iGEM 2023 — Team npu-china

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{% 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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## Notebook

#### December 5, 2022

The NPU-CHINA team was established.

#### December 25, 2022

The first topic selection meeting was held, with an initial feasibility study of the proposals being
conducted.

#### February 10, 2023

The second topic selection meeting took place, finalizing the project for this time.

#### March 14, 2023

Research and collection of data related to the project commenced.

#### April 13, 2023

Communication and exchange with the iGEM team from Northwest University.

#### May 7, 2023

Discussion on launching iGEM promotional activities within the campus.

#### May 9, 2023

The designed plasmid was handed over to Tsingke Biotechnology for synthesis.

#### May 12, 2023

The first induction expression was initiated.

#### May 31, 2023

Multiple attempts at inducing expression failed, prompting further research into genes from other
sources.

#### June 13, 2023

It was decided to use a new gene from the Paraffin-like Bacillus to construct the plasmid.

#### June 16, 2023

Exploration of conditions for the induction expression experiment began.

#### August 12, 2023

Purified expressed protein was obtained, marking the start of protein modification efforts.

#### October 4, 2023

Enzyme activity was measured.

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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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## Engineering

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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## Experiments

#### I. Preparation of Reagents:

1. LB Broth Medium and Antibiotics:

- Prepare LB broth medium based on the "Molecular Cloning Laboratory Manual"
(by J. Sambrook and D.W.
Russell). For every liter of medium, add 10g of tryptone, 5g of yeast extract, and 10g of NaCl to 950mL
of deionized water (make up to 1L). For solid medium, add an additional 15-20g of agarose, then
autoclave at 121°C, 0.1Mpa for 20 minutes.

- Prepare a stock solution of ampicillin: dissolve 50mg of ampicillin in 1mL
of sterile broth. When
using, add 100μL of this stock solution to 100mL of medium.

- Prepare IPTG stock solution: dissolve 1mol of IPTG in 1mL of sterile
water. When using, add 10μL of
this stock solution to 100mL of medium.

#### II. Transformation of Target Gene into Competent Cells:

1. Take BL21(DE3) competent cells from -80°C and quickly thaw them in ice (100 μL). Add 10 μL of target
DNA. Gently mix by tapping the bottom of the EP tube (avoid pipetting), and let it sit on ice for 30
min.

2. Heat-shock the tubes in a 42°C water bath for 60 s, then immediately transfer to an ice bath for 3
min (avoid shaking the tubes as it may reduce the transformation efficiency).

3. Add 500 μL of antibiotic-free sterile broth (LB) to the tubes, mix, and then incubate at 37°C, 180
rpm for 60 min for cells recovery.

4. Centrifuge at 5000 rpm for 1 min, retain 100 μL of the supernatant, gently resuspend the cell
pellet, and plate it on LB agar containing ampicillin.

5. Invert the plates and incubate overnight at 37°C.

#### III. Induction Expression:

1. Transfer a portion of the grown cells at a 1/100 ratio to 500 mL expression medium (LB). Incubate on
a shaker at 37°C, 220 rpm. Measure the OD600nm hourly. After the first hour, measure every 30 min and
save 1 mL of culture each time.

2. When the OD reaches 0.6-0.8, add IPTG.

3. For IPTG: dissolve 0.0715 g of IPTG in 1.5 mL of sterile water. Add 500 μL to each of the 500 mL
culture flasks.

4. Continue incubation for 12 h.

#### IV. Centrifugation and Lysis:

1. Transfer the culture to centrifuge tubes and centrifuge at 10000 ×g, 4°C, for 3 min to pellet the
cells.

2. Resuspend the pellet in lysis buffer at a ratio of 10 mL buffer per 1 g of wet cell weight.

3. Let the mixture sit on ice for 30 min.

4. Lyse the cells using an ultrasonicator (50% power; 3 s on, 5 s off, for 15 min).

5. Centrifuge the lysate at 10000×g, 4°C for 15 min. Discard the pellet and filter the supernatant
using a 0.45 μm filter.

#### V. Affinity Chromatography:(Note: All buffers should be at 4°C or in an ice bath)

1. Equilibrate the column with 3 column volumes of deionized water.

2. Balance the chromatography column with equilibration buffer (3-5 column volumes).

3. Load the filtered lysate onto the column.

4. Wash away non-specifically bound proteins with equilibration buffer (3 mL, 7 times).

5. Elute the target protein with elution buffer (3.5 mL, once).

6. Clean the column with elution buffer containing 500 mM imidazole (3-5 column volumes).

7. Rinse the column with deionized water (8-10 column volumes).

8. Store the column in 20% ethanol (3-5 column volumes) and seal. Keep at 4~8°C.

#### VI. Desalting:

1. Clean the desalting column with 4 column volumes of deionized water.

2. Equilibrate the column with 4 column volumes of desalting buffer.

3. Load 1.5 mL of the protein solution onto the column.

4. Elute the protein using 2 mL of desalting buffer.

5. Equilibrate the column again with 3-5 column volumes of desalting buffer.

6. Repeat the desalting process for the next sample.

7. Clean the desalting column with 3-5 column volumes of deionized water.

8. Store the desalting column in 20% ethanol (3-5 column volumes) and seal. Keep at 4~8°C.

#### VII. SDS-PAGEPreparation and Components of SDS-PAGE

##### 1. Gel Casting:

Prepare 10 mL of 10% separating gel (as per table). Mix immediately and pipette slowly between the
glass plates from the top-left corner, filling until about 1.5 cm from the top edge.

##### Note:

- Ensure no leakage; if so, stop and re-seal the mold.

- Act quickly during gel preparation; add TEMED just before pouring. Pour
slowly to avoid bubbles.

- A clear interface between the gel and the water indicates good
polymerization.

##### 2. Water Seal:

Carefully add a layer of distilled water on top of the separating gel. Leave it
vertically at room temperature for around 30 minutes. Once the gel is set, remove excess water with
filter paper.
**Note**
This step ensures an even gel surface and helps in the polymerization by excluding air.

##### 3. Stacking Gel:

Prepare 5 mL of 5% stacking gel (as per table). Pour the gel until it reaches
the
edge of the front glass plate. **Note**: Ensure no bubbles at the comb's insertion point and that the
comb's
bottom is level.

##### 4. Gel Setting:

After the gel sets, remove the glass plates. Carefully remove the comb and the
sealing
tape. Place the glass plate with the thinner one facing inward on the electrophoresis frame. Fill the
tanks with electrophoresis buffer. **Note**: Handle the glass plates with care to avoid breakage.

##### 5. Sample Loading:

a. Mix 10 μL protein sample with 10 μL of sample buffer. Heat in boiling water
for
3-5 minutes and let it cool to room temperature.

b. Load 10-15 μL of the sample, ensuring no dispersion.

c. Load 5 μL of protein marker. **Note**: Remove any air bubbles in the wells and avoid piercing the
gel with the pipette.

##### 6. Electrophoresis:

Connect the electrophoresis device: the anode to the positive terminal and the cathode to the negative.
Start electrophoresis at 80 V, then increase to 120 V once the samples enter the separating gel.

Stop when the bromophenol blue band is about 1 cm from the gel bottom.

##### 7. Staining:

Remove the securing plate, separate the glass plates, and cut off the stacking gel.

Mark one end of the gel. **Note**: Handle the gel delicately.

##### 8. Gel Staining:

Immerse the gel in Coomassie blue and stain overnight.

After staining, pour off the stain, briefly rinse the gel, capture the image, and record the results.

#### VIII. Bradford Protein Concentration Measurement:

##### 1. Standard Curve Preparation:

Add reagents to a 96-well plate as mentioned in the table.

Mix well and measure absorbance at 595 nm. Plot a standard curve.

##### 2. Sample Measurement:

Mix sample with G-250 solution. After 2 minutes, measure OD595.

Calculate protein concentration and recovery rate using the standard curve.

#### IX.Enzyme Activity Measurement:

1. Mix 3 mL of pH 7.4, 1% soluble starch solution with 2 µg of commercial α-amylase in a centrifuge
tube.

Immediately start the reaction in the spectrophotometer.

2. For another sample, mix reaction buffer (with 20 mM substrate starch concentration) with a
pre-filtration protein sample. Shake vigorously and start the reaction in the spectrophotometer.

3. Continuously measure absorbance at 250 nm for 180 seconds. Analyze the rate of change of Abs vs.
time.

4. Calculate enzyme activity, turnover number, specific activity, purification fold, and yield from the
linear section of the curve.

#### X. Michaelis Constant Measurement:

1. Add buffers, substrate, and enzyme solutions to eight centrifuge tubes as described. React in a 25°C
water bath for 3 minutes, add the colorimetric agent, and boil for 5 minutes. After cooling, start the
measurement using a microplate reader.

2. Calculate the reaction rate at different substrate concentrations. Perform non-linear regression
using
Graphpad Prism software.

#### XI. Effect of pH on Enzyme Activity:

1. Add 2.4 mL of reaction buffer at varying pHs (5.5, 6, 7, 7.4, 8, 9, 10) and a substrate
concentration of 20 mM to seven centrifuge tubes. Then add 20 µg of enzyme.

2. Continuously measure absorbance at 250 nm for 180 seconds.

3. Calculate the specific enzyme activity at different pHs. Plot the results to determine the optimal
pH for enzyme activity.

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