iGEM 2019 — Team Shanghai_HS_United

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# Team:Shanghai HS United/Notebook

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# Megamenu

## Notebook

1. July 5th 2019

Purpose:
Have a detailed understanding to the equipment, the chemical, and the principle

What we do?

1.Sterilization

* (1)Place the equipment in to the High-Pressure Steam Sterilization Pot* (2)Set the temperature to 121 Celsius
  * (3)Make sure there are enough water in the Pot
  * (4)Close the cap and start the sterilization

2.Preparation of LB broth (500ml)

|  |  |
| --- | --- |
| Liquid LB broth | Solid LB broth |
| -NaCl 5g | -NaCl 5g |
| -Peptone 5g | -Peptone 5g |
| -Yeast extraction 2.5g | -Yeast extraction 2.5g |
| -Water 500ml | -Agar 12g |
|  | -Water 500mll |

3.PCR amplification

(1) Reaction system:

|  |  |
| --- | --- |
| -Water 28 µL | -Buffer 10µL |
| -Template 5µL | -dNTP 4µLv |
| -Primer-F 1µL | -Primer-R 1µL |
| -DNA polymerase 1µ |  |

(2) Place the mixture in PCR

|  |  |
| --- | --- |
| Liquid LB broth | Solid LB broth |
| -NaCl 5g | -NaCl 5g |
| -Peptone 5g | -Peptone 5g |
| -Yeast extraction 2.5g | -Yeast extraction 2.5g |
| -Water 500ml | -Agar 12g |
|  | -Water 500mll |

4.Gel electrophoresis

(1)Preparation of gel:
1˚ Mix 50ml of 5X TAE to 0.75g of Agarose
2˚ Heat the mixture until transparent
3˚ Cooling for 5 minutes, and add 5µL gel red
4˚ Pour it in the gel model, and place the comb on it
5˚ Wait the gel until dry and take it off
(2)Place the gel in electrophoresis apparatus
(3)Add 1X TAE until gel is submerged in the TAE
(4)Mix the PCR production with loading buffer and drop the mixture into the hole of gel
(5)Make sure that the DNA run from cathodes to anode, so that our gel is in the right direction
(6)Start gel electrophoresis in 120v, and wait for about 20 minutes.
(7)Put the gel under the UV light, and observe it

![](https://igem.siqint.com/zhuwen/images/home/4.1.png)

5.The recycle of PCR

(1)Add our original PCR production into microfuge tube
(2)Add buffer PCR-A 15µL in microfuge tube
(3)Place the microfuge tube in the centrifuge tube.
(4)Place the centrifuge tube and microfuge tube together in the centrifuge, and start centrifuge with revolving speed of 12000 rotation/minutes for 1 minute.
(5)Pour the waste in centrifuge tube
(6)Add 400µL of buffer W2, place it in centrifuge, and repeat the method again
(7)Place it in the centrifuge again without add any buffer
(8)Add 30µL water (65˚C)
(9)Use spectrophotometer to measure the concentration of our DNA solution

6.Transformation

(1)Mix our plasmid DNA with E. coli DH10B
(2)Place the mixture on the ice
(3)Wait for 30 minutes and start heat shock (90 seconds, 42˚C)
(4)Place the mixture immediately on the ice for 2 minutes
(5)Add the mixture into liquid LB broth, and place it on incubator (37˚C) for 45 minutes

7.Culture our E. coli on the agar plate

(1)Remove the extra liquid LB broth from the tube of DNA
(2)Mix the remain, and it on the solid LB broth
(3)Blent the liquid on LB broth until dry

July 6th 2019

Purpose:
Investigating the difference between LAMP amplification and PCR amplification

1.PCR amplification for the failing sample of previous day

(1) Reaction system:

|  |  |
| --- | --- |
| -Water 28 µL | -Buffer 10µL |
| -Template 5µL | -dNTP 4µL |
| -Primer-F 1µL | -Primer-R 1µL |
| -DNA polymerase 1µL |  |

(2) Place the mixture in PCR

2.Trial of LAMP amplification

(1)Reaction system:
-Water 4.6µL        -Template 2µL
-Mixture of primer 2µL -dUTP 1.4µL
-mixture of buffer, DNA polymerase, and dNTP 10µL
(2) Place the mixture in the water bath (65˚C) for 50 minutes (that condition is only a trial)
(3) Place our mixture in 85˚C water when times up

3.Gel electrolysis for the product of LAMP amplification and PCR amplification

![](https://igem.siqint.com/zhuwen/images/home/4.2.jpg)

4.Recycle of DNA of the product of PCR amplification

5.Culture the E. coli in liquid LB broth

(1)Add 2ml liquid LB broth in each of 2 test tubes
(2)Use micropipette select two transformational E. coli, and put one in each test tube

July 7th 2019

Purpose:
Storage our target DNA, and try different condition for LAMP amplification

1.Seamless assemble for carrier DNA and fragment DNA

(1)Calculate the amount of carrier DNA and fragment DNA we need. The total amount should be 7µL
(2)Add 2µL buffer, and 1µL enzyme
(3)Place the mixture in the 37˚C water bath for 30 minutes

2.Transform our target DNA (the combination of carrier DNA and fragment DNA) in the E. Coli DH10B

3.Extract the plasmid of the E. coli from previous day

(1)Place them in the centrifuge
(2)Pour away the supernatant liquid, and add 250µL of Buffer S1 in the precipitate.
(3)Place the mixture in the centrifuge, and pour away the supernatant liquid. Add Buffer S2, and wait for about 5 minutes
(4)Add 350µL Buffer S3 in the mixture, and put the mixture in the centrifuge for 10 minutes.
(5). Recycle the DNA and measure the concentration

4.Test of the best concentration for LAMP amplification

(1)Prepare different concentration gradient of our template DNA solution (from 10���-2 to 10-9)
(2)Do Lamp amplification, and analyze result by using gel electrolysis

![](https://igem.siqint.com/zhuwen/images/home/4.3.png)

The result of gel electrolysis shows that there is no stripe in the concentration gradient of 10-7 to 10-8, but there is a stripe in the concentration gradient of 10-9 and control group (one without template) As a result, there must be a contamination of unrelated DNA

July 8th 2019

Purpose:
Investigating how Cas12a work in our gene engineering, and using test paper to test whether our product of LAMP amplification is positive or negative

1.Test the E. coli colony by using PCR amplification

2.Cas12a reaction

(1)Prepare 3 tubes. Label one with water, one with the template without stripe, and one with template with stripe
(2)Reaction system:
-Water 14.5 µL        -10X buffer 2µL
-FnCas12a enzyme 0.5µL        -crRNA 0.5µL
-FITC 2µL
-Template with stripe/ Template without stripe/Water 5µL
(3) Place the test paper in each tube

![](https://igem.siqint.com/zhuwen/images/home/4.4.png)

As the result shown, the tube with template with stripe show a positive result on 14NT test paper, but there is a test paper show a false positive result. As a result, we should add UDG to prevent it

July 12th 2019

Purpose：
-Figure out whether primer 4 (a kind of primer mixture) is the best primer for LAMP amplification or primer 5.
-Figure out the least concentration gradient for a LAMP amplification to operate.

1.LAMP amplification for different conditions for swine blood

(1)Conditions:
Temperature: 65˚C, 67˚C
Time: 30 minutes, 50 minutes
Type of primer: Primer 4, Primer 5
(2)Gel electrolysis

![](https://igem.siqint.com/zhuwen/images/home/4.5.png)

According to the gel electrolysis we found that all groups with primer 4 had a stripe in the condition of 65 ° 30mins and 67 ° 50mins, while only one stripe with primer 5 was appeared. It can be proved that primer 4 is more suitable for our LAMP amplification.

2.LAMP amplification for 8 different concentration gradients of swine blood with UDG to test if the contamination can be remove by adding UDG

(1)Conditions:
Concentration gradients: 10-3, 10-4,10-5,10-6,10-7,10-8,10-9
(2)Gel electrolysis

![](https://igem.siqint.com/zhuwen/images/home/4.6.png)

July 13th 2019

Purpose:
Improve the experiment from previous day by adding some new conditions

1.LAMP amplification for swine blood
(1)New conditions:
Temperature: 63˚C, 65˚C
(2)Gel electrolysis

![](https://igem.siqint.com/zhuwen/images/home/4.7.png)

July 15th 2019

Purpose:
Using test paper to detect the African swine fever in real blood of swine

1.LAMP amplification for swine blood (the same experiment to the last time)

![](https://igem.siqint.com/zhuwen/images/home/4.8.png)

![](https://igem.siqint.com/zhuwen/images/home/4.9.png)

![](https://igem.siqint.com/zhuwen/images/home/logo1.png)©2019 Microsoft Corporation. All rights reserved.


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# Team:Shanghai HS United/Experiments

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# Megamenu

## Experiments

PCR

Materials:

|  |  |
| --- | --- |
| Buffer | 10 μL |
| dNTP. | 4 μL |
| Enzyme. | 1 μL |
| Template. | 5 μL |
| (PSB1K3-J23100-B0034-ter) |  |
| Water. | 28 micro-liters |
| Primer1. | 1 μL |
| Primer2. | 1 μL |
| (PSB1K3-R) |  |

Procedure:

1.Add all the materials together:

•Firstly, use pipette to straw 28 μL pure water from the pure water bottle to one microcentrifuge tube.
•Secondly, use pipette to straw 10 μL buffer to the previous tube.
•Thirdly, use pipette to straw 4 μL dNTP to the previous tube.
•Next, use pipette to straw 5 μL template to the previous tube.
•Then, add 1 μL of each primer to the previous tube.
•Finally, use pipette to straw 1 μL of enzyme to the previous tube.

2.Place the tube to the PCR machine to react at a different temperature for each stage.

![](https://igem.siqint.com/zhuwen/images/home/5.1.jpg)

Foster and transform the E.coli bacteria

Materials:

|  |  |
| --- | --- |
| NaCl. | 4 grams |
| Tryptone. | 5 grams |
| Yeast. | 2.5 grams |
| Water. | 500 milliliters |

Procedure:

1.Make the lysogeny broth (LB)

•Firstly, use balance to measure 4 gram of Sodium chloride and add to a conical flask.
•Secondly, use balance to measure 2.5 gram of yeast and add it to the conical flask.
•Thirdly, use balance to measure 5 grams of tryptone and add it to the conical flask.
•Use measuring cylinder to measure 500 milliliters of water and add to the conical flask. Stir the mixture in the conical flask with a glass rod.

2.Culture the bacteria with lysogeny broth (LB)

•Firstly, add 5 μL of antibiotic to the lysogeny broth (LB).
•Secondly, pour the LB liquid to a petri dish until the liquid fill the surface of the dish and cool them in the 37-degree-environment for 1 hour.
•Secondly, use pipette to straw 1micrometer of bacteria and add to the petri dish.
•Thirdly, use a clean plastic stick to spread the liquid on the original petri dish until the surface is completely smooth and even. Put the dishes in the 37-degree-environment for 24 hours.

3.Transformation of E. coli bacteria

•Firstly, use a pipette with a tube to closely touch the E. coli bacteria on the petri dish and mix it with plasmid in the tube.
•Secondly, put the tube into the ice for 20-30 minutes.
•Thirdly, put the tube back to the 42 degrees water bath for 90 seconds and ice them again for 2 minutes.
•Next, pour the lysogeny broth (LB) into the mixture in the tube and put it on the shaking machine for 45 minutes at 37 degree Celsius.

Extract the plasmid DNA

Materials:

|  |  |
| --- | --- |
| Buffer. | 1.230 milliliters |
| Pure water. | 30 μL |

Procedure:

1.Firstly, straw all the liquid in the PCR tube except the precipitation we have used into a filter tube.
2.Secondly, use pipette with a tube to straw 130 μL of washing buffer to the filter tube which we have just used.
3.Next, put the filter tube into centrifuge and spin for 1 minutes.
4.Then, throw away the liquid in the bottom of the tube and add 700 μL of washing buffer to the filter tube.
5.Thirdly, put the filter tube into centrifuge and spin for 1 minutes. And add 400 μL of washing buffer to the filter tube again.
6.Fourthly, change the bottom tube into a new one while keep the filter.
7.Finally, add 30 μL of pure water at 65 degree Celsius to the new tube.
8.Lastly, put the filter tube into centrifuge and spin for 1 minutes and throw away the filter tube. The bottom tube is the recycle of DNA.

LAMP

Materials:

|  |  |
| --- | --- |
| Water. | 4.1 μL |
| DUTP. | 1.4 μL |
| Mixture. | 10 μL |
| (Buffer, DNTP, enzyme included) |  |
| Template. | 2 μL |
| UDG. | 0.5 μL |
| Primer. | 2 μL |
| (Suggested using number 4 primer) |

Procedure:

1.Add all the materials together:
•Firstly, use pipette to straw 4.1 μL pure water from the pure water bottle to one tube.
•Secondly, use pipette to straw 1.4 μL dUTP to the previous tip.
•Thirdly, use pipette to straw 2 μL primer to the previous tube.
•Next, use pipette to straw 2 μL template to the previous tube.
•Then, add 0.5 μL UDG to the previous tube.
•Finally, use pipette to straw 10 μL of mixture to the previous tube.
2.Put all the tubes we have made into the water bath at 65 degrees for 30 minutes.
3.Do the following Cas12a procedure to detect if the solution includes the virus.

Fluorescence probe （fam）

Materials:

|  |  |
| --- | --- |
| Water. | 14.5 μL |
| Buffer. | 2 μL |
| Enzyme. | 0.5 μL |
| Template. | 0.5 μL |
| (From solution we made after LAMP) |  |
| Detector. | 2 μL |
| crRNA. | 0.5 μL |

Procedure:

1.Add all the materials together:
•Firstly, use pipette to straw 14.5 μL pure water from the pure water bottle to one tube.
•Secondly, use pipette to straw 2 μL buffer to the previous tube.
•Thirdly, use pipette to straw 0.5 μL enzyme to the previous tube.
•Next, use pipette to straw 0.5 μL template to the previous tube.
•Then, add 0.5 μL crRNA to the previous tube.
•Finally, use pipette to straw 2 μL detector to the previous tube.
2.Straw 1 μL of the mixed solution in the tubes to the ELIASA Plate.
3.Detect the solution using ELIASA machine to check if the fluorescence value is within the range.

Cas12a detection

Materials:

|  |  |
| --- | --- |
| Water. | 14.5 μL |
| Buffer. | 2 μL |
| Cas 12A Enzyme. | 0.5 μL |
| Template. | 0.5 μL |
| (From solution we made after LAMP) |  |
| FITC Detector. | 2 μL |
| CRRNA. | 0.5 μL |

Procedure:

1.Add all the materials together:
•Firstly, use pipette to straw 14.5 μL pure water from the pure water bottle to one tube.
•Secondly, use pipette to straw 2 μL buffer to the previous tip.
•Thirdly, use pipette to straw 0.5 μL enzyme to the previous tube.
•Next, use pipette to straw 0.5 μL template to the previous tube.
•Then, add 0.5 μL CRRNA to the previous tube.
•Finally, use pipette to straw 2 μL FITC detector to the previous tube.
2.Put the solution into the environment of 37 degrees for 45 minutes.
3.Detect the solution using Cas12A test paper. If the solution contained the virus, there will be two red lines shown on the test paper；on the contrary, only one red line will be shown if the solution do not have the bacteria.

Extract the plasmid DNA:

Materials:

|  |  |
| --- | --- |
| Washing Buffer 1. | 600 μL |
| Washing Buffer 2. | 700 μL |
| Pure water. | 60 μL |
| Ethanol | 250 μL |
| S1 | 250 μL |
| S3 | 350 μL |

Procedure:

1.Firstly, add 250 μL ethanol to the washing buffer 2 using pipette.
2.Secondly, use pipette with a tube to straw 250 μL of S1 to the tube contained the E. Coli bacteria.
3.Next, straw 350 μL of S3 using pipette and add it to the E. Coli tube.
4.Then, centrifuge using the machine for 10 mins.
5.Thirdly, straw the non-precipitation solution into a mini probe using pipette.
6.Fourthly, add 500 μL of W1 and centrifuge for 1mins.
7.Fifthly, add 700 μL of W2 and centrifuge for 1mins again.
8.After that, put the filter tube into centrifuge and spin for 1 minutes and throw away the bottom tube.
9.Then, add 60 μL of water at 65 degrees and wait for one minute.
10.Lastly, measure the concentration of the solution in the tube and mark the information on the tube for user in the future.

DNA assembly:

Materials:

|  |  |
| --- | --- |
| Water. | 10 μL |
| DNA sector. | 1.5 μL |
| Template. | 5.5 μL |
| Buffer. | 2 μL |
| Enzyme Ezmax (Shanghai Tolobio company). | 1 μL |

Procedure:

1.Calculate the amount we require for DNA sector and template. As we need to ensure that the ratio for DNA sector and template is five to one and the total amount for the DNA sector and template is seven μL.
2.Add all the materials together: (this procedure should be done on ice)
•Firstly, use pipette to straw 10 μL pure water from the pure water bottle to one tube.
•Secondly, use pipette to straw 2 μL buffer to the previous tip.
•Thirdly, use pipette to straw 5.5 μL template to the previous tube.
•Next, use pipette to straw1.5 μL of DNA sector to the previous tube.
•Finally, use pipette to straw 1 μL of enzyme to the previous tube.
3.Put all the tubes we have made on ice for 30 minutes.
After the reaction, wait for 5 more minutes to stop the reaction and take it out.

![](https://igem.siqint.com/zhuwen/images/home/logo1.png)©2019 Microsoft Corporation. All rights reserved.

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:Shanghai HS United/Results

Result

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# Megamenu

## Result

To achieve the ultimate goal to reach optimal simplicity, immediacy, and accuracy, our iGEM team decides to use the LAMP + Cas12a + lateral flow method to amplify potential ASFv-affected genetic segments from the pig population. The final results of our wet-lab experiments show that our attempts on improving the current detection method is feasible.

Components of the Reagent (LAMP amplification)

There are six essential components required by the reagent needed for the LAMP amplification: the DNA template, ddH2O, primers, dUTP, UDG, and a LAMP mixture (containing buffer, BST 2.0 enzyme, and dNTP). To reach optimum accuracy, sensitivity, and specificity for the detection, the most suitable primers and the amount of components must be selected first.

Selecting target DNA

First, annealing short oligos (see figure feature) were used to mimic ASFv DNA target. Then, Cas12a detection reactions were performed by real-time fluorescent detector. We found the target-4 site had the best response speed and strength.

![](https://igem.siqint.com/zhuwen/images/home/6.1.png)

![](https://igem.siqint.com/zhuwen/images/home/6.2.png)

Selecting Primer

Under series of experiments and the final gel electrophoreses examination, primer LAMP-primer-4 finished amplification with higher quality compare to primer LAMP-primer-5 and with shorter time (30 mins), therefore primer LAMP-primer-4 is selected as primer used later on. (The sequence of primers, please see Design)

![](https://igem.siqint.com/zhuwen/images/home/6.3.png)

Adding UDG— to prevent aerosol contamination

Throughout the course of the experiment, we have encountered one set of results displaying massive amount of successful amplification, even under conditions where amplification is not supposed to occur (when the template is ddH2O), which suggests that that round of experimentation might be contaminated by aerosol particles.
After adding the UDG to the original reagent and repeating the process of the experiment, the results under gel electrophoreses proves that UDG can effectively reduce potential contamination from the aerosol particles as the set of conditions with ddH2O as the template has failed to amplify. However, during our final experimentation, aerosol particles seem to have once again affected the amplification under the presence of UDG, therefore this is an issue we aim to resolve in the ultimate set of kit or product we design to make our detection method practical in real life.

![](https://igem.siqint.com/zhuwen/images/home/6.4.png)

Specificity and Sensitivity

Moreover, the most effective set of conditions is set with the template with the concentration gradient at 10-5, temperature at 65 ℃, and 30 minutes. Although other sets of condition have also presented effective results, one core feature of our way of detection is that it requires the minimum amount of time needed with accurate results, therefore the optimum set of conditions is determined with the concentration gradient at 10-5, temperature at 65℃, and 30 minutes.

![](https://igem.siqint.com/zhuwen/images/home/6.5.png)

Our iGEM team added ASFv DNA segments into the clean blood from pigs and diluted the samples into different concentrations. The diluted samples are tested under the same LAMP process and the results show that this amplification process still effectively amplifies samples with the concentration of 10-7nM.

![](https://igem.siqint.com/zhuwen/images/home/6.6.png)

![](https://igem.siqint.com/zhuwen/images/home/6.7.png)

To test our detection method’s specificity, the original targeted DNA template with ASFv was changed into two types of different bacteria DNA templates (Escherichia coli and Staphylococcus aureus). The results display by the test strips determine that the LAMP amplification method only proliferates the targeted ASFv templates, but not other bacteria DNA, proving that the method’s specificity solely on ASFv DNA is achieved.

![](https://igem.siqint.com/zhuwen/images/home/6.8.png)

Cas12a Reaction and lateral flow

To examine the Cas12a reaction, we added crRNA, FnCas12a, the LAMP products, FITC-N14-Biotin, 10×NEB Buffer 3.1, and ddH2O to create a 20 μl reagent.
After reacting under 37 ℃ for 10 minutes, the reagent is tested by the fluorometer. From the results, Cas12a can detect concentration similar to that detected directly by the LAMP fluorescent — 10-7 nM.
Base on the LAMP product above, we take a control and templates with concentration of 10-7 nM and conduct the same Cas12a reaction. After the reaction, we add buffer MGCB into the reagent and insert the HybriDetect dipstick (test strip). As the results, the blood samples containing designed ASFv DNA segments display 2 strips on the dipstick, meaning a positive result proving the sample is affected by the ASFv.
We tested all above LAMP products (gel electrophoresis samples) for lateral flow test, it showed the same results.

![](https://igem.siqint.com/zhuwen/images/home/6.9.png)

Significance of the Results

The results of our experiments prove that the sensibility, specificity, and rapidity we desire is reached. However, the accuracy we aim to achieve still needs future improvements to be made on the product we design.

Future Plans

Our iGEM team designed a model of our future product that can be put into use in real life. The kit contains the mixture of reagents without the template, which would be added in by the pig owners with the samples from the pigs’ blood or fodder. To maximize the reduction of contamination from aerosol particles, we wish to create the kit to be completely enclosed since the environment and personnels operating the detection might not be necessarily professional or well-prepared.

![](https://igem.siqint.com/zhuwen/images/home/6.10.jpg)

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