Team:Shanghai City/Design
Design
Our iGEM team is dedicated to DNA encryption and storage of information and secret communication. The following four levels describe our topic and compare it with previous technologies.
1. The first encryption level is the combination of DNA encryption and computer technology. How do you translate that information into the sequence of DNA (ATCG)? We adopted the method corresponding to the password table or further strengthened combined with computer science. We also designed a 4W (who, where, when, what) one-step information assembly method similar to golden gate.
2. The second level is the application of steganography. Clelland et al. first used biosteganalysis to hide a meaningful piece of DNA in genomic DNA, then decoded it by polymerase chain reaction (PCR) using a pair of primers (keys), and then sequenced. We also tested this.
3. The third level is encryption or steganography of the secret key (in this case, the Primer) itself. (1) Encryption: we perform asymmetric secret key encryption similar to computer Encryption on two primers.(2) Steganography: on the basis of CADS method (long primers), add more complex information interference items, including single and double stranded DNA, etc.
4. The fourth level is the preservation of DNA. The DNA is stored on paper, making it harder for a thief to decipher. As far as I know, this is the first time DNA has been used for encryption on paper.
◦ Cloning
▶ The process of transformation
▶ Blue white screening
▶ Checking the bacteria
◦ Principle
◦ Encryption
▶ Physical protection
▶ DNA sequence
▶ Mixed primer
▶ Mixed information
▶ Computer encoding
◦ Contrast
Cloning+transformation
the experiment is designed to obtain and confirm the right DNA segment in a situation where the segments are mixed; and cloning is where it starts. E-Coli (DH5a) bacteria is chosen to be plasmid in the experiment due to its stability and the fact that it's easier to duplicate. The right and wrong DNA segments, known as connection products are injected into the E-Coli which is known as competence. Subsequently, in order to provide a comfort environment for E-coli to coalesce with the DNA segments, this mixed products first is placed on the ice for 30 mins, then through a 90s of thermal excitation at 42˚c to form small pores for the segments to enter smoothly. Next, we place the added E-Coli in a 37˚c environment with continually
gentle shaking for 45 minutes, due to the reason that E-Coli was fragile originally, providing a comfy circumstance can activate the growth of E-Coli. After the waiting, placing the reagent on the Petri dish in a relative sterile working space, leaves E-Coli to grow overnight.
figure 2: the demonstration of how blue-white screening work
Blue- white screening:
The blue-white screening technique is a method to separate the ones contain DNA segments and ones that's not and the wrong ones. In our experiment if E-Coli is connected to the right DNA segments, the dots will be white, which is the color of E-Coli itself. Whereas, if the colored dot is blue, which is a indicator of inactivation, it means either E-coli didn't connect to any segments or the wrong ones. However, there is exception, which E-Coli connected to both the right and wrong segments, in order to increase the chance of getting the right DNA segments, we picked out 64 single white bacterial colonies as samples for use to test, which is not touched with other colonies due to the reason that just single colony contains millions of bacterias and accuracy.
figure 3 is the process of PCR and components
The process of checking contains two parts, PCR (polymerase chain reaction) and electrophoresis. PCR is a method to get large number of copies of segments through the changes of temperature in the PCR machines. The process is a preparation for electrophoresis, since the segments will show as bars in the electrophoresis machine, and the larger the number the more clear it can be see in the graph.
figure 4, an example of electrophoresis graph/bar
electrophoresis:
Electrophoresis is a way that involved the migration and the separation of the charged ions under the influence of electric current. The method relates to a fuhkd under the influence of the spatial electric field(positive and negative), which will separated the particles which the positive ones will attracted too one side, while negative particles will be on the other side.
In our project, five methods are mixed to protect the information. The first layer is physical protection, which is on the paper; DNA is invisible. Therefore, if no one tell you there is DNA on this paper, it's not easy to find DNA reagent. The second layers is the DNA sequence, DNA (Deoxyribonucleic acid) is made up of molecules called nucleotides, which contains a phosphate group, a sugar group and a nitrogen group. The four types of nitrogen group are adenine(A), thymine(T), guanine(G) and cytosine(C). The order of these badges is determined by the DNA’s instruction. Three nitrogen bases will form codon, which will represent a letter or number on the keyboard.
Then we will employ those codon formed letter to write a message which will form a sequence.
Making our information more complex and safe, we will add mixed letters (nitrogen bases) in the sequence. The next step is to add primer, which will be applied to decode the mixed information, in the reagent. However, if we only contain one primer that will be too easy to solve, more primers(fake and the real) will annex in the regents. With will the pieces of information and primers, eventually it will be coded in the computer.
How to obtain the right information?
Due to the complication of this reagent, this mixed sequence will be sent to scientific laboratory to resolve, which will require special protein, Cas12a, to mixed with the crRNA and primers after decoding through the computer. Under the special characteristics of Cas12a, which will protect the main information and cut the useless information out with the primers and crRNA. The information will be extract through right portion of Cas12a, and crRNA.
Our technique is an more advanced technique to conserve information than other others. The original method that most people are using today is to mix DNA with the 293T (junk information) together, which will be replicate multiple times; and whoever is getting the information can use the primer(short nucleic acid sequence that provides a starting point for DNA synthesis) to match the primer with information and obtain informations. Yet, this method isn't secured anymore since a hacker can employ the primer to get the information directly, but our method is safer since few complicated methods are mixed together to set obstacles for the people who not suppose to obtain this information.
Key Reference
Li, S. Y.; Liu, J. K.; Zhao, G. P.; Wang, J., CADS: CRISPR/Cas12a-Assisted DNA Steganography for Securing the Storage and Transfer of DNA-Encoded Information. ACS synthetic biology 2018, 7 (4), 1174-1178.
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