iGEM 2022 — Team subcat-china

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{% block title %}Notebook{% endblock %}
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Notebook

August

2nd

1. Preparation of LB Medium
2. Incubate strains containing plasmids

3rd

Pick the mono-colony and incubate in LB liquid culture medium

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-notebook01.jpg)

4th

1. Plasmids extraction
2. PCR amplification for lipase
3. Double enzyme Digestion and gel extraction
4. T4 DNA ligase to construct the plasmids
5. Transform the plasmids into DH5α competent cells and coat them on the solid LB medium plates

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-notebook02.jpg)

5th

1. mono-colony identification and inoculate into liquid LB culture medium
2. agarose gel electrophoresis

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-notebook03.png)

6th

1. Plasmid extraction and double-enzyme digestion
2. gel electrophoresis and send for Sanger sequencing

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-notebook04.png)

7th

Transform the correct plasmids into BL21(DE3) competent cells and coat on the solid LB medium plate

8th

Inoculate mono-colony into liquid LB medium, 37℃, 200rpm overnight

9th

1. Transfer the overnight cultured medium into 1L LB liquid medium
2. Add IPTG to induce protein expression when OD600 is arranging 0.4-0.6, and culture at 37℃, 170rpm

10th

1. SDS-PAGE electrophoresis solution preparation
2. Protein purification

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-notebook05.jpg)

11th

Measure the standard curve of enzyme activity

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-notebook06.jpg)

12th

Detection of lipase activity at different pH values

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-notebook07.jpg)

13th

Detection of lipase activity assay at different temperatures

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-notebook08.jpg)

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{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
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![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-team.jpg)
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Engineering

Introduction

Lipase is widespread in the yeast, bacteria, fungi, animals, plants, and the human body, its molecular weight,
generally between 30 and 40 kDa, can is a kind of catalytic material such as triglyceride hydrolysis into glycerol
the floorboard of the enzyme. Lipase catalyzed triglyceride hydrolysis, mainly produces glycerol, fatty acid,
glycerin monoester, and diester. Lipase participates in the process of fat metabolism in cells and effectively
avoids the adipose content being too low or too much of cell metabolism disorderly or lesions. In modern society,
due to excessive intake of fat, people start to suffer from excessive fat in the body that cannot be utilized by our
body will cause different diseases such as overweight, pancreatitis, etc.
In our experiment, we use a genetic engineering approach to construct an E. coli system capable of overexpressing
lipase. These lipases will work in our small intestines. Therefore, our body will be more capable of breaking down
fats (mainly triglycerides).
Through lipase, which could transfer fat into fatty acids and glycerol in the cell class material for the body to
use, the body’s pancreatic lipase activity increased significantly and can increase the body fat digestion
efficiency.

Design

The lipase gene is amplified in the experiment using the genomes of Lactobacillus Plantarum W1-lipase and
Pseudomonas SP-lipase as templates. Then we insert the lipase gene into the plasmid pET28a to construct the E. coli
expression vector. The expression vector was then transformed into E. coli BL21 (DE3), and the lipase gene was
heterologously expressed in E. coli. Finally, we test the activity of the lipases.

Build

In order to build our plasmids, we let the synthetic company synthesize two target gene fragments (W1-lipase and
SP-lipase). We digested the target fragments and the pET28a vector with XhoI and HindIII (Figure 1), and we used T4
DNA ligase to ligate the fragments and the vector. Then we transformed the recombinant plasmids into E. coli DH5α
competent cells and coated on the LB (Kanamycin) solid plates. We send the constructed recombinant plasmid to a
sequencing company for sequencing, The returned sequencing comparison results showed that we have successfully
obtained two recombinant plasmids.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-engineering01.png)

Figure 1. Gel electrophoresis results of target gene fragments.
A. double-enzyme digested with the W1-ligase,
B. double-enzyme digested with the SP-ligase,
C. double-enzyme digested with the pET28a vector.

We verified the colonies through colony-PCR, and then we inoculated single colonies and we send the constructed
recombinant plasmid to a sequencing company for sequencing.
The returned sequencing comparison results showed that there were no mutations in the ORF region, and the plasmid
was successfully constructed (Figure 2). So far, we have successfully obtained two recombinant plasmids, which were
respectively on the pET28a vector, which can be used to express lipase proteins.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-engineering02.jpg)

Figure 2. Mapped the sequencing data to the recombinant plasmid.
A. the sequencing data of pET28a-SP-lipase,
B. the sequencing data of pET28a-W1-lipase.

As a result, the amplified target gene SP-lipase and the double digested vector pET28a were ligated with T4 ligase
to obtain recombinant plasmid pET28a-SP-lipase, so that the recombinant protein had 6×His tags at the carboxyl
terminus which could be used to purify the corresponding proteins.

Testing Approach

a) Protein expression and purification

The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and cultured overnight in the medium
containing resistance. When the OD600 was around 0.4-0.5, the IPTG was added to induce the expression of recombinant
protein W1-lipase/SP-lipase, and then the strains were cultured at 16℃ for 20h. After that, the collected bacterial
solution was cracked by Ultrasonic crushing and then transferred to a pre-balanced Ni-NTA affinity column. The
recombinant protein was bound to a nickel column and then eluted by imidazole.

b) Functional test of lipases

In order to measure the standard curve of the activity of lipases, we chose p-nitrophenol as the substrate and
detected its absorbance value of it when adding lipases. 0.02789g of p-nitrophenol (p-np) was weighed and dissolved
in 100mL of solution B, and stored in a brown reagent bottle after configuration and stored at 4°C.
0.02, 0.04, 0.06, 0.08, 0.12, 0.16mL of p-nitrophenol solution (2mmol/L) was diluted to 4mL, and the absorbance
value at 410nm was measured successively. Add reaction ingredients according to the table below:

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-engineering03.jpg)

The standard curve was drawn with p-nitrophenol (0.01, 0.02, 0.03, 0.04, 0.06, 0.08, mmol/L) as the abscissa and
absorbance value Y as the ordinate (Figure 3).

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-engineering04.png)

Figure 3. the standard curve of p-nitrophenol

According to the standard curve determination method, the standard curve is drawn as shown in Figure 5. Regression
coefficient R2=0.9979, the results are credible.

c) Measure the activity of lipase at different pH

Esterase activity was assayed in the pH range from 3.0 to 12.0, and at temperatures of 25 to 70°C. Enzyme
thermostability was measured by incubation of the enzyme in 50 mM sodium phosphate buffer (pH 9.0) at 25-70°C for 5
min, 15 min, 30 min, and 1, 2, 4, 6, and 20 h. After incubation, the residual activity of lipase was measured as
described above. To test the effects of metals, ions, and additives on the activity of the esterase, lipase was
incubated in their presence at a final concentration of 1 mM for 5 min at room temperature. Then, the substrate
(p-nitrophenyl acetate) was added, and the reaction mixture was incubated at 37°C. The experiments were performed in
triplicate.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-engineering05.png)

Figure 4. the enzyme activity of lipase at different pH

As shown in Figure 4, when changed the pH value of the buffer, the activity of lipase is changed compared with the
negative control, and the W1-lipase and SP-lipase showed no obviously different. And when the pH value is 9, the
lipase exhibited the highest activity.

d) Measure the activity of lipase at different temperature

When pH=9, the recombinant enzyme activity reached the highest, 36.-40U/mL, and decreased when pH=9, so the optimal
pH of the recombinant enzyme was 9. According to the standard curve, the enzyme activity at the optimum pH and
different temperatures are shown in Figures 5. At 40℃, the recombinant enzyme activity reached the highest, 36-40U/m
L, and decreased when the temperature was higher than 40℃. Therefore, the optimal temperature for the recombinant
enzyme was 40℃, but it had higher activity at 30-40 ℃.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-engineering06.png)

Figure 5. the enzyme activity of lipase at different temperatures

As shown in Figure 5, when changed the reaction temperature, the activity of lipase is different compared with the
negative control. And when the temperature is around 35℃, the SP-lipase showed the highest activity, while 40℃ may
be the optimum temperature for W1-lipase.

How we learn from our project

We have already collected the figures from our experiments. Lipase participates in the process of fat metabolism in
cells, so it could be used to make dietary supplements. In our project, we tested the activity of lipase through the
strain lysate. From the results, we can find that SP-lipase has a strong effect on catalyzing triglyceride
hydrolysis, and produces glycerol, fatty acid, et.al.
In the future, with people's attention to diet health, dietary supplements must play a key role in society. Lipase,
which we verified its activity in our project, provides a good choice of dietary supplements. We believe that lipase
could be used for industrial production and could even be used in people’s daily life.

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{% block title %}Experiments{% endblock %}
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Experiments

1. The preparation of the whole experiment

1.1. Preparation of Competent Cell Culture via Solid and Liquid LB Culture Medium

- Mix 2.5 g of tryptone, 1.25 g of yeast extract, 1.25 g of sodium chloride, and 250 mL deionized water into a
250 mL beaker to make the liquid LB culture medium
- Measure 0.5g of agarose into a separate 200 mL beaker
- Transfer 50 mL of the liquid culture medium from step 1 into a 200 mL beaker to create the 1% LB solid culture
medium
- Transfer the 50ml of LB culture medium and the remaining liquid culture medium to suitable containers for
high-pressure sterilization
- Sterilize 4 culture medium plates and 4 disposable streaking tools with UV light
- After cooled to 50℃, distribute 50ml of sterilized liquid LB culture medium into 4 sterilized culture medium
plates to be solidified into 4 solid LB culture medium plates

1.2 strain incubation

- Transfer samples of Top10 competent cell culture from the freezer into an ice box
- Use a streaking tool to dip into the cell culture and inoculate the culture medium with the competent cell
culture
- Label the 4 inoculated LB culture medium plates and invert them for incubation
- The next day, use a pipette tip to take 6 colonies of competent cell culture from the solid LB culture medium

(- 4 single cell colony samples from the BL21 LB petri dish)
(- 2 single cell colony samples from the DH5α LB petri dish)

- Add 1 mL of sterilized liquid culture medium to each of the 6 samples transferred into 1.5 mL centrifuge
tubes
- culture at 37℃, 210 rpm for 90 min
- Place into a makeshift icebox for 30 min
- Transfer the six 1 mL samples into 50 mL centrifuge tubes and centrifuge at 4℃，2700 g for 10 min
- Discard supernatant liquid
- Add 300 μL of glycerol and 300 μL of deionized water into each 50 mL centrifuge tube
- Evenly distribute the solution into 100 μL tubes (fill 24 BL21 microfuge tubes and 20 DH5α microfuge
tubes)
- Store in the freezer

1.3 Liquid LB Culture Medium

- Transfer the liquid culture medium into four 50 mL centrifuge tubes each filled with 25mL
- Use a pipette to fill each cylindrical centrifuge tube with 10μL of antibiotic solution (1:1000 ratio) and 8.3
μL of pET28 cell culture (1:3000 ratio)
- Place 4 cylindrical tubes in a shaker at 150 rpm for overnight incubation at 37℃

2. Preparation of desired DNA segment

2.1 PCR Amplification of Desired DNA Segment

- Initial denaturation of the designated DNA strand at 94℃ for 5 min
- Run a thermocycler for 30-35 cycles:

(-denaturation to separate DNA strands at 94℃ for 30 sec

- primer annealing at 58℃ for 30 sec
- extension at 72℃ at 1min/kb to synthesize a new DNA chain)

- Final extension of the DNA strand at 72℃ for 5 min

2.2 Agarose Gel Electrophoresis

- Mix 0.5 g agarose and 50 mL 1 × TAE buffer in a 200 mL beaker
- Melt the solution in a micro-oven for 1 min
- After the solution is cooled to 55℃, add 5 μL nucleic acid dye
- Pour the agarose solution into the casting tray and wait for 15-20 min for the agarose gel to cool
completely

2.3 DNA Gel Extraction

- Extract the designated section of DNA from the agarose gel using a small razor blade
- Weigh and place each agarose sample into a 1.5 mL microfuge tube
- Add 1 mL and 600 μL buffer B2 to the two samples respectively
- Place the two samples in the 50℃ water bath for 10 min
- Transfer the contents of the microfuge tubes into separate absorption tubes
- Centrifuge at 12000 rpm for 30 sec
- Discard the liquid
- Transfer the remaining contents of the microfuge tubes into the absorption tubes
- Centrifuge at 12000 rpm for 30 sec
- Discard the liquid and add 500 μL washer solution to the absorption tube
- Centrifuge at 12000 rpm for 30 sec and discard the liquid
- Repeat steps 10-11
- Centrifuge the absorption tube at 12000 rpm for 30 sec
- place the absorption tube in a clean 1.5 mL microfuge tube and add 35 μL Elution buffer to the center of the
absorption tube
- Centrifuge at 12000 rpm for 1 min and seal the 1.5 mL microfuge tubes to be placed in the freezer

3. Gene recombination

3.1 Preparation of Recombinant Plasmid

(pET28 DH5α, W1-pUC57, W1-pET28 a, sp-pUC57, sp-pET28)
- Add 20 mL liquid culture medium to five labeled 50 mL centrifuge tubes
- Add 10 μL of plasmids containing strains culture medium to the designated 50 mL centrifuge tubes
- For the 2 tubes with W1-pUC57 and sp-pUC57 tubes add 20 μL Ampicillin
- For the tubes with pET28 DH5α, sp-pET28, and W1-pET28 a, add 20 μL Kanamycin
- Place the 50 mL centrifuge tubes in a 37℃ shaker for overnight incubation

3.2 Double Enzyme Digestion

- Add 1 μL of each enzyme into two 1.5 mL microfuge tubes
- Add 5 μL of buffer to each microfuge tube
- Add 1 μg of extracted DNA to the microfuge tube (one tube with sample 1 pUC19-75, the other with sample 3 pUC19
donor)
- Add 12 μL of deionized water to the microfuge tubes
- Place in 37℃ water bath for 1 hour
- Place in 65℃ water bath for 30 min to stop reaction process

3.3 Plasmid Extraction

pET28 DH5α, W1-pUC57, W1-pET28 a, sp-pUC57, sp-pET28
- Take the five 50 mL centrifuge tubes out of the shaker and centrifuge at 3000 × g for 15 min
- In separate tubes set up 5 absorption tubes and add 500 μL buffer S
- Centrifuge at 12000 rpm for 1 min
- Discard the supernatant liquid and add 1 mL deionized water to the 5 samples
- Transfer the 5 samples to 1.5 mL centrifuge tubes and centrifuge at 12000 rpm for 2 min and discard the
supernatant
- Add 250 μL buffer SP1 to the centrifuge tubes (which contain RNase A) to resuspend the pellets
- Add 250 μL buffer SP2 to the centrifuge tubes and slowly invert and revert the centrifuge tubes 5-10 times to
mix the solution
- Add 350 μL buffer SP3 to the centrifuges tube and slowly invert and revert the centrifuge tubes 5-10 times to
mix the solution
- Centrifuge at 12000 rpm for 10 min
- Transfer the supernatant liquid to the absorption tubes
- Centrifuge the absorption tubes at 12000 rpm for 30 sec
- Discard the filtrate and add 500 μL of buffer DW1 into the absorption tube
- Centrifuge at 12000 rpm for 30 sec, discard liquid and add 500 μL of washer solution into the absorption
tube
- Centrifuge at 12000 rpm for 30 sec and discard the liquid
- Repeat the previous 2 steps
- Centrifuge at 12000 rpm for 60 sec
- Place the column in a clean 1.5 mL centrifuge tube and add 30 μL Elution buffer to the absorption tubes
- Centrifuge at 12000 rpm for 1 min

3.4 Recombination of pUC19-75D

- Transfer 2 μL of the sample to a new 1.5 mL centrifuge tube
- Add 1 μL pEasy Blunt Clone Vector to each sample
- Add 2 μL of deionized water
- Place in 37℃ water bath for 10 min
- Take out and place into ice bath

3.5 Transformation of pUC19-75D, pET28 DH5α, W1 pUC57, W1 pET28 a, sp pUC57

- Take one tube of 100 μL competent cell culture BL21 from the freezer and place it in ice box for 5-10 min
- Add 50 μL competent cell culture to each 5 μL plasmid sample
- Place in the ice bath for 30 min
- Place in 42℃ water bath for 90 sec
- Immediately put in the ice bath for 3-4 min to cool down

4. The treatment of the product of gene recombination

4.1 The cultivation of the bacteria solution which contains lipase

- The transformation solution was inoculated into 200mL of LB medium containing resistance and cultured overnight
on a shaker (37°C, 170rpm);
- 0.1mM IPTG was added for induction when the OD600 is arranging 0.4-0.6, and then the culture was continued at
37°C and 170rpm for 3-4h;
- After centrifuging at 3000g, 120 μL PMS, 3mL cell lysate, and 240mg lysozyme were added, and the bacteria
solution which contains lipase was obtained by ultrasonic crushing.

4.2 Purification of protein

- Using the equation Mass (mg) = Concentration (mM/mL) × Volume (mL) × Molecular Weight to preparing the Buffer A
(50 mM Tris- HCI，300 mM NaCl，10 mM imidazole，pH7.6), B (50 mM Tris-HCI，300 mM NaCl，250 mM imidazole，pH7.6), and C
(50 mM Tris-HCI，300 mM NaCl，10% glycerin，pH7.6)
- The crude enzyme solution was centrifuged (4500rpm, 15min, 4°C), and the supernatant was added to the Ni-NTA
column equilibrated with Buffer A. After washing the unbound protein with Buffer A, the target protein was eluted
with Buffer B.
- Proteins were finally stored in Buffer C.

4.3 Detection of expression (SDS-PAGE electrophoresis)

- Sample preparation

    5× sample buffer :pH=6.8, 0.06mol/L (0.4mol/L) Tris-HCl buffer, containing 25%(50%)
glycerol, 2%(10%)SDS, 5%(volume fraction) β-mercaptoethanol and 0.1% bromophenol blue.
    Sample denaturation: The protein solution was mixed with 5× sample buffer at a ratio of
4:1(volume ratio) (if the protein solution concentration is too high, needs pre-desalting treatment), after
heating in a 100°C water bath for 3 to 5min, so that the proteins were fully denatured and then centrifuged at
10000 rpm for 5min, and the supernatant was taken for SDS-PAGE.

- Gel preparation

    Separation gel: (1.5M Tris, pH8.8) 90.9g Tris was taken in 400mL deionized water and
stirred on a magnetic stirrer until completely dissolved and then the volume was fixed to 500mL. The pH was
adjusted to 8.8, filtered, and stored in an ice box at 4°C.
    Concentration gel: (1.5M Tris, pH6.8) take 90.9g Tris in 400mL deionized water, stir on a
magnetic stirrer, until completely dissolved, and then the volume is fixed to 500mL. The pH was adjusted to 6.8,
filtered, and stored in an ice box at 4°C.

- The preparation of electrophoresis liquid

    In protein electrophoresis buffer (5×), 15.1g Tris, 94G glycine, and 5G SDS were added to
800mL deionized water and stirred on a magnetic stirrer until completely dissolved, and then fixed the volume to
1L. Stored it at room temperature without pH adjustment and filtration. Taking 100mL, add 400mL of deionized
water, dilute to 1×, and use.

- The process of electrophoresis

    Protein sample loading: According to the protein concentration, an appropriate amount of
processed protein samples was added into the electrophoretic well cell in turn, and an appropriate amount of
protein standard marker was added into another free well cell.
    Start electrophoresis: Switch on the power supply, first adjust the voltage to 75-80V, and
then adjust to 120V when the sample reaches the separation gel, maintaining a constant voltage.
    End of electrophoresis and peeling gel: when bromophenol blue migrates to the bottom of
the gel, stop electrophoresis; The protein glue was removed and the bromophenol blue strip separating the bottom
of the gel was cut off.
    Staining: Protein glue was stained for 1 hour using Coomassie R250 staining solution
Decolorization: The stained protein glue was decolorized with decolorization solution until the background was
clean and the protein band was clearly visible for photo analysis.
    Decolorization: The stained protein glue was decolorized with decolorization solution
until the background was clean and the protein band was clearly visible for photo analysis.

5. Examination of the activity of the lipase in different environments

5.1 Measure the standard curve of enzyme activity

- Standard liquor of p-nitrophenol (2mmol/L)

    0.02789g of p-nitrophenol (p-np) was weighed and dissolved in 100mL of solution B (buffer
with different pH), and stored in a brown reagent bottle after configuration and stored at 4°C.

- Standard curve measurement

    0.02, 0.04, 0.06, 0.08, 0.12, 0.16mL of p-nitrophenol mother solution (2mmol/L) was
diluted to 4mL with solution B (buffer of different pH), and the absorption value at 410nm was measured
successively. The standard curve was drawn with p-C-nitrophenol (0.01, 0.02, 0.03, 0.04, 0.06, 0.08, mmol/L) as
the abscissa and absorbance value Y as the ordinate.

5.2 Detection of lipase activity at different pH values

- Solution preparation

    Solution A: 30.0mg of p-nitrobenzoitolate was mixed with 10.00mL isopropanol and stored at
4°C in A brown bottle (a 15mL foil-coated centrifuge tube can be used).
    Solution B: Add 10μl of TritonX-100, then add 100 ml of disodium hydrogen
phosphate-hydrochloric acid buffer (pH 3-12) and mix, and store at 4°C.

- Determination of lipase activity

    1mL~1.2mL of solution A and 9mL ~ 10.8mL of solution B(different pH) were mixed in
centrifuge tubes, filled and mixed, kept warm in the 37°C water bath for 5min, and evenly divided into 3 to 4 test
tubes, each 2.7mL, which is the system solution; 100 μl crude enzyme solution was added into each test tube
quantitatively. In the control experiment, the inactivated enzyme solution (boiling at a high temperature for
5min) was added, and the reaction solution was immediately after the enzyme solution was added. The reaction was
performed at 37°C for 10 to 15min and was terminated by the addition of 95% ethanol. Absorbance values were
measured at 410nm and 310nm, with two to three replicates for each sample and averaged.

5.3 Detection of lipase activity assay at different temperatures

- Preparation of solution

    The pH of the buffer solution was the optimum pH measured by it.

- Determination of lipase activity

    1mL~1.2mL of solution A was added to 9mL~10.8mL of solution B (optimal pH) in a centrifuge
tube, filled and mixed, kept warm in the 37°C water bath for 5min, and divided into 3 or 4 test tubes, each 2.7mL,
namely the system solution. The crude enzyme solution plus 100 μl was added to each test tube, the inactivated
enzyme solution (boiling water for 5min) was added to the control group, and the enzyme solution was added as the
reaction solution. A gradient was set every 5°C from 25°C to 70°C for 10 to 15min. The reaction was terminated by
the addition of 95% ethanol, and the absorbance was measured at 410nm and 310nm, with two to three replicates for
each sample, and the average value was taken.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block header\_content %}
![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-team.jpg)
{% endblock %}
{% block page\_content %}

Results

1. Construction of the lipase expression plasmids

We amplified the lipase gene from the lactobacillus Plantarum lipase gene W1-ligase
and pseudomonas lipase gene SP-ligase, and inserted them in the XhoI and HindIII
sites of pET28a, respectively (Figure 1).

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-result01.png)

Figure 1. W1-lipase and SP-ligase expression plasmids in this project

In order to build our plasmids, we let the synthetic company synthesize two target
gene fragments (W1-lipase and SP-lipase). We digested the target fragments and the
pET28a vector with XhoI and HindIII (Figure 2), and we used T4 DNA ligase to
ligate the fragments and the vector. Then we transformed the recombinant plasmids
into E. coli DH5α competent cells and coated on the LB (Kanamycin) solid plates.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-result02.png)

Figure 2. Gel electrophoresis results of target gene fragments.
A. double-enzyme digested with the W1-ligase,
B. double-enzyme digested with the SP-ligase,
C. double-enzyme digested with the pET28a vector.

We verified the colonies through colony-PCR (Figure 3), and then we inoculated
single colonies and we send the constructed recombinant plasmid to a sequencing
company for sequencing.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-result03.png)

Figure 3. verification of the recombinant plasmids through colony-PCR.
A. pET28a-W1-lipase,
B. pET28a-SP-lipase.

The returned sequencing comparison results showed that there were no mutations in
the ORF region, and the plasmid was successfully constructed (Figure 4). So far, we
have successfully obtained two recombinant plasmids, which were respectively on the
pET28a vector, which can be used to express lipase proteins.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-result04.png)

Figure 4. Mapped the sequencing data to the recombinant plasmid.
A. the sequencing data of pET28a-SP-lipase,
B. the sequencing data of pET28a-W1-lipase.

As a result, the amplified target gene W1-lipase/SP-lipase and the double digested
vector pET28a were ligated with T4 ligase to obtain recombinant plasmids
pET28A-W1-lipase and pET28A-SP-lipase, so that the recombinant protein had 6-His
tags at the carboxyl terminus which could be used to purify the corresponding
proteins.

2. Protein lipase expression

The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and cultured overnight in the medium
containing resistance. When the OD600 was around 0.4-0.5, the IPTG was added to induce the expression of recombinant
protein W1-lipase/SP-lipase, and then the strains were cultured at 16℃ for 20h. After that, the collected bacterial
solution was cracked by Ultrasonic crushing. SDS-PAGE was used to analyze the recombinant proteins. Figure 5 showed
the electrophoretic results of the protein gel.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-result05.png)

Figure 5. SDS-PAGE detection of lipase protein

3. Lipase activity detection at different pH and temperature

a) Standard curve measurement

In order to measure the standard curve of the activity of lipases, we chose p-nitrophenol as the substrate and
detected its absorbance value of it when adding lipases. 0.02789g of p-nitrophenol (p-np) was weighed and dissolved
in 100mL of solution B, and stored in a brown reagent bottle after configuration and stored at 4°C.
0.02, 0.04, 0.06, 0.08, 0.12, 0.16mL of p-nitrophenol solution (2mmol/L) was diluted to 4mL, and the absorbance
value at 410nm was measured successively. The standard curve was drawn with p-nitrophenol (0.01, 0.02, 0.03, 0.04,
0.06, 0.08, mmol/L) as the abscissa and absorbance value Y as the ordinate (Figure 6).

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-result06.png)

Figure 6. the standard curve of p-nitrophenol

According to the standard curve determination method, the standard curve is drawn as shown in Figure 6. Regression
coefficient R2=0.9979, the results are credible.

b) Measure the activity of lipase at different pH

Esterase activity was assayed in the pH range from 3.0 to 12.0, and at temperatures of 25 to 70°C. Enzyme
thermostability was measured by incubation of the enzyme in 50 mM sodium phosphate buffer (pH 9.0) at 25-70°C for 5
min, 15 min, 30 min, and 1, 2, 4, 6, and 20 h. After incubation, the residual activity of lipase was measured as
described above. To test the effects of metals, ions, and additives on the activity of the esterase, lipase was
incubated in their presence at a final concentration of 1 mM for 5 min at room temperature. Then, the substrate
(p-nitrophenyl acetate) was added, and the reaction mixture was incubated at 37°C. The experiments were performed in
triplicate.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-result07.png)

Figure 7. the enzyme activity of lipase at different pH

As shown in Figure7, when changed the pH value of the buffer, the activity of lipase is changed compared with the
negative control, and the W1-lipase and SP-lipase showed no obviously different. And when the pH value is 9, the
lipase exhibited the highest activity.

c) Measure the activity of lipase at different temperature

When pH=9, the recombinant enzyme activity reached the highest, 36.-40U/mL, and decreased when pH=9, so the optimal
pH of the recombinant enzyme was 9. According to the standard curve, the enzyme activity at the optimum pH and
different temperatures are shown in Figures 3-10 (right). At 40℃, the recombinant enzyme activity reached the
highest, 36-40U/m L, and decreased when the temperature was higher than 40℃. Therefore, the optimal temperature for
the recombinant enzyme was 40℃, but it had higher activity at 30-40 ℃.

![](https://static.igem.wiki/teams/4279/wiki/t-subcat-china-result08.png)

Figure 8. the enzyme activity of lipase at different temperatures

As shown in Figure8, when changed the reaction temperature, the activity of lipase is different compared with the
negative control. And when the temperature is around 35℃, the SP-lipase showed the highest activity, while 40℃ may
be the optimum temperature for W1-lipase.

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