Team:Shanghai City/Experiments
Part 1
DNA storing on paper& Dissolving DNA storing on paper
Introduction:
It is the level of encryption to store the DNA on the paper. After dropped on the paper, the DNA solution will evaporate and DNA inside which is kept on the paper. As it is hard to test out whether or not there is DNA on a certain paper, this method for storing DNA is safe. What’s more, paper is cheap and normal, which means everyone can get and carry it easily. Additionally, DNA has a high solubility, which means that as long as recipients immerse the paper they get into water, the DNA can solve into it and become solution state again, which is easy for the recipients to extract and test.
Our target is to find out whether storing DNA on paper is practical and which kind of paper, which way to store the DNA can have the best effect.
Experiment 1:
Reagents:
Experimental steps:
1.Using scissors to cut the filter paper into four triangular pieces. 1 μL of template DNA solution at 0.1 times the original concentration and at the original concentration were used to wet the tip of the four filter paper pieces. Dry it.
2.Configure the solution according to the concentration of reagent in the table above (excluding template DNA).
3.The reagent was divided into four tubules, each tube containing 24 μL mixing liquid.
4.Cut off the air-dried paper tips with scissors and put them into four small tubes.
5.Put the tube containing the paper into the PCR instrument to amplify.
Experiment results:
Electrophoresis proved that PCR could successfully amplify the DNA coated on paper. However, not all of the four produce visible bands. Only one of the two that contain 0.1 times the original concentration of template DNA does not show any bond, and one of the solution with the original concentration of template DNA does not show any bond as well.
Experiment reflection:
Firstly, there may be some errors during the experiments, so we decided to repeat the experiments again to test it.
Secondly, it is possible that the paper containing in the tube during PCR makes it less successful. There is to factors that may result into it:
1.During PCR, the temperature in the instrument is varied, which may make certain chemical element in the paper become less stable, and it disturbs the result of PCR.
2.The paper absorbs some water and make the total volume of the liquid in the tubes become less than 25 μL. As the PCR instrument in our lab require that the volume of the liquid inside it be within 25 μL and 50 μL, too little liquid may negatively influence the result of the experiment.
For the first reason, we repeated the experiment again, and the results are still unpredictable. As a result, we design another experiment.
Experiment 2:
Reagents:
Experimental steps:
1.Prepare 10 times the volume of the above solution (except template DNA) according to the above table.
2.Divide the solution into 12 tubules, in which each of the 6 tubes contains 24 ul, labeled as 7-12, and the other 6 tubes each contains 48ul, labeled as 1-6.
3.Template DNA 10ul at 0.1 times concentration.
4.1 times the concentration of template DNA 1ul each was added into 7-9 tubes.0.1 times the concentration of template DNA was added to 10-12 tubes, 1ul each.
5.Cut the filter paper into six similar triangles, marked as 1-6.
6.Add 1ul template DNA solution with original concentration on the tip of 1-3 filter paper; Add 1ul 0.1 times template DNA solution to the tip of 4-6 filter paper.
7.put 1-6 pieces of paper into 1-6 tube, making sure that the tip of the paper immersed in the liquid. Let them stand for 10s.
8.The paper was taken out and 12 tubes of liquid were put into the PCR instrument to amplify the DNA.
9.The amplified DNA was examined by electrophoresis.
Experimental results:
As can be seen in figure 1, except the first one, all 11 tubes are banded and the results show that the PCR amplified segments are correct. (M represents marker)
Experiment reflection:
The results showed that PCR could be performed correctly with the paper removed. But the strips produced by dropping a DNA solution onto a sheet of paper and then eluting the DNA on it are darker than they would be if they were added directly to a liquid. This may be caused by:
1.The DNA on the paper cannot be completely eluted, there are some remnants. 2.Paper absorption caused by part of the solution volume is less, resulting in the dark band.
Then, we decided to test which kind of paper has the best effect of storing DNA and we designed the following experiment:
Experiment 3:
Reagents:
Experimental group:
Experimental steps:
1.Cut 7 sheets of paper into long triangles.
2.Drop 2ul true template DNA and 2ul false template DNA on the tips of 12 sheets of paper respectively, and smear them with the tip of a gun to make them absorbed.
3.Prepare 14-tube solution according to the above ratio (except template DNA).
4.The 14-tube solution was placed into PCR for amplification.
5.PCR results were detected by electrophoresis.
Experimental results:
Figure 2
From the results of glue running, it can be seen in figure 2 that groups 3,6 and 7, namely, stored DNA best in paper jam (white), IGEM sticker and solution.
The PCR fragment sequencing
Experiment reflection:
The brightness of electrophoretic bands is limited by many factors. The reason for the difficulty in storing DNA in groups 1 and 2, the white and yellow notebooks, may be that the notebook pages have a low water absorption capacity, making it difficult for DNA to adhere to the paper. However, in group 5, the DNA storage paper group, the dark band may be due to the strong water absorption of the DNA storage paper, resulting in a small amount of residual solution, which leads to the insignificant band of electrophoresis.
Part 2
Disturbing DNA (HEK-293T) & Amplifying DNA with true primers
Introduction
Basically, storing information in DNA is to design certain specific base-pair sequences through certain cryptosystem, including binary system and so on. In order to avoid other people getting and stealing the information, that is, the correct piece of DNA, hide the significative piece into a large amount of disturbing DNA is important. In this experiment, we put the significative piece of DNA into the HEK-293T DNA. The true fragment of DNA is only 164 bp, while the disturbing DNA is about three billion (3 000 000 000) bp. As a result, it is nearly impossible to get the right piece from such a large amount of meaningless DNA without the key——primers. During PCR, two primers can work as keys to target the true piece and amplify it. After amplified, the true piece can be easily got and sequenced.
Experiment:
reagents
Experimental steps:
1.Use scissors to cut the filter paper into triangular pieces. 2 μL of template DNA solution at 0.1 times the original concentration and 0.1 times the concentration were used to wet the tip of the four filter paper pieces. Let it air dry.
2.Two samples of 0.3ul 293T DNA were collected and dropped on two pieces of paper tips with the original concentration over the original concentration and 0.1 times the concentration of template DNA. Let it air dry.
3.Configure the solution according to the concentration of reagent in the table above (excluding template DNA).
4.The reagent was divided into four tubules, each tube containing 24 μL liquid.
5.Cut off the air-dried paper tips with scissors and put them into four small tubes.
6.Put the tube containing the paper into the PCR instrument to amplify.
Experiment result:
The result of the electrophoresis, that all the bands locate at around 100 bp, shows that the disturbing DNA will not influence the decryption of the message with the correct primers.
Part 3
Disturbing primer & Cas12a protein
Introduction:
Because of the disturbing DNA, a pair of true primers, which are keys, is needed. Thus, it is important to make it safe to transport the keys. Because of it, we design certain way to encrypt the primers. In order to decode it, we use a special protein called cas12a protein.
Cas12a protein contains a unique trait, which they will crush all the single-stranded DNA. However, recent study found out that this protein can bind with a special kind of RNA, called crRNA, which can also bind with certain single-stranded DNA whose base pairs sequence specifically matches the sequence of crRNA. As a result, cas12a protein, crRNA and certain single-stranded DNA form a ternary complex. After it, cas12a protein will crush all other single-stranded DNA except this DNA binding with it, which is the true DNA.
In order to get the crRNA that specifically match with the true primer, we need to transcribe the template crRNA, which is T7-crRNA-F, into the crRNA that specifically match with the true primers.
Experiment:
Reagents:
Experiment steps:
1.T7-cRNA-F and buffer were added to the two tubes that eliminated the RNA enzyme according to the above table.
2.Add crRNA-RSA-primer-F-R and crRNA-RSA-primer-F-R into those tube 1 and tube 2 respectively.
3.After mixing, anneal them in the instrument.
1.Mix the reactants above (except the templates), and divide it into 2 tubes.
2.Add template 1 and template 2 into the two tubes separately.
3.Put the 2 tubes into culture room in 37℃ for a night for reaction.
4.Purify the RNA in 2 tubes, naming them crRNA-RSA-primer-F and crRNA-RSA-primer-R.
False primer
In this part of experiments, we add some false primers into the true primer solution. If the false primers are kept during PCR, both the wrong message and right message will be amplified. As a result, they can be easily disturbed by the wrong information and the correct ones are protected. We design the experiment to test in which condition the cas12a protein can work most successfully.
Experiment 1:
Reagents：
For Cas12a reaction (20 μL):
For PCR system (25ul):
Experiment steps:
1.Purify/dilute the crRNA.
2.Making the base for electrophoresis.
3.Dilute the concentration of the initial primer to 300 ng/ μL by employing the function: [(ng/ μL * the amount you extract) / the desired concentration] - the amount you extract = the amount of H2O you add.
4.We will be mixing the materials for Cas12a reaction. One does not contain Cas12a and crRNA, and the one mix with Cas12a and crRNA.
5.We heat the initial primer for fifteen minutes at 75 Celsius, which we activate, cut, and separate the DNA inside the test tube and ready to pair.
6.After heating, we will first mix buffer, dNTP, templet, and DNA enzyme (considering the wastage, we need to make the mixture enough for 7 test tubes. Then evenly distribute 7 μL to each test tube and trash the excessive amount), then add 6 different primers to each test tube. Then put in the shaking machine to get rid of the air bubble.
7.Since initially the amount of Cas12a was added differently between two groups, we will end up with 12 test tube that need to go through PCR.
8.PCR consists five stages. (step 2 - 4 will repeat about thirty cycles because this is the actual step that DNA are reproducing)
9.Inject group 1’s six test tube into the base of the electrophoresis, then inject marker to separate with group 2’s six test tube.
10.Let the electrophoresis run for thirty minutes, then put the base under the microscope.
Experiment results:
Figure 3
The results in figure 3 showed that the false information and no CAS12a protein groups were significantly displayed, consistent with the false sequence hypothesis, and the correct information was displayed in the first group. But there was a problem in the Cas12a group. In theory, the correct information should be clear after the reaction of Cas12a.However, the results of electrophoresis showed that although the bands were in the right position, they were not obvious, indicating that Cas12a protein excised the wrong primer and left the right primer. But the electrophoretic bands were lighter, and the bands in the sixth group were lighter than those in the fifth group. Such results showed that the concentration of Cas12a protein was too high, and when the irrelevant primers were cut off, part of the correct primers were also cut out, resulting in too weak bands.
There are four reasons may cause this results:
Too much cas12A
Too little crRNA
Too low primers
Inappropriate reaction time
Basing on the reasons, we designed the next experiment:
Experiment 2：
1. Reduce Cas12a to 0.5 microliter and put it into 37 ℃ constant temperature water bath for 15 minutes
2. Reduce Cas12a to 0.5 microliter, and put it into 37 ℃ constant temperature water bath for 10 minutes
3. Reduce Cas12a to 0.5 microliter and put it into 37 ℃ constant temperature water bath for 5 minutes
4. Increase the crRNA concentration to 1.5 microliter and put it into the water bath at 37 ° c for 15 minutes
5. Increase the crRNA concentration to 1.5 microliter and put it into the water bath at 37 ° c for 10 minutes
6. Increase the crRNA concentration to 1.5 microliter and put it into the water bath at 37 ° c for 5 minutes
7. Increase the concentration of each primer to 3 microliters, and put it into the water bath at 37 ℃ for 15 minutes
8. Increase the concentration of each primer to 3 microliters, and put it into the water bath at 37 ℃ for 10 minutes
9. Increase the concentration of each primer to 3 microliters, and put it into the water bath at 37 ℃ for 5 minutes
10, do not change yesterday's system preparation, put into 37 degrees Celsius water bath pot for 15 minutes
11, do not change yesterday's system preparation, put into 37 degrees Celsius water bath pot for 10 minutes
12, do not change yesterday's system preparation, put into 37 degrees Celsius water bath pot time for 5 minutes
Ps: in all the 12 groups mentioned above, only single variable except Cas12a exists in the preparation system. All the reagents are kept in a cooler. (RNA degrades easily at room temperature, so it is put in the refrigerator.)
Experiment steps：
1.Twelve groups of Cas12a protein systems are respectively formulated. The concentration of components in each system can be referred to experiment1
2.The PCR system was prepared, and 12 groups of prepared test solutions were added into the PCR tubes with corresponding serial Numbers as primers.
3.Put all 12 test tubes into PCR and wait for the reaction to end.
4.Preparation of agarose gel. PCR reagents were mixed with 3 microliters and loaded into the gel for electrophoresis.
5.After the electrophoresis, put it into the imager for imaging observation.
Experiment results:
Figure 4
According to the figure 4, for the primer with 0.1 micromole, only a false primer was found, but no stray bands were found. All primers with 0.2 micromole showed bands, but all primers except true primers showed stray bands, and the second band of mixed primers was not clear. The emerging bands of 0.5 micromole primers were relatively clear, except for true primers, false primers and mixed primers with obvious stray bands. All primers except true primers had stray bands, mixed primers had weak stray bands, and false primers still had obvious stray bands. The experiment cannot be considered successful, but the results are obvious. Most of the problems are false primers, and the annealing temperature will be increased again, or the bands of false primers may be unclear.
Long primer
Long primers are similar to the false primer. However, being different with the false primers which is separated from the true primers, long primers are the combination of the true primers and the false primers. Its structure is shown on the graph below.
The A and D are the true primers, while the B and C are the false primers. If the long primer is kept during PCR, C and B will connect with the DNA and function. Thus, false piece of DNA sequence will be amplified and only the wrong information can be get. However, after the true primer, which is A and D, binding with the cas12a protein, the protein will destroy the rest part of the long primer and keep the correct part.
Experiment:
Reagents:
For cas12a reaction:
Reaction time: 15min, 20min.
For PCR system:
Experimental steps:
1.Add the above reagent (except crRNA) to the 4 tubes of RNA enzyme removal under ultra-clean platform
2.crRNA was added to 4 tubes on the ice.
3.Put 1,2 tubes in a 37℃ water bath pot for 15 minutes; Bathe three or four tubes at 37 degrees Celsius for 20 minutes.
4.Put the 4 tubes of water into 75 degrees Celsius immediately after the bath, and inactivate cas12a protein.
5.The mixed solution for PCR (24 μL system) of 4 tubes was prepared and 10 μL was taken from tubes 1,2,3,4 and added into 4 PCR tubes.
6.The four-tube liquid PCR was used to amplify the DNA.
7.PCR results were analyzed by electrophoresis.
Experimental results:
Figure 5
From figure 5, it can be seen that all of the four group get a clear band. However, the correct band should be around 100 bp, and the band shown is not at the right position, which means that the PCR amplified the wrong piece of DNA sequence.
Experiment reflection:
One possible reason that cause the failure is that during cas12a protein’s reaction, the false primer, which is B and C, are not destroyed totally. Even a slight residue of the wrong primer will result in wrong information. In order to solve this problem, we decide to re-edit the long primer and increase the length of the true primers. In other words, besides the part that will match with the crRNA, an additional part is created. As a result, even if the reaction of cas12a protein cannot completely destroy the unmatched piece of DNA sequence, it is more likely that the false primers are destroyed.
Disturbing double-strained DNA
The disturbing double-strained DNA containing wrong information is added into the primer solution, and it can match with the true primer and. Thus, if it is not removed before PCR, the wrong piece of DNA sequence will be amplified and it makes it hard to distinguish the true information from it. To decode it, we use a special kind of enzyme called DNA enzyme I. This kind of enzyme will degrade DNA. However, when cas12-a protein wraps the correct single-stranded DNA, the single strand will not be degraded by DNA enzyme I.
Experiment:
Reagents：
Experimental steps:
1.Add the above reagent to the 2 tubes that remove RNA enzyme under the super clean blench (except crRNA, add DNA enzyme I to tube 1, and do not add to tube 2, each tube is 120ul).
2.crRNA was added to two tubes on the ice.
3.Put 2 tubes in a 37℃ water bath pot for 15 minutes.
4.Put two tubes of water bath into 75 degrees Celsius immediately after finishing, and inactivate cas12a protein and DNA enzyme I.
5.mixed solution for PCR (50ul system) was configured according to the above ratio.
6.Divide mix solution into 10 tubes.
7.Add 20ul tube 1 liquid and 20ul tube 2 liquid to 5 tubes respectively.
8.Cut 4 kinds of paper into 8 long strip triangles.
9.Drop 2ul true template DNA and 2ul false template DNA on the tip of 8 sheets of paper respectively, and smear them with the tip of a gun to make them absorbed.
10.Poke 8 strips into the tube with the head of the gun, let stand for 10 seconds, and then take them out.
11.Add 2ul true template DNA and 2ul false template DNA to the remaining 2 tubes.
12.Put 10 tubes of liquid into the PCR instrument to amplify.
13.The amplification results were examined by electrophoresis.
Experimental results:
Figure 6
As can be seen from the figure 6, in the 6-10 without adding DNA enzyme I (right), due to the interference of double-stranded DNA, no distinct bands could be amplified. In the 1-5 added with DNA enzyme I (left), due to the degradation of double-stranded DNA by DNA enzyme I, although some nonspecific bands were produced, distinct bands appeared.
Experiment reflection:
The presence of the correct bands indicated that the time of DNA enzyme placement, reaction temperature and other conditions were basically appropriate. Some possible reasons for the occurrence of stray bands are:
(1) the production of non-specific bands caused by the wrong part of primer connection.
(2) the protein Cas12a and DNA enzyme I failed to completely destroy the false primer, leading to the amplification of the wrong DNA.