iGEM 2021 — Team Shanghai_Metro_Utd

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# Team:Shanghai Metro Utd/Notebook

Shanghai\_Metro\_Utd

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Project Notebook

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# Team:Shanghai Metro Utd/Engineering

Shanghai\_Metro\_Utd

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Engineering

Background

Parkinson’s disease affects more than 4 million people worldwide. In the United
States, Parkinson’s disease occurs in approximately 13 per 100,000 people, and about 60,000 new cases are
identified each year. With more people living longer than before, the number of people who can get this
disease is expected to increase in the coming decades. Parkinson's disease is highly senile, and this
disease has seriously threatened the health of elderly people. This disease leads to a gradual loss of
physical motor function, lethargy, inconvenience, and slowness of movement. At the same time, patients will
also have different degrees of depression and anxiety symptoms. There is an urgent need for medical
treatments that are both effective and affordable for the majority of the aged population. However, the
current medication cannot satisfy the demand for most aged population, since it only relieves the symptom
and maintains the life quality of the patients to some extent.

Design

During our process of reading massive public literature on the matter, we came
across a relatively new target, angiogenin (ANG) protein, a secreted ribonuclease. According to the
literatures, angiogenin has been recognized to play important roles in various physiological and
pathological processes, especially in neurodegenerative diseases. The loss-of-function mutation of the ANG
gene is likely to be an important cause of certain nervous system diseases such as Parkinson’s disease.
Therefore, we realized that the delivery of active angiogenin protein could reduce neurodegeneration and
could possibly be used as a clinical treatment once we correctly inject the protein.

In our project, we aim to create a recombinant bacteria that can secret functional
ANG protein, which could possibly serve as an in vitro method to massive produce ANG for clinical use.

Build

pET-28a-rANG which produces ANG protein is shown in Figure 1.

![](https://static.igem.org/mediawiki/2021/0/05/T--Shanghai_Metro_Utd--engineering01.jpg)
Figure 1. Schematic map of pET-28a-rANG plasmid and its construction.

Test

We have obtained Freeze-dried powder of the ANG protein produced by our engineered
Escherichia coli (Figure 2).

![](https://static.igem.org/mediawiki/2021/f/f9/T--Shanghai_Metro_Utd--engineering02.jpg)

The activity of recombinant ANG protein was determined by the RNA degradation
assay. The result indicated that ANG protein possesses ribonuclease activity as we expected.

![](https://static.igem.org/mediawiki/2021/1/1b/T--Shanghai_Metro_Utd--engineering03.jpg)

Email us at:*ashleyyang48@gmail.com;alexlin711@gmail.com*

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# Team:Shanghai Metro Utd/Experiments

Shanghai\_Metro\_Utd

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EXPERIMENTS

Experiment 1：Gradient PCR (20μl)

Protocol:

1. Mix the following reagents

![](https://static.igem.org/mediawiki/2021/8/84/T--Shanghai_Metro_Utd--experiments01.jpg)

2. PCR

![](https://static.igem.org/mediawiki/2021/7/7d/T--Shanghai_Metro_Utd--experiments02.jpg)

Experiment 2：Bacterial colony PCR (20μl)

Protocol:

1. Isolating Single Colony from LB Plate Covered by Kanamycin

1) Mark and number the bacterial colony on the plate.

2) Pick a monoclonal population from the plate by a sterile
pipette tip and place it in a test tube with LB 4 mL of antibiotic-free LB liquid medium.

3) Place the tube in a shaking incubator at 37℃ for 12 h.

2. PCR

![](https://static.igem.org/mediawiki/2021/c/c4/T--Shanghai_Metro_Utd--experiments03.jpg)

Experiment 3：Gel Electrophoresis (2% Agarose)

Protocol:

1. Weigh 0.6g agarose.

2. Add 1960 mL distilled water to dilute 40ml 50X TAE buffer into 2L 1X TAE buffer
solution.

3. Measure 30 mL 1X TAE buffer solution.

4. Mix agarose with prepared TAE buffer solution.

5. Heat in microwave for 3 min until the solution clarifies.

6. Cool down the solution at room temperature until hands can be comfortably kept
on the flask.

7. Shake gently to mix well and pour the agarose solution into the gel box and
wait for it to solidify.

Experiment 4：LB Culture Medium

1. Liquid 500 mL

![](https://static.igem.org/mediawiki/2021/c/ce/T--Shanghai_Metro_Utd--experiments04.jpg)

2. Solid 200 mL

![](https://static.igem.org/mediawiki/2021/a/af/T--Shanghai_Metro_Utd--experiments05.jpg)

Experiment 5：Plasmid Extraction (Carrier plasmid PET28a)

![](https://static.igem.org/mediawiki/2021/6/6e/T--Shanghai_Metro_Utd--experiments06.jpg)

Protocol:

1. Add 500μL of Buffer BL to the spin column. Centrifuge at 13,400×g for 1 min.
Decant the filtrate in the collection tube.

2. Add 5-15ml overnight LB culture. Centrifuge at 13,400×g for 1 min. Collect the
supernatant.

3. Add 500μl of Buffer P1. Resuspend the bacterial pellet. Besure that the bacteria
pellet is completely resuspended.

4. Transfer to the microfuge tube. Add 500μl Buffer P2 and gently inverting the
tube for 6-8 times until a clear solution is formed.

5. Add 500μl Buffer P4 and gently inverting the tube for 4-6 times and sit quietly
for 10 min and centrifuge at 13,400×g for 10 min.

6. Add the supernatant to the filter tube CS and centrifuge at 13,400×g for 2 min.

7. Add 0.3× isopropanol to the filtrate and gently mixing and transfer to CP4 spin
column.

8. Centrifuge at 13,400×g for 1 min and discard the filtrate.

9. Add protein PD and centrifuge at 13,400×g for 1 min and discard the filtrate.

10. Add 600 μl buffer PW and sit quietly for 2-5 min and centrifuge at 13,400×g for
1 min

11. Repeat step 10 and centrifuge at 13,400×g for 1 min

12. Place CP4 in a clean microfuge tube and add 30μL heated buffer TB to the
absorption film and sit quietly for 2 min and centrifuge at 13,400×g for 1 min and collect the solution in
the collection tube.

Experiment 6: Bacterial Transformation

Protocol:

1. Take out the competence from the fridge (-81℃) and put it in an ice box.

2. Due to the experimental requirement, we add 9μl TB to dilute 1μl 262.5ng/μl
plasmid into 26.25ng/μl.

3. Add 1μl diluted plasmid into 50μl competence, and put it in the ice box for 30
minutes.

4. Transfer the sample to the 42℃ water bath for 90s.

5. Immediately iced the sample for 5 minutes.

6. Add 500μl LB solution to the sample and cultivate it at 37℃ for 40 minutes.

7. Centrifuge the sample at 800xg for 5 minutes.

8. Use an alcohol burner to sterilize glass rods and set the glass rods aside for
5~10 minutes.

9. Discard 450μl supernatant with a pipette.

10. Mix the the precipitate with the supernatant left in the tube.

11. Use a pipette to transfer the mixture onto a board with 10㎍/mL Kanamycin

12. Use the glass rod to spread the mixture evenly on the board and cultivate the
mixture on the inverted board for 12~15hs.

Experiment 7: Restriction Enzyme Digestion

Protocol:

1. Cut vector

1) Mix the following reagents:

![](https://static.igem.org/mediawiki/2021/c/ce/T--Shanghai_Metro_Utd--experiments07.jpg)

2) Centrifuge for a few seconds

2) Centrifuge for a few seconds
3) PCR for 1h under 37℃.

2. Cut targeted gene

1) Mix the following reagents:

![](https://static.igem.org/mediawiki/2021/8/8b/T--Shanghai_Metro_Utd--experiments08.jpg)

Experiment 8: DNA Gel Extraction (Performed
according to AxyPrep DNA Gel
Extraction Kit)

1. Reagent:

![](https://static.igem.org/mediawiki/2021/e/ee/T--Shanghai_Metro_Utd--experiments09.jpg)

2. Caution:

1) Cutting the DNA-containing gel
into small pieces (DNA-containing gel can be seen under the UV light) to decrease the surface area and
increase the extraction rate. Do not expose DNA-containing gel under the UV light for long periods of time
to reduce UV damage to DNA.

2) The gel must be completely
dissolved for better extraction rate.

3) The eluent should be pre-warmed
to 65°C to increase the elution efficiency.

3. Preparation Before Experiment:

1) Before using, ethyl alcohol
should be added to Buffer W2.

2) Make sure that the pipette tip
and microfuge tube are without nucleic acid contamination and no precipitations are in Buffer.

3) Adjust the water bath to 65°C.

4. Protocol:

1) Excise the DNA-containing
agarose gel slice with a clean, sharp scalpel under ultraviolet illumination. Briefly place the excised gel
slice on absorbent toweling to remove redundant buffer. Mince the gel into small pieces, transfer to a 2.0
ml microfuge tube.

2) Add a 250 μL Buffer BL.
Resuspend the gel in Buffer BL by vortexing and heating at 65°C. Intermittently vortexing (every 2-3
minutes) until the gel is completely dissolved (typically, 6-8 min later) and the color of the dissolved gel
should be light yellow.

3) Transfer the solution to EC and
centrifuge at 12,000 xg 22°C for 1 min and discard the collection tube.

4) Add 700 μL Buffer W2 to EC and
centrifuge at 12000 xg 22°C for 1 min and discard the collection tube.

5) Repeat step 4.

6) Transfer EC to the empty
collection tube and centrifuge at 12000 xg 22°C for 2 mins.

7) Transfer EC to the clean 1.5 ml
microfuge tube and stew for 2 mins with lids opened and add 50 μL pre-warmed Eluent and stew for 2 mins,
then centrifuge at 12000 xg 22°C for 2 mins.

8) Transfer the Eluent in the 1st
microfuge tube to the 2nd microfuge tube and stew for 2 mins with lids opened and centrifuge at 12000 xg
22°C for 2 mins.

9) Repeat step 8 for until all
solutions are eluted.

Experiment 9: SDS-PAGE (Performed according to
Biochemistry Experiment and
Guidance of China Pharmaceutical University)

1. Mix the following reagents: (TEMED is added last)

1) Monomer running gel

![](https://static.igem.org/mediawiki/2021/d/d7/T--Shanghai_Metro_Utd--experiments10.jpg)

2) Monomer Stacking gel

![](https://static.igem.org/mediawiki/2021/4/45/T--Shanghai_Metro_Utd--experiments11.jpg)

2. Protocol:

1) After the glass plates are
aligned, put them into the rubber frame and clamp them (the plates must be aligned to avoid leakage). Then
clamp the clip.

2) Use pipette to extract 4 ml of
running gel solution and release it into spaces between parallel glass, until it reaches 5 mm below the
line. Create a flat surface above the running gel solution by adding a water layer on top of the solution.
Allow the gel to polymerize for at least 30 min.

3) When there is a refracting line
between running gel and water layer, pour off the water. Wash the gel for several times with deionized water
and blot the water with filter paper.

4) Pour the stacking gel above the
running gel.

5) Immediately insert the comb to
create sample wells in the stacking gel before it polymerizes (keep the comb level). Be sure that the comb
is clean and that no air bubbles form. Allow the gel to polymerize for at least 30 minutes.

Experiment 10: Protein Induction and Expression

Protocol:

1. Pick a single colony of the transformed BL21 in 4 ml LB (containing 100 μg/ml
kanamycin), and culture it overnight at 37°C.

2. Inoculate the bacterial colony into 1L LB (containing 50 μg/ml kanamycin), and
continue to cultivate it until the value of OD is 0.6 (with LB as the control).

3. Add IPTG to a concentration of 1 mmol/L.

4. Induce expression at 37°C for 3 h.

5. Centrifuged at 4°C at 4000 rpm for 10 min.

6. Collect bacterial pellets.

Experiment 11: Preparation and dissolution of inclusion bodies

Protocol:

1. Add 50 ml of buffer (50 mmol/l Tris-HCl, pH 8.0, 2 mmol/L EDTA) to the E. coli
collected from centrifugation.

2. Vortex the bacterial suspension.

3. Fissure the bacteria with a High Pressure Homogenizer.

4. Centrifuge at 4℃ at 12000 rpm for 20 min and collect the precipitate.

5. Resuspend the inclusion bodies with 50 ml inclusion body washing solution (50
mmol/l Tris-HCl, pH 8.0, 2 mmol/L EDTA, 1% Triton X-100), and centrifuge at 4°C at 12000 rpm for 20 min.

6. Repeat step 5 and collect inclusion bodies.

7. Conduct 15% SDS electrophoresis to analyze the bacterial liquid before and after
induction, inclusion bodies and supernatant after broken bacterial cells.

8. Add the above inclusion bodies to 20 ml of inclusion body denaturation solution
(7 mol/L guanidine hydrochloride, 0.15 mol/L GSH, 0.1 mol/L Tris-HCl, pH 8.1, 2 mmol/L EDTA)

9. Stir and dissolve in an anaerobic environment for 2 hours.

10. Centrifuge at 4000r for 20 minutes to take the supernatant, which is the
denatured protein solution.

Experiment 12: Protein Refolding

Protocol:

1. Slowly drop the denatured protein solution into 500 ml refolding buffer (0.5
mol/L L-Arginine, 0.6 mmol/L GSSG, pH 8.0) which has been filtered with 0.22 μm microporous filter before
use.

2. After all the denaturation solution is added to the refolding buffer, continue
to stir the refolding buffer for about 1 h.

3. Place the solution overnight at 4°C, and dilute to 2 L with ddH2O.

Experiment 13: Protein Purification

Protocol:

1. Pack SP Beads 6FF gel stored in 20% ethanol on the column (about 10 ml).

2. Equilibrate 3 column volumes with buffer A (10 mM Tris-HCl, pH 8.0, 0.2 M NaCl).

3. Load the refolded protein solution filtered by a 0.22 μm filter membrane and
pass it through the column.

4. Analyze the solution before and after passing the column by SDS-PAGE.

5. Wash with buffer A for about one column volume and collect the washed solution.

6. Elute with buffer B (10 mM Tris-HCl, pH 8.0, 0.8 M NaCl) for more than 5 column
volumes, and continuously collect the eluted solution for analysis by SDS-PAGE.

7. Concentrate the eluted protein solution with an ultrafiltration tube and replace
it with ddH2O.

8. Freeze the protein solution into a solid with liquid nitrogen.

9. Lyophilized the protein solid with a freeze dryer to obtain a protein powder.

Experiment 14: Protein Activity Verification I - RNA Extraction

Protocol:

1. Resuspend the HEK293-GFP cell pellet with 2 ml Trizol to a 15 ml centrifuge
tube.

2. Leave it at room temperature for 5 min.

3. Add 0.4 ml of chloroform and shake for 15 seconds.

4. Leave it at room temperature for 5 min

5. Centrifuge it at 12000 rpm at 4°C for 15 min. (At this time, the liquid is
divided into three layers, the lower red phase, the middle organic phase, and the upper colorless phase.)

6. Carefully transfer the upper colorless phase to a clean 1.5ml Ep tube. (The
volume of the upper colorless water sample layer is about 60% of the volume of Trizol added.)

7. Add an equal volume of pre-cooled isopropanol.

8. Leave it at room temperature for 10 minutes to precipitate RNA.

9. Centrifuge at 12000 rpm at 4°C for 10 minutes. (At this time, the sediment can
be seen at the bottom of the tube. If the there is a large amount of sediment, you can see the white
sediment in the form of flakes, and if there is less, you can see granular sediment.)

10. Carefully remove the supernatant.

11. Add 2 ml of 75% ethanol, blow with a gun several times.

12. Centrifuge at 4°C at 7500 rpm for 5 min.

13. Discard the supernatant and wait till the ethanol evaporates and dries.

14. Add 40μl of enzyme-free water to resuspend all RNA, and measure the RNA
concentration and OD value with Nanodrop.

15. Store the RNA at -80℃.

Experiment 15: Protein Activity Verification II - RNA Degradation

Protocol:

1. Replace the electrophoresis solution in the electrophoresis tank with a new one.

2. Prepare 3 reaction systems with PCR tubes.

1) 50 μl: 0.33 mmol/L Hepes, 0.33
mol/L NaCl, 883.82 ng/μl total RNA, 10 μmol/L rANG;

2) 50 μl: 0.33 mmol/L Hepes, 0.33
mol/L NaCl, 883.82 ng/μl total RNA, 1 μmol/L rANG;

3) 50 μl: 0.33 mmol/L Hepes, 0.33
mol/L NaCl, 883.82 ng/μl total RNA, 0.2 mg/ml RNaseA.

3. Metal bath at 37°C for 1 h.

4. Take 50 μl and add 10 μl 6X gel loading to electrophoresis. (1% agarose gel
electrophoresis, 140 V, 40 min)

Experiment 16: Interaction study between heparin and Angiogenin

Protocol:

1. Add 1mg/ml Heparan Sulfate to the transparent flat bottom 96-hole plate, cultivate for 2 hours.
2. Wash the plate with PBS 3 times, 5 minutes each time.
3. Add 3% concentration of BSA, react for 1 hour. Add 100ul angiogenin with different concentrations to each
hole, react for 1 hour. Wash the plate with PBS for 3 times.
4. Add anti-His (1:2000) coupled with HRP, react for 1 hour. Wash the plate with PBS 6 times.
5. Add TMB, react for 30 minutes. Add 2 mol H2SO4.
6. Use SpectraMax M5e to measure the absorption value.

Email us at:*ashleyyang48@gmail.com;alexlin711@gmail.com*

Follow us on Wechat: *血管马力Bloody
Marry*

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# Team:Shanghai Metro Utd/Results

Shanghai\_Metro\_Utd

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Results

Overview

As previously mentioned, we are interested in the property of angiogenin, and we
hope to get its prokaryotic expression.

Section 1 is made up of Plasmid Extraction, Restriction Enzyme Digestion,
Transformation, and Recombination. Section 2 is made up of Protein Inducible Expression, Preparation and
Dissolution of Inclusion Body, Renaturation, Purification, and Activity Validation.

Results

**Plasmid Construction**

![](https://static.igem.org/mediawiki/2021/2/22/T--Shanghai_Metro_Utd--result01.jpg)

We extracted pET-28a plasmid for the use in the later part of restriction enzyme
digestion and transformation.

![](https://static.igem.org/mediawiki/2021/7/71/T--Shanghai_Metro_Utd--result02.jpg)
Figure 2: the resulting concentration of the extracted plasmid in Figure 1

We measured the concentration of the extracted pET-28a plasmid. According to the
concentration, we took a
certain volume for enzyme digestion.

![](https://static.igem.org/mediawiki/2021/1/19/T--Shanghai_Metro_Utd--result03.jpg)
Figure 3: Construction flowchart of the target plasmid pET-28a-rANG (by Snapgene)

![](https://static.igem.org/mediawiki/2021/f/fc/T--Shanghai_Metro_Utd--result04.jpg)
Figure 4: the scanning gel electrophoresis map of the plasmid vector after enzyme digestion

As seen in figure 4, lane 1 is the pET-28a without enzyme digestion and lane 2 to 9
is the result for pET-28a after enzyme digestion. The bands of our plasmid vectors after enzyme
digestion(5311bp) showed at 5000 bp around which are correct and the bands at 300 bp around were the SUMO
fragments that were cut by enzyme digestion. After that, we could further conduct the E. coli
transformation.

![](https://static.igem.org/mediawiki/2021/3/3f/T--Shanghai_Metro_Utd--result05.jpg)
Figure 5: The gel electrophoresis map of PCR

Lane 1 to 6 is the result of PCR. We got rANG band at around 400bp (369bp). Enzyme
digestion was conducted and it was linked with digested pET-28a.

![](https://static.igem.org/mediawiki/2021/5/5d/T--Shanghai_Metro_Utd--chg2.jpg)
![](https://static.igem.org/mediawiki/2021/3/3e/T--Shanghai_Metro_Utd--chg3.jpg)
Figure 6. E. coil having the desired pET28a-rANG

The plates in figure 6 (1) showed monoclonals of pET28a-rANG constructs.
The pET28a-rANG was constructed successfully which has been proved by sequencing as shown in figure 6 (2).

**Recombination E. coli**

![](https://static.igem.org/mediawiki/2021/a/a1/T--Shanghai_Metro_Utd--result07.jpg)
Figure 7: the scanning gel electrophoresis map of Colony PCR (the top was before and the bottom was
after)

Lane NC to XCN6 and lane XCN7 to N6 are the results of colony PCR. We get the band
531bp at 500bp around. It indicates that the obtained recombinant monoclonals were positive monoclonals
containing the pET-28a-rANG recombinant plasmid.

SDS PAGE

![](https://static.igem.org/mediawiki/2021/7/7e/T--Shanghai_Metro_Utd--result08.jpg)
Figure 8. SDS-PAGE Analyzing the proteins before and after induction, within inclusion bodies, and in
supernatants.

The results for SDS-PAGE shows that the induction was successful, and the target
protein (about 15KDa) was
mainly in the inclusion body.

**Protein Purification**

![](https://static.igem.org/mediawiki/2021/1/1d/T--Shanghai_Metro_Utd--chg4.jpg)
Figure 9. SDS-PAGE and stained with Coomassie Brilliant Blue after protein purifying: (1) first
attempt; (2) second attempt

Based on figure 9 (1), there was a band at 15 KDa before passing through the column
but disappeared after passing through the column, indicating that the protein had hung the column. But we
notice that there is a miscellaneous band with a small amount. In order to obtain the purified protein, we
repeated the protein purification experiments and finally obtained the more purified protein as seen in
figure 9 (2) where you could only see one band at 15 KDa.

![](https://static.igem.org/mediawiki/2021/f/fd/T--Shanghai_Metro_Utd--result10.jpg)
Figure 10. First SDS-PAGE, Coomassie blue staining. Samples from left to right: marker; 1.before
column(unconcentrated); 2. after column 1(unconcentrated); 3. after column 2(unconcentrated); 4. buffer
A rinsed; 5 second elution; 6 third elution.

![](https://static.igem.org/mediawiki/2021/2/26/T--Shanghai_Metro_Utd--result11.jpg)
Figure 11. Second SDS-PAGE, Coomassie blue staining. Samples from left to right: marker; 1. second
elution; 2.third elution; 3.fourth elution; 4. fifth elution.

It can be seen from Figure 10 and Figure 11 that there is actually not much protein
after the third
elution.

![](https://static.igem.org/mediawiki/2021/9/94/T--Shanghai_Metro_Utd--result12.jpg)
Figure 12 Lyophilized protein

As seen in figure 12, the protein was successfully lyophilized into the form of
powder.

**RNA Extraction**

![](https://static.igem.org/mediawiki/2021/3/31/T--Shanghai_Metro_Utd--result13.jpg)
Figure 13. The concentration of the extracted RNA by nanodrop

**RNA Degradation Experiment**

![](https://static.igem.org/mediawiki/2021/d/d7/T--Shanghai_Metro_Utd--result14.jpg)
Figure 14. electrophoresis map after RNA degradation tests

According to the electrophoresis map, the renatured rANG has ribonuclease activity
with the capability of RNA incomplete degradation and produced a band at about 100 bp, which is consistent
with the data in the literatures (Ref. 1, 2).

![](https://static.igem.org/mediawiki/2021/9/91/T--Shanghai_Metro_Utd--result15.jpg)
Figure 15. OD450 curve of the system with heparin and rANG

The OD450 could indicate the interaction ability between heparin and
angiogenin which means that the higher the OD450 value, the greater the interaction ability
between heparin and angiogenin. This curve shows the more angiogenin we added, the higher the OD450 value,
namely the interaction ability between heparin and angiogenin. Therefore, it can be concluded that the
interaction of heparin has dose-dependent with angiogenin.

Future Plan

As we have successfully obtained the eingineered E. coli to produce the angiogenin
with well activity. We would further focus more on the improvement of the purity and yield of our protein
and more tests should be conducted to ensure the safety and stability.

Reference:

[1]孙德森,盛静浩.重组人血管生成素制备和生物活性鉴定[J].中国生物化学与分子生物学报,2015,31(12):1315-1321.

[2] Shapiro R, Riordan JF, Vallee BL. Characteristic ribonucleolytic activity of
human angiogenin. Biochemistry. 1986 Jun 17;25(12):3527-32. doi: 10.1021/bi00360a008. Erratum in:
Biochemistry 1986 Oct 21;25(21):6730. PMID: 2424496.

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