iGEM 2022 — Team csu-china

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# Sidebars examples

[Experiments](/)

* Introduction

  + Overview
  + Updates
  + Reports
* Results

  + [引物测试](#yingwu)
  + [RPA测试](#rpa)
  + [CRISPR-Cas12a测试](#cas)
  + [探针测试](#tanzhen)
* Conclusion

  + New
  + Processed
  + Shipped
  + Returned
* Outlook

  + New...
  + Profile
  + Settings
  + Sign out

## Introduction

Our diagnostic system for parasitic diseases tests the person's circulating cell-free DNA (cfDNA) in their blood to see if they are infected with the parasite. We used the Recombinase Polymerase Amplification（RPA）to amplify cfDNA in blood. Then the amplification product was used to activate the trans-cleavage activities of CRISPR-Cas12a system, cut the nucleic acid probe we designed . Finally, the colloidal gold test stripes were used to detect the cleavage product.

## Results

### 1.引物测试

![](https://static.igem.wiki/teams/4127/wiki/yinwuceshi.png)

Figure 1． Amplification of the target fragment. Target fragments were amplified by PCR using 16 pairs of cfDNA predictive primers, the amplified products were subjected to 2 % agarose electrophoresis at 140V for half an hour.

### 2.RPA测试

![](https://static.igem.wiki/teams/4127/wiki/rpaceshi.png)

Figure 2. Recombinase polymerase amplifies the target fragment. Recombinase polymerase amplification（RPA） was performed using templates with different copy numbers（R1-R5）. Then purifying the amplification products with phenol-chloroform method and using agarose electrophoresis to test the purified DNA.

### 3.CRISPR-Cas12a测试

![](https://static.igem.wiki/teams/4127/wiki/crispr-cas12aceshi.png)

Figure 3. CRISPR-Cas12a detects amplified target fragments. (A) The fluorescence of reporters was detected under UV light. The concentration of reporter 1(8 nt) or reporter 2(15 nt) is 500 nM; Amplicon, The amplified E gene fragment; E, the sgRNA of SARS-Cov-2 E gene; N, the sgRNA of SARS-Cov-2 N gene. (B) Fluorescence reader detects different reaction. The red dashed line indicates the Max fluorescence intensity of reaction 3. \*\*\*\*, p < 0.0001.

### 4.探针测试

![](https://static.igem.wiki/teams/4127/wiki/tanzhenceshi.png)

Figure 4. DNase digests fluorescent reporter (15 nt). (A) Reporter fluorescence under UV light. Reporter concentration: R1, 500 nM; R2, 1 μM; R3, 2 μM; R4, 4 μM; R5, 8 μM; R6(NC), 500 nM, without DNase. (B) Fluorescence intensity detected by fluorescence reader. The red dashed line indicates the Max fluorescence intensity of the NC. \*\*\*\*, p < 0.0001.

## Conclusion

1. We selected several primers with better amplification efficiency among 16 pairs of cfDNA predictive primers by PCR.
2. We successfully amplified the target fragment using RPA at a low copy number (1 copy).
3. We using the Target fragment amplified by RPA to activate the trans-cleavage activities of CRISPR-Cas12a system, and to cut the fluorescent reporter we designed and synthesized.

## Outlook

We try to extract cfDNA from human blood, then detect the cfDNA using our system to diagnose parasitic diseases including the final colloidal gold test papers detection. In addition, we try to optimize CRISPR-Cas12a nucleic acid detection system by multi-sgRNA.

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{% extends "layout.html" %}
{% block title %}Engineering SUCCESS{% endblock %}
{% block lead %}Make a useful contribution for future iGEM teams. Use this page to document that contribution.{% endblock %}
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![](https://static.igem.wiki/teams/4127/wiki/cover12.jpg)
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"Ideal is the beacon. Without ideal, there is no secure direction; without direction, there is no life"

##### Engineering Success

* [The Summary](https://2022.igem.wiki/csu-china/engineerings#The%20Summary)
* [Iteration of the engineering design cycle](https://2022.igem.wiki/csu-china/engineerings#Iteration)
* [And](https://2022.igem.wiki/csu-china/engineerings#And)
* [Or](https://2022.igem.wiki/csu-china/engineerings#Or)

# The Summary

After several iterations of the engineering design cycle, we finally designed six pairs of primers based on verified hydatid DNA sequences and six sgRNAs based on PAM sequences of CRISPR-Cas12a. We mixed six primers to detect echinococcosis sequences by RPA and CRISPR coupling. The experimental results show that the method can accurately detect free echinococcosis DNA with a high detection rate.

# Iteration of the engineering design cycle

We designed 16 primer pairs for the characteristic fragments of Echinococcus and confirmed that 14 amplification of the characteristic fragments in subsequent experiments. Considering whether the corresponding fragments can design sgRNA and amplification effect, we finally selected 6 effective primers to perform multiple primer amplification, and explore the stable system of multiple primer RPA amplification, namely to ensure a single each primer concentration of 0.3 μ M.

![](https://static.igem.wiki/teams/4127/wiki/con-fig2.png)

Fig 1. RPA amplification results of 16 primer pairs

For the amplification products of the characteristic Echinococcus fragments, we designed 13 sgRNA, and the sgRNA corresponding to mgs4, mgs5, mgs6, mgs9, mgs10 and mgs14 fragments were highly active.

Since the cfDNA in the patient's blood often exists in small, randomly broken fragments, we hope to improve the true-positive rate by detecting multiple sequences simultaneously.

In our project, it is divided into AND and OR two parts.

![](https://static.igem.wiki/teams/4127/wiki/con-fig3.png)

Fig 2. CRISPR-Cas12a shear fluorescent reporter

# And

Our AND partially consists of the RPA system and the CRISPR Cas12a system. After repeated practical experiments, we have achieved the first multi-primer pairs isothermal amplification and multiple sgRNA-guided CRISPR Cas12a detection in the iGEM project since 2020.

#### 1.RPA system

| Agentia | Dosage/μl |
| --- | --- |
| Buffer R2.1(10×） | 0.6 |
| DEPC water | 3.6 |
| Cas protein (10 μM) | 0.3 |
| sgRNA (250 nM) | 1.5 |
| 37 ℃ 15 min | | | | | | | |
| 15 nt probe (10 μM) | 4 |
| add 9μl after mixing | | | | | | | |
| substrate | 1 |
| Buffer R2.1(1×） | 40 |
| 37 ℃ 30 min | | | | | | | |

#### 2.Crispr-Cas12 system

| Agentia | Dosage/μl |
| --- | --- |
| Buffer R2.1(10×） | 1.2 |
| DEPC water | 7.2 |
| Cas protein (10 μM) | 0.6 |
| sgRNA (250 nM) | 3.0 |
| 37 ℃ 15 min | | | | | | | |
| 15 nt probe (10 μM) | 3.0 |
| add 9μl after mixing | | | | | | | |
| substrate | 1 |
| Buffer R2.1(1×） | 40 |
| 37 ℃ 30 min | | | | | | | |

If the substrate was E gene,add 1μl sgRNA and 4.1μl DEPC water

The RPA and CRISPR methods shown in the table are the results of our improved system. We continued to explore the reaction time, ratio and temperature of the system, and after asking medical workers who had actually used RPA for nucleic acid detection, we developed a preincubation method for amplification.

![](https://static.igem.wiki/teams/4127/wiki/ps-1.png)

Fig 3. AND system

After several experiments, the method can significantly improve the success rate and accuracy of RPA, so that the results meet the experimental expectation. It also has a significant effect on subsequent CRISPR-CAS cutting.

By coupling RPA with CRISPR, we were able to detect cfDNA at room temperature without instrument dependence.

# Or

In the conventional amplification technique, only one pair of primers was selected. In order to improve the detection rate and accuracy as high as possible under the condition of room temperature without instruments, we screened out 6 pairs of primers with basically no interaction, and then tested them after mixing them in an appropriate proportion.

Our OR part consists of 6 primer pairs and the sgRNA targeting the corresponding sequence, and the simultaneous detection of multiple sequences increases our true-positive rate from 40% to 70%.

![](https://static.igem.wiki/teams/4127/wiki/ps-2.png)

Fig 4. OR system

As long as any pair of primers are successfully combined with the target sequence, they can be reported in the subsequent system to achieve low requirements, high coverage and high detection.

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"An experiment is worth nothing if its procedure is not written down"

##### Experiments

* [Plasmid Clone](https://2022.igem.wiki/csu-china/experimentslab#Plasmid Clone)
* [PCR Amplification](https://2022.igem.wiki/csu-china/experimentslab#PCR Amplification)
* [RPA Amplification](https://2022.igem.wiki/csu-china/experimentslab#RPA Amplification)
* [CRISPR Cas12a detection](https://2022.igem.wiki/csu-china/experimentslab#CRISPR Cas12a detection)
* [DNaseⅠsystem](https://2022.igem.wiki/csu-china/experimentslab#DNaseⅠsystem)

# 1.Plasmid Clone

#### (1) Heat shock transformation of Escherichia coli

⚫ Melt the competent Escherichia coli on the ice.

⚫ Blend the plasmid with the competent Escherichia coli,then take an ice bath for 30 minutes.

⚫ 42°C Heat for 90 s,then take an ice bath for 5 minutes.

⚫ Add 500μl nonresistant LB and put it into a 37°C rocker for 40 minutes.

⚫ Centrifuge under 7000 rpm for 1 minute.

⚫ Inhale the upper clearance and leave 40 μl heavy suspension.

⚫ Coating the board with AMP.

#### (2) mini shaking of the plasmid

Select a single coenobium, lift it with the tip,and put it into mixture mixed by 2 ml LB and 2μl AMP,then cultivate the cells by a 37°C 180 rpm rocker for 10 hours.

#### (3) large shaking of the plasmid

Cultivate the mixture mixed by 500 ml LB，500 μl AMP and 1ml shaked plasmid into a 37°C 180 rpm rocker for 16 hours.

#### (4) plasmid mini extraction

⚫ Centrifuge under 8000 rpm for 5 minute,then collect the cells.

⚫ Remove the supernatant and add the 250 μl Buffer P1 / RNase A mixture.

⚫ Add 250 μl Buffer P2,invert to mix and place at ordinary temperature.

⚫ Add 250 μl Buffer P2,invert to mix.

⚫ Centrifuge under 13000 g for 10 minute.

⚫ Place the HiPure DNA Mini ColumnⅡinto a collection tube,then transfer the supernatant to a column and centrifuge under 13000 g for 1 minute.

⚫ Discard the filtrate,place the column into the collection tube and add 500 μl Buffer PW1,then centrifuge under 13000 g for 1 minute.

⚫ Discard the filtrate,place the column into the collection tube and add 600 μl Buffer PW2,then centrifuge under 13000 g for 1 minute.

⚫ Discard the filtrate,place the column into the collection tube and add 300 μl Buffer PW2,then centrifuge under 13000 g for 1 minute.

⚫ Discard the filtrate,place the column into the collection tube and centrifuge under 13000 g for 1 minute.

⚫ Place the column into the collection tube and add 50 μl DEPC water,then centrifuge under 13000 g for 3 minute.

⚫ Collect the filtrate.

#### (5) plasmid large extraction

⚫ Add 300 ml shaked bacterial liquid into 500 ml tube.

⚫ Centrifuge under 4000 rpm for 15 minute.

⚫ Add 10 ml SolutionⅡ , mix and place at ordinary temperature for 2 min.

⚫ Add 5 ml pre-cooled N3 Buffer,mix and place at ordinary temperature for 2 min.

⚫ Discard the precipitation,then transfer the filtrate to a new 50 ml tube.

⚫ Add 0.1 x volume of ETR Solution.

⚫ Place on the ice 10 min.

⚫ 42℃ water bath for 5 min.

⚫ Centrifuge at 8000 g for 5 min at 25℃.

⚫ Transfer the supernatant to a new 50 ml tube with 0.5 x volume of absolute ethanol,mix and place at ordinary temperature for 2 min.

⚫ Place the DNA Maxi Column into a 50 m l tube,add 3 ml GPS Buffer and place at ordinary temperature for 2 min,then centrifuge at 4000 g for 3 min .

⚫ Discard the filtrate ,add 20 ml of the above-added absolute ethanol solution, and centrifuge at 5,000 g for 3 min.

⚫ Readd the filtrate to DNA Maxi Column and centrifuge at 5,000 g for 3 min.

⚫ Repeat the upper two-step process..

⚫ Add 10 ml HBC Buffer and centrifuge at 5000 g for 3 min.

⚫ Discard the filtrate,add 15 ml DNA Wash Buffer and centrifuge at 5000 g for 3 min.

⚫ Discard the filtrate,add 10 ml DNA Wash Buffer and centrifuge at 5000 g for 3 min.

⚫ Discard the filtrate,then centrifug at 5,000 g for 10 min.

⚫ Remove the DNA Maxi Column and air for 5 min.

⚫ Transfer the DNA Maxi Column to a new 50 ml tube,add 500 ml of sterile water to DNA Maxi Column and place at ordinary temperature for 2 min,then centrifuge at 10,000 g for 10 min,collect the filtrate.

⚫ Repeat the previous step three times.

# 2.PCR Amplification

#### (1) PCR system

| Agentia | Dosage/μl |
| --- | --- |
| Taq mix | 5 |
| DEPC water | 2 |
| DNA | 1 |
| F Primer (10 μM) | 1 |
| R Primer (10 μM) | 1 |
| Total | 10 |

#### (2) PCR process

| 95 ℃ | 5 min |
| --- | --- |
| 95 ℃ | 30 sec |
| 58 ℃ | 30 sec |
| 72 ℃ | 15 sec |
| 72 ℃ | 5 min |
| 10 ℃ | ∞ |

# 3.RPA Amplification

#### (1) RPA system

| Agentia | Dosage/μl |
| --- | --- |
| Buffer A | 25 |
| DEPC water | 13 |
| mix and divide into five group | | | | | | | |
| F Primer (10 μM) | 0.4 |
| R Primer (10 μM) | 0.4 |
| DNA | 1 |
| BufferB((dilute twice and add to the tube cap) | 1 |
| 39℃ 30 min | | | | | | | |

#### (2) purify the RPA product

RPA product: trichloromethane: phenol =2:1:1

Refer to the above system to add 15 μl RPA product, 7.5 μ l trichloromethane and 7.5 μ l phenol,then mix and centrifuge at 13,000 rpm for 10 min.

#### (3) recycle the RPA gel

⚫ Add sample into a 1.5 ml EP tube and use water to replenish to 100 μl .

⚫ Add Equal volume of Buffer GDP.

⚫ Add 1.5 x the above volume of absolute ethanol .

⚫ Place the column into the collection tube,add the mixture to the center of the column and centrifuge at 10,000 g for 60s.

⚫ Repeat the last step.

⚫ Discard the filtrate,set back the column to the tube and add 500 μl Buffer DW2,then centrifuge at 8000 g for 60s

⚫ Discard the filtrate,set back the column to the tube and add 25μl Elution Buffer and place for 2 min,then centrifuge at 10,000 g for 60s.

# 4.CRISPR Cas12a detection

#### (1) CRISPR Cas12a system for fluorescence detection

| Agentia | Dosage/μl |
| --- | --- |
| Buffer R2.1(10×） | 0.6 |
| DEPC water | 3.6 |
| Cas protein (10 μM) | 0.3 |
| sgRNA (250 nM) | 1.5 |
| 37 ℃ 15 min | | | | | | | |
| 15 nt probe (10 μM) | 4 |
| add 9μl after mixing | | | | | | | |
| substrate | 1 |
| Buffer R2.1(1×） | 40 |
| 37 ℃ 30 min | | | | | | | |

#### (2) CRISPR Cas12a system for test paper detection

| Agentia | Dosage/μl |
| --- | --- |
| Buffer R2.1(10×） | 1.2 |
| DEPC water | 7.2 |
| Cas protein (10 μM) | 0.6 |
| sgRNA (250 nM) | 3.0 |
| 37 ℃ 15 min | | | | | | | |
| 15 nt probe (10 μM) | 3.0 |
| add 9μl after mixing | | | | | | | |
| substrate | 1 |
| Buffer R2.1(1×） | 40 |
| 37 ℃ 30 min | | | | | | | |

In the process of using the test paper, we found that there may be a slow binding speed of the probe and colloidal gold, resulting in false positives.

So, we cut out the part of the test paper containing colloidal gold and elute the colloidal gold (50 μL DEPC water was used for each test paper).

40 μL colloidal gold solution and 40 μL digested products are mixed, and detected in 96-well plate.

C band appears within 2 minutes, and the result interpretation should be completed within 10 minutes.

# 5.DNaseⅠsystem

| Agentia | Dosage/μl |
| --- | --- |
| 15 nt probe | 1 |
| 10× Buffer(include MgCl2) | 5 |
| DNaseⅠ | 2 |
| DEPC water | add to 50 |
| 37 ℃ 15 min | | | | | | | |
| EDTA（50mM） | 1 |
| 65℃ 10 min | | | | | | | |

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{% block title %}Results{% endblock %}
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"Progress is the activity of today and the assurance of tomorrow"

##### Results

* [Verify the probe effect](#Verify the probe effect)
* [Verify the primer effect](#Verify the primer effect)
* [Find the lower
  detection limit](#Find the lower detection limit)
* [Test the feasibility
  by single sgRNA treatment](#Test the feasibility by single sgRNA treatment)
* [Test the feasibility
  by single sgRNA and multiplex sgRNA treatment](#Test the feasibility by single sgRNA and multiplex sgRNA treatment)
* [Test both positive
  and negative results with test strips](#Test both positive and negative results with test strips)
* [The specificity test](#The specificity test)
* [Multiple sgRNA can improve detection speed](#Multiple sgRNA can improve detection speed)

# Verify the probe effect

The effect of reporter type and reporter concentration on the fluorescence amount was detected by Cas protein. The E gene was digested with Cas protein by various concentrations of the 2020NBT reporter, 2022\_5nt reporter and 2022\_15nt reporter. From the result，the 2022\_15nt reporter had the best effects. After comprehensive consideration of cost and effect, we select 4 μM as actual used concentration.

![](https://static.igem.wiki/teams/4127/wiki/results1.png)

Figure 1. (A) Fluorescence was detected by UV light

![](https://static.igem.wiki/teams/4127/wiki/results2.png)

Figure 1. (B)Fluorescence was detected by Microplate Reader.

# Verify the primer effect

Hydatid characteristic sequence plasmids were purified after RPA amplification with 16 pairs of primers. The hydatid characteristic sequence plasmids after RPA amplification were purified with phenol and chloroform, and the supernatant was taken after centrifugation for electrophoresis. Based on the results, because there was no band at the expected target fragment, so the two primer pairs, mgs-2 and mgs-8, were removed.

![](https://static.igem.wiki/teams/4127/wiki/results3.png)

Figure 2. 1.5% agarose gel electrophoresis

# Find the lower detection limit

To find the lower detection limit of hydatid DNA in Cas digestion after RPA amplification.

Hydatid characteristic sequence plasmids at different copy numbers were subjected to Cas digestion after RPA amplification and treated with the 2022\_15nt probe to detect a lower limit of detection of 10 copies.

![](https://static.igem.wiki/teams/4127/wiki/results4.png)

Figure 3. Fluorescence was detected by Microplate Reader.

# Test the feasibility by single sgRNA treatment

Test the feasibility of detecting the hydatid plasmid with Cas digestion by single sgRNA treatment after RPA amplification with single primers. This indicates that RPA digestion with Cas was effective in detecting hydatid DNA.

![](https://static.igem.wiki/teams/4127/wiki/results5.png)

Figure 4. (A)Forecast result table

![](https://static.igem.wiki/teams/4127/wiki/results6.png)

Figure 4. (B)Fluorescence results Table

![](https://static.igem.wiki/teams/4127/wiki/results7.png)

Figure 4. (C)Table of results of the microplate reader

# Test the feasibility by single sgRNA and multiplex sgRNA treatment

Test the feasibility of the hydatid plasmid undergoing Cas digestion by single sgRNA and multiplex sgRNA treatment after RPA amplification with multiple sets of primers. This indicates that the detection of hydatid DNA was effective by multiple primer RPA and Cas digestion with single sgRNA or multiplex sgRNA treatment.

![](https://static.igem.wiki/teams/4127/wiki/results8.png)

Figure 5. (A)Forecast result table

![](https://static.igem.wiki/teams/4127/wiki/results9.png)

Figure 5. (B)Fluorescence results Table

![](https://static.igem.wiki/teams/4127/wiki/results10.png)

Figure 5. (C)Table of results of the microplate reader

# Test both positive and negative results with test strips

Test both positive and negative results with test strips, shown as follows,
Positive: C band has a red line and T band has a red line.
Negative: Only C band has a red line, while T band has no red line.

![](https://static.igem.wiki/teams/4127/wiki/results11.png)

Figure 6. Test strip result diagram

# The specificity test

The specificity test of the test method indicates that this test method will not detect other parasitic diseases as the positive, nor detect human DNA as the positive, and the primer sequence is specific to the sgRNA sequence.
Normal human DNA, schistosoma DNA and hydatid DNA were blindly tested, and multiple sgRNA-guided cas12a digestion was performed after multiple primer RPA amplification.

![](https://static.igem.wiki/teams/4127/wiki/results12.png)

Figure 7. Blind test results of simulated patient samples

# Multiple sgRNA can improve detection speed

The necessity of verifying the multiple primer detection method, indicating that the use of Cas digestion detection with multiple sgRNA is more convenient to improve our detection speed.
Simulated patient samples were subjected to Cas digestion with single sgRNA after RPA amplification using single primers.

![](https://static.igem.wiki/teams/4127/wiki/results13.png)

Figure 8. Single primer review results of simulated patient samples

![](https://static.igem.wiki/teams/4127/wiki/results14.png)

Figure 9.The comparison of real positive rate between single sequence detection and multiple sequence detection.

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