iGEM 2023 — Team bpi-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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![](https://static.igem.wiki/teams/4667/wiki/page-heading/349e283c5fd22318237c36e74c6235b.jpg)

### August

Seven days

Cultivation of BL21 bacteria

![](https://static.igem.wiki/teams/4667/wiki/note1-1.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-2.jpg)

![](https://static.igem.wiki/teams/4667/wiki/note1-3.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-4.jpg)

Eight days.

- Cas12a protein expression:

Increased IPTG

Testing the value of OD 600pET-28a synthesised E. coli BL21 (DE3)

Recombinant plasmid pET-Cas12a plasmid extraction:

Concentrations were measured using Nanodrop

PCR detection of pET-Cas12a plasmid

![](https://static.igem.wiki/teams/4667/wiki/note1-5.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-6.jpg)

![](https://static.igem.wiki/teams/4667/wiki/note1-7.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-8.jpg)

Nine Days.

Cas12a protein extraction:

Ultrasonic Cleavage

Nickel affinity purification

Extraction of target DNA fragments

Concentrations were measured using Nanodrop

![](https://static.igem.wiki/teams/4667/wiki/note1-9.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-10.jpg)

![](https://static.igem.wiki/teams/4667/wiki/note1-11.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-12.jpg)

Ten days

Cas12a protein electrophoresis, SDS-PAGE

In vitro transcription of pET-Cas12a

Purification of crRNA

Incubation of crRNA and Cas12a protein

- Test reaction system using pET-Cas12a plasmid

reporter is included in the system, results will be based on colour intensity

Reaction system tested using different miRNAs (miR-21 miR-16 miR-155 miR-221)

Validation using gel electrophoresis after PCR

![](https://static.igem.wiki/teams/4667/wiki/note1-13.jpg)

Eleven days.

- Incubation of crRNA and Cas12a protein

- Test reaction system using different miRNAs (miR-21 miR-16 miR-155 miR-221)

PCR followed by gel electrophoresis proved

- Test reaction system using pET-Cas12a plasmid

reporter is included in the system, results will be based on colour intensity

![](https://static.igem.wiki/teams/4667/wiki/note1-14.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-15.jpg)

![](https://static.igem.wiki/teams/4667/wiki/note1-16.jpg)

Twelve days

- Test results system pET-Cas12a plasmid

reporter is included in the system, the results will be based on the intensity of colour detected by multiple scanning rises

![](https://static.igem.wiki/teams/4667/wiki/note1-17.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-18.jpg)

![](https://static.igem.wiki/teams/4667/wiki/note1-19.jpg)
![ ](https://static.igem.wiki/teams/4667/wiki/note1-20.jpg )

Lateral chromatography test strip results.A. Negative. B. Positive. Negative: the T-line does not show any colour,

which is classified as negative, indicating that the reporter molecule has not been cleaved by Casase and Casase

has not been activated; Positive: the T-line shows colour, which is classified as positive,indicating that the

nucleic acid probe has been cleaved by Casase and Casase has been activated;

## Notebook entry：

![](https://static.igem.wiki/teams/4667/wiki/note1-21.jpg)
![ ](https://static.igem.wiki/teams/4667/wiki/note1-22.jpg )
![](https://static.igem.wiki/teams/4667/wiki/note1-23.jpg)
![](https://static.igem.wiki/teams/4667/wiki/note1-24.jpg)

{% endblock %}

{% extends "layout.html" %}
{% 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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![](https://static.igem.wiki/teams/4667/wiki/page-heading/dc080f346369ea4a21b095bf18f09f6.jpg)

### Introduction

MicroRNAs (miRNAs) are non-coding, short, single-stranded RNAs. They play a key role in the genetic regulation of many biological processes. Their aberrant expression is always accompanied by a range of human diseases, including cancer, viral infections, and tumors. Therefore, accurate and sensitive in vitro diagnosis of miRNAs are valuable for disease prevention and treatment.
miRNA is a class of short non-coding RNA molecules and its abnormal expression is closely related to a variety of diseases. Therefore, achieving the detection of miRNA for clinical diagnosis is of great significance. However, due to the low abundance and strong homology of miRNA, there is a certain level of testing required for its high sensitivity and high specificity detection. In order to achieve highly sensitive detection, researchers have proposed many nucleic acid amplification techniques, such as RCA, CHA, HCR, etc., of which HCR technology, due to its simple operation, mild reaction conditions, and other advantages, has been favored by the majority of researchers. The concept of HCR was first proposed by Dirks and Pierce in 2004, in which the target molecule triggers the alternating ring-opening self-assembly of two DNA hp into linear double-stranded DNA nanostructures containing a large number of repeating units, with the advantages of thermostability, enzyme-free, and high amplification efficiency.

### How to design plasmids

We chose the T7 promoter and T7 RNA polymerase because they have strong translational ability and are usually used for protein expression. E. coli expresses our target protein pET-Cas12a. For this purpose, we designed the DNA sequence of pET-Cas12a, inserted it into the pET-28a vector, and transformed the recombinant plasmid into E. coli BL21(DE3) for protein expression.

### How to construct plasmids

To construct our plasmid, We asked the company to synthesize DNA fragments and insert the gene for LbCas12a into the pET-28a vector. The constructed plasmid was contained in an E. coli strain, which we inoculated by streaking on LB solid plates containing the appropriate antibiotics and incubated overnight at 37°C (Figure 1).
Figure 1 BL21 colonies

##### How to Transform Plasmids

Plasmid transformation (on ice)

Ⅰ. Preparation

1. The centrifuge tube containing the plasmid was centrifuged at 1300r for 5min;

2. Plasmids were lysed with ultrapure ddH2O;

3. Take 0.5μL of the integration solution into the tube;

4. 50μL receptive cell BL21 was added into the tube and wait for 1min 30s in the tube.

II. Ice baths

1, ice bath for 30min, with constant temperature water bath at 42℃, heat shock 90s;

2. Add 800 μL LB liquid medium（Kan+） to the centrifuge tube (no resistance).

III. Coat the plate

Leave for 30 minutes The LB liquid medium (150μL) was put into the constant temperature medium containing resistance selective medium at 37℃. After the bacteria solution was completely absorbed, the Petri dish was inverted and incubated at 37℃ for 12-16 hours.

IV. Inoculating colonies

1. Dip single colonies on the cultivated plate with the tip of a pipet gun;

2. Put into a tube with 1000μL medium (liquid) and culture with a shaker;

3. 4 tubes of bacteria (two plates, one picked twice) were inserted into the four tubes with the tip of a gun, incubated at 75r at 37 ° C on the shaker, and then single colonies were picked.

V. Directly add 150μL of bacterial solution to 150 ML LB liquid medium (Kan+) at 100:1. Cultures were incubated overnight at 75r at 37° C on a shaker.

### How to Test PET-Cas12a

Protein expression and purification:

The recombinant plasmid pET-Cas12a was transferred into the expression strain BL21 sensory state, and after the colonies were identified correctly, prokaryotic expression was performed. (Figure 2).

1. BL21 colonies containing the correctly identified recombinant plasmid pET-Cas12a were inoculated into 5 mL LB liquid medium（Kan+）at 37 ℃, 200 rpm, for shock culture for 12-16 h.

2. Absorb 2 mL of the bacterial solution cultured overnight into 200 mL of LB liquid medium（Kan+）, and culture with shaking at 37 ℃, 200 rpm, until the bacterial solution is about OD600 to 0.5, about 2-3 h.

3. 1.5 mL bacterial solution was aspirated as a whole cell control before IPTG induction and stored at -20 ° C.

4. Add IPTG until the final concentration is 0.5mmol /L, 1mmol /L, and 1.5mmol /L, respectively, at 37 ℃ and 150 rpm, and oscillate for 3-6 h.

5. 1.5 mL of bacterial solution was aspirated as a whole cell control after IPTG induction and stored at -20 ° C.

6. The rest of the bacteria were centrifuged at 5000 rpm and 4 ° C for 5 min, and the bacteria were collected. After the supernatant was discarded, the bacteria were repeatedly frozen and thawed 3 to 4 times with liquid nitrogen (if liquid nitrogen was not available, the bacteria were washed twice with PBS, resuspended with 1ml lysate, and added with a final concentration of 1mmol/L PMSF solution. Crushed on ice with an ultrasonic crusher).

7. The bacteria were resuspended in 20 mL of ice-cold PBS for precipitation, and then crushed by an ultrasonic crusher on ice until the bacterial solution was translucent to avoid bubbles, and ultrasonic crushing was carried out on ice.

8. After centrifugation at 10,000 rpm at 4 ℃ for 10 min, the supernatant and precipitate were aspirated and subjected to SDS-PAGE to observe the expression and dissolution of GST fusion protein. If the expression of the pET-Cas12a fusion protein is high and present in the supernatant, proceed to the next step of purification of the pET-Cas12a using nickel column.

![](https://static.igem.wiki/teams/4667/wiki/project-engineering/1.png)

Figure 1. Results of SDS-PAGE electrophoresis condensation and HCR amplification reaction electrophoresis of Cas12a protein. A. as12a protein has a size of 130kDa. The results of SDS-PAGE electrophoresis indicated that Cas12a protein was present in our solution collected at 130kDa and was not present in nonspecific protein impurities. Thus, Cas12a protein was expressed and purified with high quality. B. lane 1： H1 (1 µM); lane 2: H2 (1 µM); lane 3:1 µM H1+ 1 µM H2; lane 4:1 µM H1+ 1 µM H2+ 50 nM miR; lane 5:1 µM H1+ 1 µM H2+ 100 nM miR; lane 6:1 µM H1+ 1 µM H2+ 200 nM miR; lane 7: 1 µM H1+ 1 µM H2+ 500 nM miR-; lane 8:1 µM H1+ 1 µM H2+ 1000 nM miR.
Then, we made a standard curve (Fig. 3) y=0.0075x+0.1205 R²=0.9868 of the obtained data of protein concentration and absorbance in response to its steady growth changes.

![](https://static.igem.wiki/teams/4667/wiki/project-engineering/2.png)

Figure 2. Standard linearity of the BCA method used to calculate the concentration of protein
Through the curve values, we measured the absorbance of Cas12a as 0.2276 (L/ (g.cm) and the protein concentration (μ g /ml). This result indicates that we obtained a sufficient concentration of Cas12a protein.

### CRISPR-CAS12a protein cleavage assay

In order to verify whether our purified crRNA-Cas12a can function as a regular reverse cleavage, we conducted relevant experiments.
The lateral chromatographic test strip used in this method has two strips, the lower quality control line and the upper detection line. The quality control line was coated with avidin (SA), the detection line was coated with sheep anti-mouse secondary antibody, and the colloidal gold was labeled with anti-FAM /FITC monoclonal antibody. The complete reporter molecule (Biotin and FAM labeled at both ends) could capture all the colloidal gold at the quality control line. When a reporter molecule was cut off by the Cas enzyme, the colloidal gold bound to the cut fragment could not be captured by the quality control line, forming a detection line. The Cas enzyme was activated by the detection line, and the positive or negative results of the test were further judged, and the constructive opinions of the patients were given.

![](https://static.igem.wiki/teams/4667/wiki/project-engineering/3.png)

Figure 3. Lateral chromatographic test strip test results. A. Negative. B. Positive. Negative:·T line does not show color and is judged negative, indicating that the reporter has not been Cas. Enzyme cleavage, Cas. enzyme is not activated; Positive :T·line color, judged as positive, indicating that there is a nucleic acid probe cut by Cas. enzyme, cas enzymes are activated;

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4667/wiki/page-heading/0253ff7cf48ffa5588d464db914d064.jpg)

### 1. Primer design

|  |  |
| --- | --- |
| ingredinents | quantities |
| H1 | 4 |
| H2 | 4 |
| miRNA | 4 |
| crRNA | 1 |
| reporter | 1 |

See the attached primer sequences

BBa\_K4667007；

BBa\_K4667008;BBa\_K4667009;BBa\_K46670010;BBa\_K4667011;BBa\_K4667012

### 2. Plasmid construction by Genscript Biotech Company

See the plasmid report from Genscript Biotech attached.
pET-Cas12 certificate

### 3 Transformation of the plasmid into BL21(DE3) E. coli

The procedure is based on the Trans5a conversion manual, with minor modifications, and is as follows.

1 10 μL of ligation product was added to 50 μL of just melted competent cells, mixed gently, and left on ice for 30min.

2. Heat shock at 42° C for 45 s, quickly transfer to an ice bath and place for 2min. Please do not shake the centrifuge tube during this process.

3. Add 500μL LB liquid medium（Kan+）, 37℃, 200 rpm, and incubate for 1h to revive the bacteria.

4, Centrifuge the bacteria at 5000 rpm for 2 minutes. Collect the bacteria and discard a portion of the supernatant. Resuspend the remaining bacteria and inoculate them onto an LB liquid medium（Kan+） Incubate the culture overnight (12-14h) in an inverted incubator at a constant temperature of 37℃.

### 4 inoculate single colonies

The methodology follows Han (2016) with slight modifications as follows:

The recombinant plasmid pET-Cas12a was transferred into the expression strain BL21 sensory state and, after the correct identification of colonies, prokaryotic expression was carried out.

1. BL21 colonies containing the correctly identified recombinant plasmid pET-Cas12a were inoculated into 5 mL LB liquid medium（Kan+）at 37 ℃, 200 rpm, for shock culture for 12-16 h.

2. Absorb 2 mL of the bacterial solution cultured overnight into 200 mL of LB liquid medium（Kan+）, and culture with shaking at 37 ℃, 200 rpm, until the bacterial solution is about OD600 to 0.5, about 2-3 h.

3. 1.5 mL bacterial solution was aspirated as a whole cell control before IPTG induction and stored at -20 ° C

4. Add IPTG until the final concentration is 0.5mmol /L, 1mmol /L, and 1.5mmol /L, respectively, at 37 ℃ and 150 rpm, and oscillate for 3-6 h.

5. 1.5 mL of bacterial solution was aspirated as a whole cell control after IPTG induction and stored at -20 ° C.

6, The rest of the bacteria were centrifuged at 5000 rpm and 4 ° C for 5 min, and the bacteria were collected. After the supernatant was discarded, the bacteria were repeatedly frozen and thawed 3 to 4 times with liquid nitrogen (if liquid nitrogen was not available, the bacteria were washed twice with PBS, resuspended with 1ml lysate, and added with a final concentration of 1mmol/L PMSF solution. Crushed on ice with an ultrasonic crusher).

7. The bacteria were resuspended in 20 mL of ice-cold PBS for precipitation, and then crushed by an ultrasonic crusher on ice until the bacterial solution was translucent to avoid bubbles, and ultrasonic crushing was carried out on ice.

8. After centrifugation at 10,000 rpm at 4 ℃ for 10 min, the supernatant and precipitate were aspirated and subjected to SDS-PAGE to observe the expression and dissolution of GST fusion protein. If the expression of the pET-Cas12a fusion protein is high and in the supernatant, proceed to the next purification test. If the expression of the pET-Cas12a fusion protein is low or in precipitation, IPTG concentration and incubation temperature should be optimized.

### 5 culture BL21 E. coli

1. Prepare LB liquid medium（Kan+）500 ml to culture Cas12a E. coli, weigh the following reagents with an electronic balance, and add them into 4 conical flasks using a spatula.Tryptone\* 5 g

|  |  |
| --- | --- |
| Tryptone\* | 5g |
| Yeast extract\*\* | 2.5g |
| Suspend with ddH2O\*\*\*\* until the volume reaches | 500 ml |

\*Tryptone is used to provide essential amino acids such as peptides and peptones to the bacteria

\*\*Yeast Extract provides a range of organic compounds for bacteria growth

\*\*\*Sodium chloride balances osmotic pressure and provides sodium ions for the bacterium

\*\*\*\*ddH2O: double distilled water

2.Autoclave all LB (lysogeny broth)

\*No need to adjust the pH as both sodium hydroxide and TRIS buffer are usually unnecessary.

3. Allocate 150ml autoclaved LB into two equal aliquots, each 75ml.

4. Add 75μl of Kan+ antibiotics into one flask, and 75μl of Amp+ antibiotics into the other (as the volume ratio between LB and antibiotics should be 1000:1). Label the flasks.

5. Prepare 4 sets of cell culture tubes, each with 3 tubes.

6. In each group of tubes, allocate 5ml of Amp+ LB into one tube，and 5ml Kan+ LB into the other two.

7. Inoculate bacterium (Add 50μl of E. coli-containing-medium into each tube, ending up with four tubes with four different types of GM E. coli), and label the date, its antibiotics, and integrated sequence on the tube. The number and types of cell culture tubes are recorded in the table below:

|  |  |  |  |
| --- | --- | --- | --- |
| Integrated sequence | Plasmid name | Corresponding antibiotics | Number of tubes |
| Cas12a | pET-28a | Kan+ | 20 |

8. Place all tubes in the shaker rock for 16 hours, and allow it to proliferate.

### 6 Purify Cas12a proteins

##### Prepare pulse buffer

Tris-glycine-sds buffer instant pellet was added to 1000ml of deionized water.

##### Dispensing gel

1. 2.5ml separation gel solution A and 2.5ml separation gel solution B were mixed

2. Add 25μl coagulant polymerization agent to mix, quickly inject the mixture into the gelatin glass plate (0.5-1cm comb tooth)

3. Insert the comb teeth and wait for solidification

##### Concentrate gel

10μl of gel polymerizer was mixed, and added to the rubber plate.

##### Whole Protein Extraction

1. Add 10µl of phosphate inhibitor and 10µl of protease inhibitor PMSF to 1ml of cold lysate, mix well and place on ice.

2. Take 1-5mL of the bacterial solution and centrifuge at 12000rpm for 1 minute, discard the supernatant.

3. Add 0.5-1ml of the new solution to the bacterial block and vortex until well mixed.

4. Transfer the liquid to a 1.5ml EP tube and centrifuge at 4℃, 12000 rpm for 30 minutes.

5. Aspirate the supernatant into a new tube (store the protein at -80°C).

6. Protein gel electrophoresis (300v,20min) was performed.

##### Collect E. coli bacteria

Purpose: to separate it from LB Centrifugation the bacteria medium at 4000 rpm

Discard the supernatant

1.Suspend E. coli within Buffer A

2.Add 5 ml of Buffer A into the sediments, allow the biomass to float

##### Ultrasound Cell Lysis of E. coli

Purpose: Break the bacteria membranes so that Cas12a proteins can be extracted and purified

1.Place the entire sample on ice (maintain a temperature of 4 °C to preserve its proteins) and place it into an ultrasound cell crusher.

2.Set the total time as 10 minutes and the interval as 5 seconds (to close for 5 seconds every time the system worked for 5 seconds). The power ratio should be at 70%.

##### Centrifugation of Ultrasound Results.

Place the sample in the centrifuge. Set the system at 4000 rpm, 20 minutes, 4°C.

After centrifugation, extract the supernatant (containing CAS 12a proteins)

##### Nickel affinity purification of Cas12a

Principle: The 6×His tail of the Cas12a protein is strongly attracted to the nickel column and is therefore not washed away by buffer A, but is washed away by buffer B. Whereas other proteins, which adhere more or less strongly than Cas12a, are either washed away by the addition of buffer A or not washed away by buffer B.

1. Mix the reagents below in the assigned portion as shown in the chart to obtain 0.5 L His buffer A

![](https://static.igem.wiki/teams/4667/wiki/project-experiments/shiyan1.png)

2. Mix the reagents below in the correct portion as mentioned in the chart below and obtain 0.25 His buffer B

![](https://static.igem.wiki/teams/4667/wiki/project-experiments/shiyan2.png)

1. Complete installing the nickel affinity purification system by stacking the nickel column onto a new microtube. Then, place it on ice.

2. Using a pipette, add 4 times the nickel column’s size worth of Buffer A to moist the column. Collect one tube of the solution under the nickel column and label it as the “first filter”.

3. Add the supernatant into the nickel column. Decant slowly and gradually. Keep the velocity of the flow at 1ml/min.

4. Add 6 ml of Buffer A into the nickel column. Keep the velocity of the flow at 1ml/min.

5. Collect one tube of the solution under the nickel column and label it as the“last filter”.

6. Repeat step 4 for two more times.

7. Add 3 ml of Buffer B into the nickel column. Keep the velocity of the flow at 1ml/min.

8. Collect two tubes of the solution under the nickel column and label them as “Cas12a protein #1” and “Cas12a protein #2” respectively. They are the target protein.

9. Add 2 ml of His Buffer B into the nickel column.

10. Collect one tube of the solution under the nickel column and label it as “Buffer B elution”.

11. Add 5 ml of Buffer A into the nickel column.

12. Collect one tube of the solution under the nickel column and label it as “Buffer A elution”.

13. Wash the nickel column by decanting 10ml of His buffer A and 5ml 20% ethanol. Measure the concentration of Cas12a protein in the resultant solution

1、Wash off any residue on the NanoDrop using ddH2O.

2、Add 2μl of Buffer B onto the probe using a pipette (As the negative control group). Record the protein concentration. Wash off any residue on the NanoDrop.

3、Add 2μl of “Cas12a protein #1” solution onto the probe using a pipette. Record the protein concentration. Wash off any residue on the NanoDrop.

4、Add 2μl of “Cas12a protein #2” solution onto the probe using a pipette. Record the protein concentration. Wash off any residue on the NanoDrop.

### 7 Incubate crRNA and CAS12a proteins and test the reaction system

![](https://static.igem.wiki/teams/4667/wiki/project-experiments/shiyan3.png)

\*Note that the volume of oligo DNA varies according to its concentration. The reaction buffer will also vary to achieve a fixed volume of 20 μl.

1. In 4 separate 200 μL centrifuge tubes, miRNAs (miR-21, miR-155, miR-155, miR-133) were added, followed by the corresponding H1 and H2.

2. Mix the above mixture by flicking several times, centrifuging slightly (avoiding vigorous vortex shaking) and repeating 3 times.

3. The solution was incubated in a gradient thermocycler at 37°C for 30 min.

4. At the end of the above reaction, 1µg of the reaction product was added to Reporter (oligo DNA) 0.6µl, crRNA 1µl, and reaction buffer 2 µl, respectively.

5. Stir the above mixture several times to mix well, slightly centrifugal (avoid vortex violent vibration), repeat 3 times

6. Place the reaction tube at 37 °C for 45 min.

7. Take 10 μL of nucleic acid amplification product and dilute it 5 times with 40 μL of ddH2O in a centrifuge tube, mix well.

8. All the diluted reaction products were dropped onto the test strip, and the test results of the interpretation area were recorded within 5~10 minutes;

9. After recording the results, seal the test strip and discard it in a safe place.

The results showed that the control line showed color, which proved that crRNA and CAS12a protein could function normally.

### 8 miRNA extraction (on ice)

1. Cells preserved in liquid nitrogen storage tanks were taken, frozen and hardened into blocks with liquid fluoride, and then ground.

2. Add 100mg/mL TRIZOL Reagent→ Add 700μL ultrapure water /70% ethanol when grinding directly proportional to cells (2 times).

3. Take 700μL of cells (supernatant as much as possible) with a pipetting gun (add to centrifuge tube ).

4. Add 700μL wash buffer I to centrifuge ~12000rpm, pour out the waste liquid in the centrifuge tube and replace it with a new collection tube.

5. Take 500 μL of washbuffer II and pour the waste liquid away.

6. Repeat step 5.

7. After discarding the waste liquid first, the centrifugal speed is 12000 rpm for 2 min, and the new collection pipe is replaced.

8. Add 30-100μL of ultrapure water and allow to stand for 1 minute.

### 9 HCR isothermal amplification reaction

1. Centrifuge H1, H2, miR at 12000rpm for 1min;

2. H1: 47.9 µl ddH2O was added first to prepare 100µM, and then gradient dilution to 1µM;

3. H2: 46.8µl ddH2O was added first to prepare 100µM, and then gradient dilution to 1µM;

4. miRNA: 100.1 µl ddH2O was added first to prepare 100µM, and then gradient dilution was carried out according to experimental requirements;

5.HCR reaction and verification method: H1 and H2 were dissolved and heated at 95℃ for 2min and cooled to a temperature of 1h;

6. The concentrations of H1 and H2 were both 1μm, and different concentrations of miRNA (0, 100 μm, 500μm, 1μm, 3μm) were added for reaction at room temperature for 4h;

7. Perform agarose gel electrophoresis (electrophoresis conditions: gel concentration :2%, voltage 120v, duration 30min);

8. Sample loading sequence (Agarose gel);

Ladder/H1+H2/H1/H2/miRNA 0.1μm H1, H2/ miRNA 0.5μm H1, H2/miRNA 1μm H1, H2/miRNA 3μm H1, H2/miRNA 10μm H1, H2/miRNA 1μm;

### 10 Electrophoresis of HCR amplification products

The 6% nondenaturing PAGE gel analysis was performed as follows: a total volume of 15 ml of PAGE gel contained 3 ml of 30% acrylamide (acrylamide: double acrylamide, 19:1), 3 ml 5 × TBE, 110 µl 10% ammonium persulfate, 10 µl N, N, N', N'-tetramethylethylenediamine (TEMED) and deionized water, immediately pour the gel into the electrophoresis tank equipment, rest until the gel is set. Then, 15 µL of sample solution was taken for loading, after which electrophoresis was carried out at 120 V for 50 min, electrophoresis was stopped, and the gel block was removed and stained in 1 × GelRed dye solution for 10 min. Afterwards, the gel was imaged in the gel imaging device.

Photogel results demonstrated that the HCR isothermal amplification reaction occurred

### 11 CRISPR-CAS12a protein cleavage assay

Principle:

The lateral chromatographic test strip used in this method has two strips, the lower quality control line and the upper detection line. The quality control line was coated with avidin (SA), the detection line was coated with sheep anti-mouse secondary antibody, and the colloidal gold was labeled with anti-FAM /FITC monoclonal antibody. The complete reporter molecule (Biotin and FAM/FITC labeled at both ends) could capture all the colloidal gold at the quality control line. When a reporter molecule was cut off by Cas enzyme, the colloidal gold bound to the cut fragment could not be captured by the quality control line, forming a detection line. Activation of the Cas enzyme was determined by the presence or absence of the detection line.
Prepare 20 μl of reaction system (note: operate on ice):

![](https://static.igem.wiki/teams/4667/wiki/project-experiments/shiyan4.png)

\* Cas12a Protein (10μM) diluted to Cas12a Protein (1μM) with Tris Buffer or ddH2O.

\*\* Add 1μl of "crRNA for Positive Control (20X)" to the positive control group, and add crRNA specific to the target sequence to the other reactions \*\*\* Add the thermostated amplification products from Step 2, if the template concentration is high, the recommended template spiking volume is 2μL, x≤5μL.

1. Lightly flick several times to mix, slightly centrifugal (avoid vortex violent vibration), repeat 3 times

2. The reaction tube was placed at 37 °C for 4 5 min.

3. 10μL nucleic acid amplification products were diluted 5 times with 40μL ddH2O in a centrifuge tube and mixed.

4. All the diluted reaction products were dropped on the nucleic acid test strip, and the test results of the interpretation area were recorded within 5-10 minutes

5. After recording the test results, seal the test strip and discard it in a safe place.

![](https://static.igem.wiki/teams/4667/wiki/project-experiments/shiyan5.png)

Negative: T line did not show color and was judged negative, indicating that the reporter was not cut by Cas enzyme and the Cas enzyme was not activated.

Positive: T-lines visible to the naked eye were judged as positive, indicating that the nucleic acid probe was cleaved by Cas enzyme, and Cas enzyme was activated.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4667/wiki/page-heading/5f34d8f4a2b8caa073d8294bd303ba2.jpg)

### 1. Plasmid construction

We have designed the pET-Cas12a expression plasmid. To construct our plasmid, we let the company synthesise DNA fragments by inserting FnCas12 into the pET28a plasmid vector. The constructed plasmids were contained in E. coli strains, we streak inoculated them on LB liquid medium plates containing corresponding antibiotics and incubated them at 37℃ overnight (Figure 1).

![](https://static.igem.wiki/teams/4667/wiki/project-results/1.jpg)

Figure 1. Incubation of plasmids containing strains. A. E. coli BL21 containing the pET28a plasmid. B. E. coli BL21 containing the pET28a plasmid. C. pET28a plasmid after single colony culture. D. pET28a plasmid after single colony culture

### 2. Purification of Cas12a protein and purified

![](https://static.igem.wiki/teams/4667/wiki/project-results/2.png)

Figure 2. Expression and purification of protein Cas12a. A. Incubation of Cas12a containing the pET28a plasmid. B. SDS-PAGE electrophoresis gels of Cas12a protein after IPTG induction at different concentrations.
The Cas12a protein has a size of 130kDa.SDS-PAGE electrophoresis results showed that Cas12a protein was present in our collected solution at 130 kDa. It was found that 1 mmol/l IPTG was the optimal induction concentration by experiment. Therefore, Cas12a protein was expressed and purified with high quality.
We tested the concentration of Cas12a protein by Bicinchoninic Acid Assay (BCA) using a SpectraMax i12x Multi-mode zymography with the absorption peak at 562 nm (Figure 3).

![](https://static.igem.wiki/teams/4667/wiki/project-results/3.png)

Figure 3. Standard linear regression line of BCA method used to calculate protein concentration. Absorbance and calculated protein concentration of Cas12a.
Through the curve values, we measured the absorbance of Cas12a as 0.2276 (L/(g.cm)) and the protein concentration (ug/ml) as 10.7549295, this result indicates that we obtained a sufficient concentration of Cas12a protein.

![](https://static.igem.wiki/teams/4667/wiki/project-results/4.png)

Figure 4. Electropherogram of the results of miRNA-initiated and HCR isothermal amplification reaction.lane 1： H1(1 µM)；lane 2： H2(1 µM)；lane 3：1 µM H1+ 1 µM H2； lane 4：1 µM H1+ 1 µM H2+ 50 nM miR； lane 5：1 µM H1+ 1 µM H2+ 100 nM miR； lane 6：1 µM H1+ 1 µM H2+ 200 nM miR； lane 7：1 µM H1+ 1 µM H2+ 500 nM miR-； lane 8：1 µM H1+ 1 µM H2+ 1000 nM miR

Electrophoretic graph of the results of HCR isothermal amplification reaction triggered by miRNA, only H1 H2 cannot trigger the amplification reaction, but it can be triggered by the addition of miRNA and the shapes of the amplification bands triggered by different concentrations of miRNA are similar, and all of them show the results of trapezoidal bands, but the higher the concentration of miRNA, the faster the reaction speed.

### 3. Colour intensity and changes in different miRNA species

![](https://static.igem.wiki/teams/4667/wiki/project-results/5.jpg)

Figure 5 Specificity results of miRNA-initiated isothermal amplification reaction
As can be learnt from the figure, the method we have developed is characterized by high specificity. Different miRNAs can target different hairpin structures, thus triggering an amplification reaction, which occurs with a clear difference between the colour change and the blank as well as other kinds of miRNAs, demonstrating good method specificity.

#### 4. Judgement of lateral chromatography test strips

![](https://static.igem.wiki/teams/4667/wiki/project-results/6.png)

Figure 6 Lateral chromatography test strip results. A. Negative. B. Positive. Negative: the T-line does not show any colour, which is classified as negative, indicating that the reporter molecule has not been cleaved by the Cas enzyme and the Cas enzyme has not been activated; Positive: the T-line shows colour, which is classified as positive, indicating that the nucleic acid probe has been cleaved by the Cas enzyme and the Cas enzyme has been activated.
This method uses lateral chromatography test strips with two bands, a lower quality control line and an upper detection line. Affinity agent (SA) is coated at the quality control line, sheep anti-mouse secondary antibody is coated at the detection line, and anti-FAM/FITC monoclonal antibody is labelled on colloidal gold. An intact reporter (labelled on both ends with Biotin and FAM/FITC) allows all of the colloidal gold to be captured at the QC line, but when a reporter is cut by the Cas enzyme, the colloidal gold that binds to the cut fragment is not captured by the QC line and forms the detection line. The presence or absence of the detection line determines whether the Cas enzyme is activated or not. In order to make our assay kits more user-friendly, we tested the isothermal amplification method for detecting miRNAs instead of the PCR amplification method. The experimental results showed that the specificity of our method was good and the detection results could be judged by the negative and positive test strips, indicating that the protocol of miRNA detection by amplification via the isothermal amplification method was feasible. This provides strong experimental data support for us to develop a portable detection kit.

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