lin and Prof .Wei who were experts in our research fields. Meanwhile, his encouragement motivated us to think wild about the application of our project. We did it .

Prof. Rao is an expert in the field of spores’ tolerance to extreme conditions. He is interested in the architecture of spore coat, formation of spore surface and killing spores with special instruments which can provide heat or high pressures. Therefore, we found somewhat similar in our interests and get in contact with him, hoping to get some inspirations from his knowledges and solve our problems regarding to safety concerns as well.

We held an online meeting with Prof. Rao and enjoyed a fruitful time! Prof.Rao works in the field of spore germination and killing spores, and he was pleasant in answering our questions regarding spore tolerance. He first introduced several mechanisms parting part and further explained the reasons from 4 points, and that is SASP protein, low water content, thick spore coat and inner layer of spore surface. Among which we are very much interested in encountering SASP protein since it plays a similar role in protecting spores genome, just like Dsup proteins.
Besides, several suggestions on protocols also had been proposed by professor Rao which influenced our project largely. We found that extract DNA from spores is very difficult without specific instruments, but it is a must-to-do since we shall finally test our goal of protecting spores’ genome by evaluating its damage rate after exposed to radiations. So we come up with the idea that we shall let spores germinate into bacteria at first in order extract DNA more conveniently. However, Prof. Rao pointed out that spore shall start repairing DNAs during germination which shall bring mistakes into our statistics. He recommended to clash spores with tiny beeds and maintain the form of spores for them nearly have no metabolism and stable enough to keep information untouched.
Issues about bio-safety were also consulted during the meeting and the discussion are showing below in the safety.

We added log formulus in our modeling to assess the viability of spores amid harsh environments, and this provides a new idea on how to calculate the number of spores we need to carry one copy of information!
We changed our protocol of “first germinate then extract“ strategy to extracting DNA from spores straight by some mechanical operations. However, we do not obtaining the proper instruments so that we failed twice. Fortunately, Prof.Rao is very pleased to help! He firstly offer to help us extracting if no proper instruments available, later, with the blckdown of pandemic around school, package is not allowed. But he still keep online conversations with us to give advice on adjusting the protocol into our labototry environments.

As we were looking for some proof for the application of our Odyssey, Professor Zuo become our savior for his abundant research experience in DNA information computing and storage.

We coincidentally saw his post online,inviting everyone who was interesting to have a academic discussion . We felt lucky to grab this chance and meet with him on Aug.26. We briefly showed him our work and asked him about the frontier news about DNA data storage. We learned that the DNA information storage was mainly used in cold data storage as those data didn’t need to be accessed frequently and was inefficient to be stored in traditional storage medium. Meanwhile , he also hints us that the biosafty is a important focus in our project.

Prof.Zuo made a deep influence in our design part , digging into our most principle questions---Why are we doing this. It turns out that our Odyssey system can be broadly used in cold data storage such as ancient antique information or ancient books. It was his patient words that inspired us to realize this point. Moreover, his opinions on biosafety such as the leaking risk of our spores was open-minded, resulting in our more attention to this area where we might ignored in the first place.

Our project wants to apply DNA information storage technology to space transportation, which requires us to find a better DNA storage algorithm. After extensive literature search, we chose Yin-Yang Code as our information storage algorithm. However, we did not understand some of the contents in the paper thoroughly; there were also errors reported during the running of the code, resulting in files that could not be converted into base sequences. This makes us wish to communicate with the authors of the Yin-Yang Code paper.

The authors of the Yin-Yang Code paper are mostly from the UW Life Sciences Institute in Shenzhen, with rich experience in synthetic biology and DNA information storage. In the communication with Dr. Ping and Dr. Shen, the authors of Yin-Yang Code, they were very positive and enthusiastic, and they replied very fast! In the first email exchange, we asked some detailed questions about the Yin-Yang code, including the reasons for the use of index and monitor, how the number of bases per segment is obtained, the error correction method of RS code, the equivalence of the 6144 coding rules, etc. Dr. Ping and Dr. Shen enthusiastically responded to the relevant programming details, deepening our understanding of the Yin-Yang code algorithm and guiding us in the use of the Yin-Yang code. In the second email exchange, we asked about the criteria for judging whether the coding rules are good or not, the error correction capability of RS code, the choice of index and RS code length, and the countermeasures for sequences that cannot be coded with exactly the right pair. Dr. Ping and Dr. Shen responded positively, which gave us a thorough understanding of the structure and framework of Yin-Yang code itself and provided us with good theoretical guidance for programming with Yin-Yang code. In the third email exchange, we introduced our project to the UW Life Sciences Institute and wanted to hear each other's opinions and suggestions. Dr. Ping and Dr. Shen were highly concerned and expressed their views on the superiority and practicality of DNA information storage in space with interstellar transportation. They suggested that we could transport a large amount of information at one time in the form of a database or library to save the cost of interstellar transportation. At the same time, they argue that the low temperature and the absence of DNA enzymes in space are favorable for interstellar transport of DNA. The irradiation conditions in space are noteworthy and tend to trigger the breakage of DNA molecules; sudden high temperatures may also lead to damage of DNA files. These suggestions provide ideas for our mathematical modeling.

We understand the use of shaded codes more thoroughly and we decided to use shaded codes with high information storage density and stable base sequences obtained to encode our deposited information. Also the suggestion of Dr. PingQuan and Dr. Yue Shen made us think that it is necessary to design and model the damage to budding spores in terms of cosmic irradiation and high temperature.

Doctor Deng is from Ting Zhu's Lab in Pecking University. Prof. Zhu is a seicentist famous for his research in DNA data storage by dextral DNA. We get in contact with Doctor Zhu to get some professional suggestions.
That's when we met Doctor Deng

During our meeting with Doctor Deng , we routinely described the designs and goals of our project. Doctor Deng responds positively and made several advice. First, our idea is interesting and novel, but we also should achieve some breakthroughs in our models and wet experiments. Second, we should evaluate the balance between the spores's ability to store information and its viability so that we can make sure this technology works. Last , the idea of store information in living creatures is fascinating ,but we still need to think systematically about the advantages so that we can better convince our potential users.

Doctor Deng provided us with so many constructive ideas that his ideas became some of our fundamental way of thinking during our projects. In wet lab and modeling ,we found it meaningful to deal with the question he raised. What's more , his opinions on our novelity made us reflect more on the initial pursuit of our project, motivating us to improve it over and over.

Prof Tao is working in ther field of the development and application of high-throughput technology, which invovled the research on affinity-maturation evolution. We get in contact with him through E-mail to humbly collect information for our models of direct evolution.

We had several E-mail contact to solve our puzzles. As we lack the basic knowledge of direct evolution, we consulted him to find out if we were using the right online tools to predict the model in the first place. Besides, he suggested us to verify our predicted model through quantitative experiment for protein-melanin &DSUP- DNA affinity.In order to standardize our experiment ,he also suggested some published standard protocol to screen the phage peptide libraries.

Prof Tao showed us the better way to conduct the direct evolution through wet experiment. Although the pandemic in September and October made his experiment impossible, we were still really grateful to his generous help.