iGEM 2021 — Team USTC

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# Team:USTC/Notebook

USTC-iGEM-Notebook

[USTC-iGEM 2021](https://2021.igem.org/Team%3AUSTC)

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# Hello, Welcome to NOTEBOOK PAGE

# Notebook

# **Week1**18/7/2021-24/7/2021

1.Chemical competency of DH5α
2.Transformation of plasmids:
    template1\_PUC19
    template2\_pRSFDuet-1
    NA-template-pUC57
    AP-template-pUC57
    for amplification
    chose Amp+ plate to cultivate
3.The four plasmids were cultured with ampicillin resistant liquid medium to amplify
4.Plasmid extraction of the aforementioned four plasmids
5.Storage of four bacterium in 60% glycerite

# **Week2&3**25/7/2021-7/8/2021

1.PCR for NA and AP dsDNA from plasmids
2.Amplification of NA and AP dsDNA
3.Survey the conditions of asymmetric PCR for NA

# **Week4**8/8/2021-14/8/2021

1.Asymmetric PCR for NA and AP ssDNA (ratio of primer:20:1)

![](https://static.igem.org/mediawiki/2021/8/84/T--USTC--result_fig1.png)

2.Examination of the ssDNA products by recombination comparsion with

![](https://static.igem.org/mediawiki/2021/6/66/T--USTC--result_fig2.jpeg)

###### Figure2: Verification of double chain assembly mode

# **Week5**15/8/2021-21/8/2021

1.Test for the qPCR device’s sensitivity of detecting the signal
![](https://static.igem.org/mediawiki/2021/f/fa/T--USTC--result_fig3.jpeg)

###### Figure3: Experimental positive control template gradient

2.Test for the relation between template concentration and signal intensity(design 1.1)
Template concentration:
10
-8mol/L, 10
-9mol/L, 10
-10mol/L, 10
-11mol/L, 10
-12mol/L, 10
-13mol/L and blank
![](https://static.igem.org/mediawiki/2021/5/5d/T--USTC--result_fig4.png)

###### Figure4: Different concentrations of template

# **Week6**22/8/2021-28/8/2021

1.Expression and purfication of tau protein
2.Survey of hardware
3.Development of software

# **Week7**29/8/2021-4/9/2021

1.Test for aptamer’s ability to block the apmlification of template and relation between template concentration and signal intensity(design 1.1)
    Six groups of different template concentration:
        10nM, 1nM, 100pM, 10pM, 1pM, 0.1pM
    Every group is divided into aptamer-with and aptamer-without
2.After several repetition, errors happen. The wrong data is below
![](https://static.igem.org/mediawiki/2021/4/45/T--USTC--eng_fig7_new.png)

###### Figure5: Strange-Fig: ContraryAptamer Repression Test

![](https://static.igem.org/mediawiki/2021/1/17/T--USTC--eng_fig8_new.png)

###### Figure6: Strange-Fig2: Confused Amplification Curves at Different Concentration

# **Week8**5/9/2021-11/9/2021

1.Verification our hypothesis about the wrong data by directly amplifing the template chainwith primer1 and aptamer and modeling.

![](https://static.igem.org/mediawiki/2021/6/60/T--USTC--eng_fig10.png)

###### Figure7: Aptamer repressive test：From left to right, the template concentration decreased in turn, and the interior of each group: primer1 + primer2; primer1+aptamer; primer1+ddH2O （except marker)

![](https://static.igem.org/mediawiki/2021/3/35/T--USTC--eng_fig9.png)

###### Figure8: The fluorescence signal caused by aptamer shedding changes with concentration

# **Week9**12/9/2021-18/9/2021

1.Test for aptamer(with repressor group)’s ability to block the apmlification of template and relation between template concentration and signal intensity(design 1.1)
    Concentration of template: 10nM B:1nM C:100pM D:10pM E:1pM F:0.1pM
![](https://static.igem.org/mediawiki/2021/a/a4/T--USTC--eng_fig5_1_new.png)
![](https://static.igem.org/mediawiki/2021/1/12/T--USTC--eng_fig5_2_new.png)

###### Figure9: Aptamer Repression Test with different concentration of template chains: A:10nM B:1nM C:100pM D:10pM E:1pM F:0.1pM （1cycle=20s the same below)

![](https://static.igem.org/mediawiki/2021/f/f4/T--USTC--eng_fig6_new.png)

###### Figure10: The template amplification curves of different concentrations showed very discrete fluorescence signals.

2.Detection of different concentration of Tau protein in aqueous solution concentration of Tau protein(design 1.1): 0ng/ml, 1ng/ml, 2ng/ml, 4ng/ml, 6ng/ml
![](https://static.igem.org/mediawiki/2021/f/f6/T--USTC--result_fig6.jpeg)

###### Figure11：Different concentrations of Tau protein in aqueous solution

3.Detection of different concentration of Tau protein in aqueous solution concentration of Tau protein(design 1.2): 0pg/ml, 1pg/ml, 2pg/ml, 3pg/ml, 4pg/ml
![](https://static.igem.org/mediawiki/2021/2/26/T--USTC--result_fig7.jpeg)

###### Figure12: Different concentrations of Tau protein in aqueous buffer

# **Week10**19/9/2021-25/9/2021

1.PCR for template 1 and template 2 dsDNA from plasmids
2.Amplification of template 1 and template 2 dsDNA
3.Asymmetric PCR for template 1 and template 2 ssDNA (ratio of primer:20:1)
![](https://static.igem.org/mediawiki/2021/1/1f/T--USTC--eng_fig17b_new.png)

###### Figure13: Asymmetric PCR Result of template 1

![](https://static.igem.org/mediawiki/2021/b/b8/T--USTC--eng_fig15_0.png)

###### Figure14: Asymmetric PCR Result template 2

4.Test for the relation between template concentration and signal intensity(design 2.0)
Template concentration:10
-8mol/L, 10
-9mol/L, 10
-10mol/L, 10
-11mol/L, 10
-12mol/L and 10
-13mol/L
![](https://static.igem.org/mediawiki/2021/0/00/T--USTC--eng_fig18_new.png)

###### Figure15: The template fluorescence signal without protein and aptamer changes with concentration.

# **Week11**26/9/2021-2/10/2021

1.Detection of different concentration of Tau protein in aqueous solution
concentration of Tau protein(design 1.2):
0pg/ml, 1pg/ml, 2pg/ml, 3pg/ml, 4pg/ml
![](https://static.igem.org/mediawiki/2021/2/26/T--USTC--result_fig7.jpeg)

###### Figure16: Different concentrations of Tau protein in serum buffer

2.Detection of different concentration of Tau protein in aqueous solution (Elisa kit)
![](https://static.igem.org/mediawiki/2021/f/fa/T--USTC--result_fig8.png)

###### Figure17: Tau protein concentrations and absorbancy

Equation:Y=0.00339X+0.08339

# **Week12**3/10/2021-9/10/2021

1.Hardware design
![](https://static.igem.org/mediawiki/2021/d/d4/T--USTC--hardware_fig3.jpeg)

###### Figure18: Explosion diagram of the qPCR device

2.Last preparation for iGEM

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# Team:USTC/Engineering

USTC-iGEM-Engineering

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# Hello, Welcome to ENGINEERING PAGE

# Design 1.0

First, we want to construct a molecular switch that does not amplify when there is no target protein. And when there is a target protein, the amplification reaction will start due to the binding between aptamer and the target. The probe has low fluorescence signal in the absence of protein and strong fluorescence signal in the presence of protein. So we designed the molecular switch as shown in Figure 1.

![](https://static.igem.org/mediawiki/2021/7/77/T--USTC--eng_fig1.png)

Figure1: Scheme 1.0 molecular switch design：Green: Fluorescence group Red: Quencher Black and yellow: block . The probe sequence is the same as the top chain so it can play a role as primer as well. The yellow chain is aptamer which bind to the top strand by hydrogen bond instead of forming phosphate diester bond. The incomplete part is the complementary sequence of the two primers.

When there is no protein, due to the blocking effect of aptamer block group, DNA polymerase cannot amplify, resulting in no exposure of probe and primer binding sequence and no fluorescence signal generation. When there is the protein, the aptamer is separated from the double strand, and the DNA polymerase extends the bottom chain completely, exposing the binding sequence of primers and probe, so as to start the amplification reaction, and the fluorescence signal rises.

# Build 1.0

In order to realize the construction of molecular switche, we first need to obtain the top chain (no aptamer chain，NA chain for short with block modification), the bottom chain (the chain wit aptamer ,AP chain for short), and the aptamer (with block modification). Naturally, we think that we can use asymmetric PCR to obtain ssDNA[1]. We only need to use different primer ratios and temperature so that we can confirm the most suitable conditions for asymmetric PCR.

However, NA chain not only needs to get a long single stranded DNA, but also needs to be modified by adding a repressor group at its end, which can not be done by asymmetric PCR. Direct chemical synthesis will have a great probability of base synthesis errors. Therefore, we want to use TDT enzyme and ddNTP to achieve this function.

# Test 1.0

We tried to use TDT enzyme to add an unrecoverable ddNTP to ssDNA made by asymmetric PCR. This repressive approach was inspired by Sanger sequencing[2].but we found that this amount is very difficult to control, which is easy to cause residual ddNTP in the reaction solution, which will have unpredictable adverse effects on our experiment. Moreover, the efficiency of TDT enzyme is not high. It usually needs a single chain for more than 1h, and the single chain is usually not very stable[3]. We prefer that it can be quickly assembled into the switch we need.

Another consideration is that if TDT enzyme is used, we cannot determine how many NA chains are modified. Because modified or unmodified chains cannot be separated. If modified NA chains participate in the reaction, it will cause serious false-positive consequences.

At the same time, according to the modeling analysis, we found that using the probe as the primer will consume a large amount of probe, and the repressor group generated after the probe is cut by the enzyme may have a certain impact on the amplification of DNA polymerase. Therefore, probes should not be used as primers, but additional primers should be set.

In view of so many problems, we rejected this scheme through research, so how can we design to avoid these troubles

In fact, according to our design, the chain that opens the reaction is the AP chain, because the AP chain has both primer1 binding sequence and primer2 sequence. The problem is that the NA chain will expose the binding sequence of primer2 with the AP chain as the template out of control, resulting in abnormal opening of the reaction. Therefore, this problem can be solved only by making primer2 complementary sequence of the AP chain unable to be copied. Adding a repressor group at the end of NA is the most common idea, but this idea was rejected. It seems that the experiment has encountered a bottleneck.

We thought hard about this repression method, and suddenly a flash of light came: the reason why the NA chain can start to react uncontrollably is that it has a bare 3 'end, and this bare 3' end happens to be the primer2 complementary sequence. If we add the bare 3 'end into a complete double chain and let the real primer2 sequence become a ring and stay quietly, won't it have the same effect?

So we create a new design.

# Design 1.1

![](https://static.igem.org/mediawiki/2021/4/4b/T--USTC--designfig2.png)

Figure2: Scheme 2.0 molecular switch design: purple: NA chain blue: AP chain pink: aptamer

Both NA chain and AP chain are supplemented with a long complementary sequence (yellow in the figure), so that the primer sequence of AP chain will be twisted into a stem ring. In this way, whether the reaction is turned on or not depends entirely on the AP chain.

Generally, the reaction system stays quietly. In the presence of tau protein, the aptamer is detached from the AP chain., The polymerase extends the AP chain completely. The complementary sequence of primer1 is exposed. When primer1 starts copying with the AP chain as the template, it will open the prime2 sequence that becomes the stem ring, so that the prime2 complementary sequence can be copied. The amplification reaction began！

Now its time to build our switch.

# Build 1.1

We successfully made ssDNA by asymmetric PCR (Fig. 3). After calculating the concentration, mix 1:1, and cool naturally to room temperature after constant temperature water bath for 5min.

![](https://static.igem.org/mediawiki/2021/8/85/T--USTC--eng_fig3.png)

Figure3: Asymmetric PCR Result

![](https://static.igem.org/mediawiki/2021/f/f5/T--USTC--eng_fig4.png)

Figure4：Verification of double chain assembly mode

Since we didn't know whether we get correct ssDNA and whether our assembly method can really turn ssDNA into dsDNA, we thought of a clever way to verify it.

We amplified the Na chain and its complementary chain by asymmetric PCR, mixed them 1:1, and cooled them naturally in a constant temperature water bath at 98 ℃. The AP chain performs the same operation. Then 3% agarose gel electrophoresis was used to add AP double stranded, NA double stranded, AP recombination double strands and NA recombination double chains from left to right shown as Fig4. The gel diagram is completely consistent, which shows that the amplified single chain is correct and the way of molecular assembly is feasible

# Test 1.1

We tested this design by using different template concentration with or without aptamer. As shown in Fig. 5, all concentration gradients show the same results: the fluorescence signal is significantly lower in the presence of aptamer than in the absence of aptamer, indicating that the aptamer has a good blocking effect on RPA.

![](https://static.igem.org/mediawiki/2021/a/a4/T--USTC--eng_fig5_1_new.png)

![](https://static.igem.org/mediawiki/2021/1/12/T--USTC--eng_fig5_2_new.png)

Figure5: Aptamer Repression Test with different concentration of template chains: A:10nM B:1nM C:100pM D:10pM E:1pM F:0.1pM （1cycle=20s the same below）

![](https://static.igem.org/mediawiki/2021/f/f4/T--USTC--eng_fig6_new.png)

Fig6: The template amplification curves of different concentrations showed very discrete fluorescence signals.

We repeated the experiment many times and got the same experimental results. But when we were about to finish this part and move on to the next part, we suddenly found the opposite result.

![](https://static.igem.org/mediawiki/2021/4/45/T--USTC--eng_fig7_new.png)

Figure7: Strange-Fig: ContraryAptamer Repression Test

![](https://static.igem.org/mediawiki/2021/1/17/T--USTC--eng_fig8_new.png)

Figure8: Strange-Fig2: Confused Amplification Curves at Different Concentration

At first, we thought we had mistaken the positive group and the negative group when doing the experiment. But in the following repeated experiments, it was still the opposite result. And obviously, this statement could not explain why the curves become chaotic when the template concentration was different.

We became very depressed. If we could not overcome this step, there was no need to carry on the subject. What’s more sad was that the degree of curve confusion was different every time the test was repeated. Some curves were low last time, but the next time they ran to the top.

These curves were like naughty children, changing their positions. They had a good time, but these data deeply hurt our hearts.

# Learn

After No.N （many times）failure, we made up our mind to analyze why the data was so bad.
The first is why the signal of the group with aptamer is higher than that of the group without aptamer.

Aptamers do not catalyze chemical reactions, at least not at present. Therefore, there must be other unknown factors in the aptamer group, resulting in more double chains that can be detected by the probe. When we thought about it carefully, we feltl something wrong. The primers that can amplify were quantitative. Then the amplification intensity of the two groups was at most the same. Why was the group higher?

Things seemed to have come to a dead end. But we suddenly realized something we had never realized before. The aptamer itself is an oligonucleotide! What are oligonucleotides? They are primers!

The previous aptamer did not play this function due to the existence of its repressor group. However, our aptamer has been in solution for 4 months, and has experienced repeated freezing and thawing and temperature rose and fell. It was likely that the repressor group was no longer reliable. Once the repressor group falls down, the aptamer becomes a primer that can amplify the AP chain. At the same time, the amplified sequence happens to have a probe recognition site!

In this way, our AP chain has a new primer different from primer2, and the double chain copied by this primer is far shorter than the previous chain, but it can also be recognized by the probe to produce fluorescent signals. It is not difficult to understand that the fluorescence signal enhancement caused by this short double chain is much faster than the previous long chain. Therefore, there are two kinds of double chains in the solution. The fluorescence signal should be based on the long double chain spreading value, plus the short double chain spreading value. Therefore, the fluorescence signal in the presence of aptamer is higher than that in the absence of aptamer. However, the enhancement effect of short double chain is affected by many factors: The aptamer concentration is always several times of the equivalent concentration of the template, but the concentration of primer2 is always constant. Therefore, the lower the concentration is, the less the total number of short double chains is compared with that of long double chains. When the template concentration decreases, the total concentration of various double chains decreases, but the increasing effect of short double chains on long double chains is more significant. These factors constitute an antagonistic relationship, which makes the fluorescence signal become a multivariate function with the change of concentration, so the curve becomes very chaotic.

For this situation, our team did a modeling analysis, making some changes based on the second model modeled by RPA (see the Model section of the Wiki for details).Firstly, the fracture of the aptamer in the actual experiment is simulated. In the actual experiment, the fracture of the aptamer used in each experiment is different, so random numbers are used to simulate the fracture of the aptamer in each experiment. We also added a side effect caused by aptamer fracture.

![](https://static.igem.org/mediawiki/2021/7/75/T--USTC--eng_tab1.png)

Table1: Parameter description

Our model uses Miltonian equation to calculate the amplification rate of short double strands in RPA.And python was used to build the model, calculate the data, and finally draw the image as shown below.

![](https://static.igem.org/mediawiki/2021/3/35/T--USTC--eng_fig9.png)

Fig9：The fluorescence signal caused by aptamer shedding changes with concentration

The model roughly explains why the experimental results are disordered, and because the repressor group of aptamer may fall off over time, the results will be difficult to repeat.

# Test 1.2

In order to test this conjecture, we did a simple test: directly amplify the template chain with primer1 and aptamer. If most blocks are good, the amplification results should not be seen in theory. On the contrary, if you see a double chain shorter than the template, most blocks should fall down.

![](https://static.igem.org/mediawiki/2021/6/60/T--USTC--eng_fig10.png)

Fig10: Aptamer repressive test：From left to right, the template concentration decreased in turn, and the interior of each group: primer1 + primer2; primer1+aptamer; primer1+ddH2O(except marker)

As you can see, there are clear short double stranded fragments, so the aptamer should be broken.So we re purchased the aptamer again and obtained the same results as we want.

After that, we determined the tau protein. Please see result part for the detailed experimental results.

[Results(click here)](https://2021.igem.org/Team%3AUSTC/Results)

You think it's over? On the contrary, it gives us new ideas.

# Design 1.2:

![](https://static.igem.org/mediawiki/2021/8/88/T--USTC--eng_fig11.png)

Fig11: Schematic diagram of trans detection

Compared with the previous scheme, we abandon the block on prime2 and aptamer and directly use aptamer to amplify short double strand. There is no doubt that a stronger fluorescence signal will be obtained. At the same time, tau protein will bind free aptamers, which will reduce the number of aptamers for amplification and reduce the fluorescence intensity. The higher the tau concentration, the higher the decrease of fluorescence intensity.

The advantage of this method over the previous method is that it does not need aptamer to block the amplification of polymerase, but changes the fluorescence signal value through the identity of primers. The higher the protein concentration, the lower the signal, which is a little unusual. However, there is no doubt that it is beneficial for low concentration protein, because its fluorescence intensity will not be too low to detect. This detection is just the opposite of the previous situation, so the previous scheme is called cis detection, and this scheme is called trans detection.Of course, this method also has some shortcomings. It loses the effect of amplifying the signal of the previous scheme, and the detection range may be reduced.

# Build 1.2

With basis of previous work, the construction of new molecular switch was much easier.Asymmetric PCR is the same as the previous protocol. When assembling the double chain, using aptamer without repressor group ,with the same proportion and method as before.

# Test 1.2.1

![](https://static.igem.org/mediawiki/2021/9/90/T--USTC--engineering100.jpg)

Fig12.a: Expected results of trans detection

center center center center center center center center center center center center center center center center center center center center center center center

![](https://static.igem.org/mediawiki/2021/4/46/T--USTC--eng_fig12b_new.jpeg)

Fig12.b：Real results of trans detection

Wow, perfect discrete curve! But wait a minute, why is it contrary to our prediction?

# Learn

In this part, we hope to make some prediction and corresponding analysis on the possible results：

1. The fluorescence curves of proteins with different concentrations may be closer. This is due to the excessive concentration of aptamer. This happens because the decrease of aptamer concentration caused by protein binding is insignificant for the whole fluorescence reaction.

2. The range of protein concentrations detected is not large: because the binding is too tight, this is different for different proteins. For those with particularly strong binding, it can be subdivided in a limited concentration range to obtain more sensitive results.

3. It does not necessarily change according to the desired concentration gradient: for the template that has become a complete long double chain, there is an aptamer sequence on it, so tau protein can bind to these AP chains, which may promote the release of another template chain. From this point of view, tau still promotes the fluorescence value. So we can find a monotonic interval by testing different concentrations to solve this problem.

As you can see, our experimental results get such a curve. The curve is very discrete, but it changes according to the CIS result. It even looks better. It is a little interesting. We conducted repeated experiments and obtained similar results, indicating that the amount of aptamers may be excessive, or tau tends to bind to tau proteins on the AP chain rather than free ones. We believe that this phenomenon is not universal, so it may need to be retested by changing the combination of aptamers and proteins. This scheme needs further testing and we will do it after the competition.

So far, both cis and trans methods have been completed. Although the trans method had a little problem, it is not too big. But is this over?

# Design 1.3

In the previous scheme 1, only the AP chain really works, and NA only plays a blocking role, which is actually a waste for molecular design. So we naturally thought that we could use the sandwich of two aptamers. Thus, the following design is achieved:

![](https://static.igem.org/mediawiki/2021/7/7a/T--USTC--eng_fig17.png)

Fig13：Principle 1.3

It is better to choose different sequences for the two aptamers, so as to prevent the formation of complementarity in the chain. The principle is basically the same as scheme 1.1, except that when tau protein exists, it can bind to two aptamers or release two single chains.

# Learn

The advantage of this method is to improve the utilization of DNA strand. The coordination of the two aptamers can make the fluorescence signal of the reaction higher and the amplification effect better. However, the superposition of two de repression effects of two aptamers may cause high background value, so the actual results need to be tested.

# Design 1.4

With cis double aptamer sandwich, it is natural to think of trans design:

![](https://static.igem.org/mediawiki/2021/a/a7/T--USTC--eng_fig18.png)

Fig14：Principle 1.4

center center center center center center center center center center center center center center center center center center center center center center center

# Learn

The advantage of this method is to improve the utilization of DNA strand. The coordination of the two aptamers can make the fluorescence signal of the reaction higher and the amplification effect better. However, the superposition of two de repression effects of two aptamers may cause high background value, so the actual results need to be tested.

The single chains of NA and AP were obtained by asymmetric PCR, and the aptamers were purchased directly. Building switch is not difficult.

It seems that the scheme has been difficult to modify, but the inspiration has not stopped.

# Design 2.0

In the previous design, aptamers and DNA are bound by hydrogen bonds. We thought that if they can be bound by phosphodiester bonds, the background value is likely to be reduced. Of course, in order to achieve this goal, we need to use the new secondary structure to realize the function, so we have the following design:

![](https://static.igem.org/mediawiki/2021/c/c6/T--USTC--eng_fig13.png)

Fig15: Scheme 2.0 molecular design

In the presence of tau protein, it binds to the aptamer, resulting in the opening of amplificationr. After the 3 'end exposed above is extended, the primer binding sequence can be copied. After one round of amplification, amplificaitonf will also be opened, and the binding sequence of the probe and another primer will be exposed to start exponential amplification.

![](https://static.igem.org/mediawiki/2021/5/51/T--USTC--eng_fig14.png)

Fig16: Schematic diagram of scheme 2.0

# Build 2.0

Through endonuclease, we obtained the required template double strand, and then obtained the corresponding template single strand by asymmetric PCR. The results were verified by agarose gel electrophoresis.

![](https://static.igem.org/mediawiki/2021/b/b8/T--USTC--eng_fig15_0.png)

Fig17.a:Template2 PCR

center center center center center center center center center center center center center center center center center center center center center center center

![](https://static.igem.org/mediawiki/2021/1/1f/T--USTC--eng_fig17b_new.png)

Fig17.b template1-PCR

# Test 2.0

The theoretical expectation and actual results are shown in the figure.

![](https://static.igem.org/mediawiki/2021/0/00/T--USTC--eng_fig18_new.png)

Fig18：The template fluorescence signal without protein and aptamer changes with concentration.

center center center center center center center center center center center center center center center center center center center center center center center

![](https://static.igem.org/mediawiki/2021/e/e0/T--USTC--eng-fig19a_new.png)

Fig19.a: Expected results of fluorescence intensity with concentration without tau protein.

center center center center center center center center center center center center center center center center center center center center center center center

![](https://static.igem.org/mediawiki/2021/9/97/T--USTC--eng_fig19b_new.png)

Fig19.b:Expected result of tau protein detection

# Learn

It can be seen from the image that the value of fluorescence intensity is very low, far less than scheme 1.0. This may be due to the short priming sequence, which is not enough to recruit enough DNA polymerase to complete amplification after double strand opening. The length of the priming sequence can be appropriately increased to enhance the fluorescence signal value.

Since the first concentration gradient experiment did not get particularly good results, we did not have time to continue. Because of its high complexity and many secondary structures, the possible experimental results are difficult to predict. We'll finish the plan later.

# Summary

In short, we have experienced sufficient engineering cycle design, designed many possibly successful schemes, and successfully constructed a working molecular switch (1.1) to realize the detection function. We will test the remaining schemes one by one after the game in order to get the best experimental results. In fact, we still have several proposals under consideration, but because of the space problem, we will not list them one by one here. The process we show here is the process of our engineering cycles, hoping to show how we modify the experimental scheme according to the experimental results and continuous learning, and finally obtain the target molecular components. We enjoy this method of analyzing and solving problems, and we can also find the fun of exploration from new design ideas.

If you also have good ideas about our captamer technology, please contact us.

# Reference

1. S. Ashley, S. Bradburn, C. Murgatroyd, A meta-analysis of peripheral tocopherol levels in age-related cognitive decline and Alzheimer's disease. Nutr Neurosci 24, 795-809 (2021).

2. N. J. Ashton et al., Plasma p-tau231: a new biomarker for incipient Alzheimer's disease pathology. Acta Neuropathol 141, 709-724 (2021).

3. S. Fossati et al., Plasma tau complements CSF tau and P-tau in the diagnosis of Alzheimer's disease. Alzheimers Dement (Amst) 11, 483-492 (2019).

4. Y. Gao, L. Tan, J. T. Yu, L. Tan, Tau in Alzheimer's Disease: Mechanisms and Therapeutic Strategies. Curr Alzheimer Res 15, 283-300 (2018)

5. R. Gorkin et al., Centrifugal microfluidics for biomedical applications. Lab Chip 10, 1758-1773 (2010).

6. K. R. Jacobs et al., Correlation between plasma and CSF concentrations of kynurenine pathway metabolites in Alzheimer's disease and relationship to amyloid-beta and tau. Neurobiol Aging 80, 11-20 (2019).

7. S. Janelidze et al., Plasma P-tau181 in Alzheimer's disease: relationship to other biomarkers, differential diagnosis, neuropathology and longitudinal progression to Alzheimer's dementia. Nat Med 26, 379-386 (2020).

8. S. M. Krylova et al., Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures. FEBS Lett 579, 1371-1375 (2005)

9. C. Li, J. Gotz, Tau-based therapies in neurodegeneration: opportunities and challenges. Nat Rev Drug Discov 16, 863-883 (2017).

10. D. Li, M. M. Mielke, An Update on Blood-Based Markers of Alzheimer's Disease Using the SiMoA Platform. Neurol Ther 8, 73-82 (2019).

11. S. Lisi et al., Non-SELEX isolation of DNA aptamers for the homogeneous-phase fluorescence anisotropy sensing of tau Proteins. Anal Chim Acta 1038, 173-181 (2018)

12. E. Nichols et al., Global, regional, and national burden of Alzheimer's disease and other dementias, 1990–2016: a systematic analysis for the Global Burden of Disease Study 2016. The Lancet Neurology 18, 88-106 (2019).

13. O. Piepenburg, C. H. Williams, D. L. Stemple, N. A. Armes, DNA detection using recombination proteins. PLoS Biol 4, e204 (2006).

14. B. Shui et al., Biosensors for Alzheimer's disease biomarker detection: A review. Biochimie 147, 13-24 (2018).

15. J. Simren, N. J. Ashton, K. Blennow, H. Zetterberg, An update on fluid biomarkers for neurodegenerative diseases: recent success and challenges ahead. Curr Opin Neurobiol 61, 29-39 (2020).

16. O. W. Stringer, J. M. Andrews, H. L. Greetham, M. S. Forrest, TwistAmp® Liquid: a versatile amplification method to replace PCR. Nature Methods 15, 395-395 (2018).

17. D. Tao et al., Development of a Label-Free Electrochemical Aptasensor for the Detection of Tau381 and its Preliminary Application in AD and Non-AD Patients' Sera. Biosensors (Basel) 9, (2019).

18. I. T. Teng et al., Identification and Characterization of DNA Aptamers Specific for Phosphorylation Epitopes of Tau Protein. J Am Chem Soc 140, 14314-14323 (2018).

19. J. Wu, X. Liu, L. Wang, L. Dong, Q. Pu, An economical fluorescence detector for lab-on-a-chip devices with a light emitting photodiode and a low-cost avalanche photodiode. Analyst 137, 519-525 (2012).

20. F. B. Yang, J. Z. Pan, T. Zhang, Q. Fang, A low-cost light-emitting diode induced fluorescence detector for capillary electrophoresis based on an orthogonal optical arrangement. Talanta 78, 1155-1158 (2009).

21. Marimuthu Citartan1 , Thean-Hock Tang1\*, Soo-Choon Tan2 , Chee-Hock Hoe1 , Rajan Saini3 , Junji Tominaga4 and Subash C.B. Gopinath4\*,Asymmetric PCR for good quality ssDNA generation towards DNA aptamer production.Songklanakarin Journal of Science and Technology 34(2):125-131

22. DNA恒温快速扩增试剂盒（荧光型）使用说明书

23. Terminal Deoxynucleotidyl Transferase protocol

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:USTC/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:USTC/Results

USTC-iGEM-Results

[USTC-iGEM 2021](https://2021.igem.org/Team%3AUSTC)

* Nav-bar
* Team
  + [Members](https://2021.igem.org/Team%3AUSTC/Members)
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* Project
  + [Description](https://2021.igem.org/Team%3AUSTC/Description)
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  + [Model](https://2021.igem.org/Team%3AUSTC/Model)
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* [Safety](https://2021.igem.org/Team%3AUSTC/Safety)
* [Awards](https://2021.igem.org/Team%3AUSTC/Awards)

# Hello, Welcome to RESULTS PAGE

# Asymmetric PCR

Under the condition of primer ratio of 20:1, asymmetric PCR products were obtained. Electrophoresis was performed at 130V for 30min to obtain the test gel pattern. (Fig 1)

![](https://static.igem.org/mediawiki/2021/8/84/T--USTC--result_fig1.png)

Fig 1 3% agarose gel electrophoresis of NA chain and AP chain. Because, AP dsDNA is too short, some protective bases were added to the AP double chains originally designed on the plasmid . And those redundant bases were removed by PCR. So there are three bands on the gel map in addition to primers.

Except for marker, the first four channels are NA chains （none aptamer chain）and the last four channels are AP (chain with aptamer)chains. The length of NA double strand is 206bp, while the length of AP double strand is 139bp. The position of double chain is undoubtedly correct, but the movement of single chain can not be judged by marker alone. To make sure we get the right single chain, we used the same asymmetric PCR method to obtain complementary chains of NA and AP ssDNA. We put the NA chain and its complementary chain together, 98 ℃ for 5min, and cooled naturally in a water bath to get recombinant double chains. So was the AP chain. We performed an electrophoresis to compare the original NA and AP double strands and the recombinant double strands. (Fig 2)

![](https://static.igem.org/mediawiki/2021/6/66/T--USTC--result_fig2.jpeg)

Fig 2 original double strands and recombinant double strands

The left two channels are original AP and NA chains, the next two channels are recombinant chains. From the figure we can find that the two NA chains are at the same position, the two AP chains as well. It proves that the single strands obtained by asymmetric PCR are correct.

# RPA

## Positive normal template

Before the formal experiment, in order to test the resolution ratios that the RPA kit can achieve, we carried out RPA experiment with the positive normal templates. (Fig 3)

![](https://static.igem.org/mediawiki/2021/f/fa/T--USTC--result_fig3.jpeg)

Fig 3 different concentrations of positive normal templates

When the concentration of positive normal templates is 0.4pM, the curve can be significantly different from that of no template chains. Besides, the RPA kit can detect a difference of 0.3pM. The resolution required for our experiment is within the range that the kit can achieve, which indicates that our experiment can be continued.

## Aptamer-free

We use fluorescence quantitative PCR instrument to detect the real-time fluorescence signal of our system. Every 20 seconds the signal strength will be recorded. According to our design, the template can be amplified by enzymes in the absence of aptamer. In order to verify the correctness of this design, we selected different concentrations of aptamer-free template for RPA amplification and got the fluorescence intensity curves with time change. (Fig 4)

![](https://static.igem.org/mediawiki/2021/5/5d/T--USTC--result_fig4.png)

Fig 4 different concentrations of template chains

The concentrations of template chains are 10-8, 10-9, 10-10, 10-11, 10-12 and 10-13 mol/L. The lowest curve represents blank control group. It can be seen from the figure that except for the 10-12 and 10-13 groups, the fluorescence intensity of template chains with different concentrations is obviously different, and the signal is positively correlated with chain concentration. The two nearly overlapping curves indicate that fluorescence signal intensity cannot show significant difference when the concentration of template chain is too low, so we only selected the data with concentration of 10-12mol/L for analysis in these two groups. All other curves are above the blank control group, confirming the validity of those data.

According to our design, the probe can combine with complete template chain obtained from RPA. At the same reaction time, the higher the concentration of template chains is, the more sites the probe can bind, which leads to a higher signal. Our image fully illustrates that the vacant part of template chain will be filled, and complete AP complementary chain will be obtained under the action of chain 1 primers and enzymes, so that the binding site of chain 2 primer will appear. In other words, the image shows that the template can amplify without block and the difference in fluorescence intensity is consistent with our hypothesis.

## With aptamer

To confirm that the aptamer can bind to the single-strand portion of template and block the amplification of template chain in the absence of tau, we conducted a comparative experiment with different concentrations of aptamers and without aptamers. (Fig 5)

![](https://static.igem.org/mediawiki/2021/9/95/T--USTC--result_fig5.png)

Fig 5 different concentrations of template chain A:10-8mol/L B:10-9mol/L C:10-10mol/L D:10-11mol/Lp E:10-12mol/L F:10-13mol/L

The concentrations of the template chains we chose are still from 10-8 to 10-13 mol/L. In the groups with aptamers, we added twice the template concentration of aptamers to ensure more template bind aptamers. The gray curve is aptamer-free while the red curve is with aptamer, and the blue curve is blank group. We can easily make a distinction between the curves representing aptamer and aptamer-free. At any concentration, the curves representing aptamer-free is above the curves representing aptamer, while the blank control group is the lowest in the figures. Both curves are above the blank control group, proving that our data are valid. It means that the aptamer can successfully bind to the template strand, and the block can inhibit the DNA polymerase from completing the template strand. The amplification reaction is blocked and reflected in the fluorescence signal intensity.

Those figures above preliminarily prove the feasibility of our experiment. According to experiment data, when the concentration of template is 10-10mol/L, the comparison between the presence and absence of aptamers is obvious enough, and there is no need to select template chain with a higher concentration. Therefore, further experiments on Tau protein were carried out under the condition of template chain of 10-10 mol/L. In addition, it is proved that the experiment can achieve good results under the condition of two-equivalent aptamer, so the two-equivalent aptamer was also selected in the subsequent experiments.

## With tau

According to our experimental design, when tau protein is present, it will bind to aptamer with block so the aptamer will release from the template chain. Then RPA reaction begins. Therefore, we added different concentrations of tau protein into the reaction system for experiments (Fig 6).

![](https://static.igem.org/mediawiki/2021/f/f6/T--USTC--result_fig6.jpeg)

Fig 6 different concentrations of tau protein in an aqueous solution

The experimental results shows that the fluorescence intensity will increase as the tau protein concentration grows. This suggests that tau protein can bind to aptamer with blocks and take the aptamer and blocks away from the NA and AP chains. It can be seen from the figure that when the concentration of tau protein is between 4pg/ μL and 6pg/ μL, the fluorescence intensity of the reaction takes a leap. We suspect that this is because tau protein may bind to the aptamer sequence on the template chain, resulting in the change of nucleic acid conformation. This will promote the opening of hydrogen bonds, which to some extent plays a role in unzipping the DNA. This phenomenon is especially obvious when the concentration of tau protein is high enough.

To verify that our design can be used in a real blood test, we replaced water with a serum buffer. Under the same condition, we got the figure of different concentrations of tau protein and fluorescence intensity. (Fig 7)

![](https://static.igem.org/mediawiki/2021/2/26/T--USTC--result_fig7.jpeg)

Fig 7 different concentrations of tau protein in a serum buffer

![](https://static.igem.org/mediawiki/2021/4/4c/T--USTC--results_add.png)

Fig 8 Fluoresence-Concentration Linear Fitting (Standard Curve)

From top to bottom, the concentration of tau proteins represented by the curves decreases sequentially. The fluorescence intensity at 20 min into the reaction was plotted against the concentration, and a good linear relationship was obtained. This result shows that our design is not just a theory. The device is not limited to aqueous laboratory solutions, but can be used in actual blood tests. In the actual test we can use the standard curve (Fig 8) for quantitative determination of proteins. This is important for the early detection of Alzheimer's disease because it allows for the detection of blood markers on a large scale with extremely fast response times and precise measurement accuracy.

## Compared with ELISA

The concentration of tau protein was detected by ELISA kit and we obtained the relation between the absorbance of the system and the concentrations. (Fig 9)

![](https://static.igem.org/mediawiki/2021/f/fa/T--USTC--result_fig8.png)

Fig 9 Tau protein concentrations and absorbancy
Y=0.00339X+0.08339

The liner relationship between absorbance and protein concentration is excellent, indicating that ELISA kit can accurately determine the concentration of tau protein, and its detection limit can reach at least 7.81pg/ mL, which is beyond our reach at present. However, it takes a long time about 3-4h with high requirements for the operator and every test costs a lot. Our method has the advantages of rapid detection, simple operation and low expense. According to our experiment results, good effects can be achieved after about 20 minutes of the reaction. Significant differences can be shown in the groups with different concentrations of tau protein added. Compared with ELISA kit, the two biggest advantages of our device are simple operations and short reaction time.

# Future work

At present, our experiment is limited by the affinity between tau protein and aptamer, so the minimum concentration of tau protein that can be measured by our device is limited, and the accuracy of the experiment will also be affected. Currently, the aptamer sequence we use are obtained from the literature, and the affinity with tau protein is not the highest value that the aptamers can reach today. Besides，the head and tail of the aptamer we are using are complementary, which easily leads to dimerization and even polymerization of the aptamer, affecting the experiment results. So this aptamer is not the best choice. If we can get new aptamers with a higher affinity through screening, the minimum protein concentration we can measure will continue to decrease and that we can achieve higher detection accuracy.