iGEM 2024 — Team sfls-hangzhou

{% 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 %}
{% block page\_content %}

Notebook

[Jan. 2024](#section1)
[Feb. 2024](#section2)
[Mar.-June. 2024](#section3)
[July. 2024](#section4)

Jan. 2024

Project Design & Brainstorming

Experimental design and material preparation with the instructor

Feb. 2024

Phase 1: Experimental attempts, including:
>Preparation of reagents for bacterial culture and molecular cloning

1. Preparation of LB Medium
2. Activation of pET28a Strain

![IMG_4118](https://static.igem.wiki/teams/5093/notebook/1.jpg)

>PCR and acquisition of the target gene

1. Extraction of pET28a Plasmid
2. Double Digestion with NdeI/XhoI → Target Gene/pET28a
   Plasmid
3. Agarose Gel Electrophoresis
4. Gel Extraction of the Digested Bands:
   pET28a/EC2.4.1.5/EC2.4.1.9

![](https://static.igem.wiki/teams/5093/notebook/2.jpg)

> Transformation

1. T4 DNA ligase-mediated ligation of pET28a-2.4.1.5 and
   pET28a-2.4.1.9
2. Buffer preparation
3. Heat shock transformation

![](https://static.igem.wiki/teams/5093/notebook/3.jpg)

Mar.-June. 2024

1. Learn from the theory class and prepare for the human
   practice
2. Periodic group discussions

July. 2024

Objectives of Experiment:

1. Plasmid extraction of pET28a-2.4.1.5/2.4.1.9
2. Heat shock transformation into E. coli BL21(DE3)

![](https://static.igem.wiki/teams/5093/notebook/4.jpg)

Objectives of Experiment:

1. PCR identification of monoclonal/transformed colonies in
   BL21(DE3)
2. Agarose gel electrophoresis

![](https://static.igem.wiki/teams/5093/notebook/5.jpg)

·Agarose Gel
Electrophoresis

|  |  |
| --- | --- |
| Component | Amount |
| Agarose | 0.5 g |
| 1×TAE Buffer | 50 mL |
| Nucleic Acid Stain | 3 μL |

Objectives of Experiment:

1. Preparation of seed culture fermentation broth
2. SDS-PAGE verification of protein expression

SDS-PAGE Separating Gel Concentrations and Optimal Separation Ranges:

|  |  |
| --- | --- |
| SDS-PAGE Gel Concentration | Optimal Separation Range |
| 6% PAGE gel | 70-300 kD |
| 8% PAGE gel | 30-200 kD |
| 10% PAGE gel | 20-80 kD |
| 12.5% PAGE gel | 15-60 kD |
| 15% PAGE gel | 10-45 kD |

![](https://static.igem.wiki/teams/5093/notebook/6.jpg)

Objectives of Experiment:

1. Protein expression and purification
2. Thin layer chromatography
3. Construction of Digestion Model and Detection of Sucrose Changes and Product Formation in Food Digestion Mixture.

|  |  |  |  |
| --- | --- | --- | --- |
| 2% Sucrose Treatment | | Trehalose Treatment | |
| Number | Sample Amount - Time | Number | Sample Amount - Time |
| 1 | 1-5 | 26 | 1-5 |
| 2 | 1-10 | 27 | 1-10 |
| 3 | 1-15 | 28 | 1-15 |
| 4 | 1-30 | 29 | 1-30 |
| 5 | 1-60 | 30 | 1-60 |
| 6 | 5-5 | 31 | 5-5 |
| 7 | 5-10 | 32 | 5-10 |
| 8 | 5-15 | 33 | 5-15 |
| 9 | 5-30 | 34 | 5-30 |
| 10 | 5-60 | 35 | 5-60 |
| 11 | 10-5 | 36 | 10-5 |
| 12 | 10-10 | 37 | 10-10 |
| 13 | 10-15 | 38 | 10-15 |
| 14 | 10-30 | 39 | 10-30 |
| 15 | 10-60 | 40 | 10-60 |
| 16 | 15-5 | 41 | 15-5 |
| 17 | 15-10 | 42 | 15-10 |
| 18 | 15-15 | 43 | 15-15 |
| 19 | 15-30 | 44 | 15-30 |
| 20 | 15-60 | 45 | 15-60 |
| 21 | 20-5 | 46 | 20-5 |
| 22 | 20-10 | 47 | 20-10 |
| 23 | 20-15 | 48 | 20-15 |
| 24 | 20-30 | 49 | 20-30 |
| 25 | 20-60 | 50 | 20-60 |

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{%
endblock %}
{% block page\_content %}

Engineering

[Overview](#section1)
[Cycle1: BBa\_K5093002 (pET28a-EC.2.4.1.5)](#section2)
[Cycle2: BBa\_K5093003 (pET28a-EC.2.4.1.9)](#section3)
[Other Test](#section4)

Overview

In our project, we
developed a capsule containing two catalytic enzymesdextransucrase and
inulosucrase that are coded by genes
EC.2.4.1.5 and EC.2.4.1.9 to catalyse the
conversion from sucrose to two types of soluble dietary fibres
(SDFs) dextran and inulin respectively as shown in
Figure 1. Therefore, individuals may enjoy their diets without change in flavor while reducing the human
body's effective absorption of sugars in food and increasing fiber intake. With no change to the sugar
content of the food, this initiative intends to promote bowel movement, regulate gut bacteria, and lower
the quantity of sugar absorbed by the human body into the bloodstream.

In this part, we will demonstrate the design of our plasmid, how it was built, how to test its
function and what we learned through this construction.

![](https://static.igem.wiki/teams/5093/engineering/test.png)

Cycle1: BBa\_K5093002(pET28a-EC.2.4.1.5)

Design 1:

EC.2.4.1.5 belongs to the Glucansucrase (GS) family(Fig
1). It
breaks sucrose molecules, polymerizes the D-glucose fraction, synthesizes α-glucan with different bond
compositions, branches, and chain lengths, and releases fructose molecules. With the particular
catalytic property, recombinant proteins are obtained by heterologous recombination to explore the
catalytic properties of recombinant proteins for converting sucrose to produce Dextran, a dietary
fiber.

![96fd84806c5285711295023254d178c2](https://static.igem.wiki/teams/5093/engineering/1.jpg)

Fig.1 Target gene map
of
EC.2.4.1.5

The
pET-28a plasmid is a prokaryotic high-efficiency expression vector containing the anti-kanamycin gene,
commonly used as a fusion protein in bacteria. The host cell induces the expression of this gene by
providing T7 RNA polymerase. The pET-28a composes a new vector with EC.2.4.1.5 in the construction, and
it can be extracted from Escherichia coli (E.
coli).

![808ef7ed5dc49b104a70a73f41e0315e](https://static.igem.wiki/teams/5093/engineering/2.jpg)

Fig.2 pET28a
plasmid

The template plasmid was used to
obtain the EC.2.4.1.5 gene
through polymerase chain reaction (PCR), and the pET28a plasmid was extracted from E. Coli. The
target gene and pET28a plasmid were double-digested, ligated, and transformed into E. coli DH5α. PCR
and sequenced then identified the bacterial liquid and confirmed the construction of a recombinant
plasmid. The final plasmid is the aim of our construction. We used Snapgene to create the profile of
the plasmid.

![截屏2024-07-27 13.51.07](https://static.igem.wiki/teams/5093/engineering/3.jpg)

Fig.3
pET28a-EC.2.4.1.5
plasmid

Build
1:

The biotechnology company
GeneScript synthesizes the DNA
molecules containing our desired gene. Then, they are cut with restriction endonucleases Nde1 and
Xho1 and amplified using polymerase chain reaction (PCR). The gene's length is 4400 bp. The bands
representing EC.2.4.1.5 successfully appear at their corresponding positions in the gel, as shown in
Fig 10, indicating that the cutting and amplifying are successful.

![](https://static.igem.wiki/teams/5093/engineering/4.png)

Fig.4
Double digestion of EC.2.4.1.5

A
double digestion experiment can change the vector from a ring to a line, using Nde1 and Xho1 to cut and
link the sites at the gaps at both ends of the vector.

![1601722064714_.pic](https://static.igem.wiki/teams/5093/engineering/5.jpg)

Fig.5 Double
digestion of
pET28a

Nde1
and Xho1 also cut pET28a to make complementary sticky ends. EC.2.4.1.5 is connected to the linear
plasmids with T4 ligase. Then, the heat shock conversion of the recombinant plasmids is applied to
competent E. coli DH5α.We then incubated them overnight at
37°C after streak inoculating them on LB solid medium
plates that included appropriate
antibiotics (LB-kana), as
shown in Fig 11B. Afterward, we picked
threecolonies from each petri dish and extracted their plasmids. PCR
and gel electrophoresis (Fig 6A) were run to confirm the extracted plasmids were the ones we required.
EC.2.4.1.5 (4440 bp long) appears at its corresponding position. Then, the pET28a-EC.2.4.1.5
plasmids are sent to Azenta Life Sciences for
sequencing, whose results further proved our success in constructing the plasmid (Fig 7).

![](https://static.igem.wiki/teams/5093/engineering/6.jpg)

Fig.6 A: PCR identification; B: colony
identification

After obtaining the PCR results, it
was confirmed that the bands
after its PCR was completed were consistent with the expected target bands, so we concluded that the
recombinant plasmid was successfully constructed.

The
recombinant plasmid was sent to Azenta Life Sciences for sequencing, and the sequencing results
accurately proved that the pET28a-EC2.4.1.5 plasmid was constructed successfully, as seen in the DNA
sequencing graph.

![WechatIMG156](https://static.igem.wiki/teams/5093/engineering/7.jpg)

Fig.7 Sequencing
diagram

The
verified plasmids were transformed into E.coli
BL21(DE3), incubated on LB solid medium plates (Kana+), and cultured at 37°C overnight. Four colonies were
selected for
PCR amplification to confirm success in the second transformation (Fig 8) and then transferred into 1L
fresh LB (Kana+) culture medium for the scale-up
cultivation. IPTG (0.2 mM) was
used to induce the expression of genes EC.2.4.1.5 with OD600 around 0.6-0.8 and cultured at 16℃ for 20h. The proteins
were
then extracted from the supernatant of the E.coli
BL21(DE3) after ultrasonic cell disruption and
centrifugation.

![](https://static.igem.wiki/teams/5093/engineering/8.jpg)

Fig. 8 DNA gel
electrophoresis of EC.2.4.1.5 (A) and clones of E.coli BL21(DE3) containing the gene
(B)

Test
1:

After
transforming the recombinant plasmid into BL21(DE3), we allowed it to express the protein in significant
quantities by expanding the culture and IPTG induction. The protein size on the protein strip after
sonication and fragmentation is 167kDa, which is consistent with the size of our target gene EC.2.4.1.5.
This indicates that our target protein was correctly expressed in large quantities.

![](https://static.igem.wiki/teams/5093/engineering/9.jpg)

Fig.9 Results of
SDS-PAGE
of supernatant, flowthrough, wash, and elution parts for the protein purification of EC.2.4.1.5

Learn
1:

|  |  |
| --- | --- |
| Problem | Solution |
| How to find a proper method to identify the function of the obtained enzyme | We evaluated the available equipment and materials, read relevant essays, and researched information. Finally, according to the teacher’s suggestion, we used thin-layer chromatography. |
| Why the results of SDS-PAGE were not clear | We may need to purify the protein by reconcentrating it. If the solution is too dilute, the results might not be precise. |

Cycle2: BBa\_K5093003 (pET28a-EC.2.4.1.9)

Design
2:

 EC.2.4.1.9 codes for the
enzyme inulosucrase, which transfers a
fructose group from the disaccharide sucrose to a growing inulin chain, a fiber, to produce glucose and
inulin. The
pET28a plasmid has other desirable features that make it an ideal vector besides the T7 promoter and
terminator it possesses. It has multiple restriction sites for the insertion of a new gene. Meanwhile,
it has a sequence coding for a 6×His tag between the T7 promoter and terminator, so the protein produced
can be purified via nickel column affinity chromatography.

![](https://static.igem.wiki/teams/5093/engineering/10.jpg)

Fig.10 The gene map
of
EC.2.4.1.9

In
our
project, this enzyme reduces glucose absorption by catalyzing this reaction using sucrose as its
substrate, lowering the chance of getting obese or contracting non-communicable diseases.
The template plasmid was
used to obtain the EC.2.4.1.9 gene through polymerase chain reaction (PCR), and the pET28a plasmid was
extracted from E. Coli. The target gene and pET28a
plasmid were double-digested,
ligated, and transformed into E.
coli DH5α. PCR and sequenced then identified the
bacterial liquid and confirmed the construction of a recombinant plasmid. The final plasmid is the aim
of our construction. We used Snapgene to create the profile of the plasmid.

![](https://static.igem.wiki/teams/5093/engineering/11.jpg)

Fig. 11 Plasmid map of
pET28a-EC.2.4.1.9

Build
2:

The biotechnology
company GeneScript synthesizes the DNA molecules containing our desired gene. Then, they are cut with
restriction endonucleases Nde1 and Xho1 and amplified using polymerase chain reaction (PCR). The gene's
length is 2400 bp. The bands representing EC.2.4.1.9 successfully appear at their corresponding
positions in the gel, as shown in Fig 19, indicating that the cutting and amplifying are
successful.

 ![](https://static.igem.wiki/teams/5093/engineering/12.png)

Fig.12 The results of gel
electrophoresis
of EC.2.4.1.9

Nde1 and Xho1 are
also
used to cut pET28a to make complementary sticky ends. EC.2.4.1.9 is connected to the linear plasmids
with T4 ligase. Then, the heat shock conversion of the recombinant plasmids is applied to competent
E. coli DH5α. We then incubated them overnight at
37°C after streak
inoculating them on LB solid medium plates that included appropriate antibiotics (LB-kana), as shown in
Fig 20B. Afterward, we picked three colonies from each petri dish and extracted their plasmids. PCR and
gel electrophoresis (shown in Fig 13A) were run to confirm the extracted plasmids were the ones we
required. EC.2.4.1.9 (2400 bp long) appears at its corresponding position.

 ![](https://static.igem.wiki/teams/5093/engineering/13.jpg)

Fig.13 A: Gel electrophoresis to
confirm
pET28a-EC.2.4.1.9 plasmids. B: pET28a-EC.2.4.1.9 containing strain clones.

Then, the
pET28a-EC.2.4.1.9 plasmids are sent to Azenta Life Sciences for sequencing, whose results further proved
our success in constructing the plasmid (Fig 14).

![](https://static.igem.wiki/teams/5093/engineering/14.jpg)

Fig.14 The DNA sequencing diagram for
pET28a-EC.2.4.1.9

The verified
plasmids
were transformed into E.coli BL21(DE3), incubated
on LB solid medium plates (Kana+), and cultured
at 37°C overnight. Four colonies were selected for
PCR amplification to confirm success in the second transformation (Fig 15) and then transferred into 1L fresh LB
(Kana+) culture medium for the scale-up
cultivation. IPTG (0.2 mM) was used to induce the expression of genes EC.2.4.1.9 with
OD600 around 0.6-0.8 and cultured at 16℃ for
20h. The proteins were then extracted from the supernatant of the E.coli BL21(DE3) after ultrasonic cell disruption and
centrifugation.

![](https://static.igem.wiki/teams/5093/engineering/15.jpg)

Fig. 15 DNA gel electrophoresis of
EC.2.4.1.9 (A) and clones of
E.coli BL21(DE3) containing the gene (B).

Test
2:

Nickel affinity
chromatography effectively purifies inulosucrase because the protein contains a 6×His tag. We got a more
precise result with little interference by non-specifically bound proteins. Fig 8 shows only one band
with a molecular weight of 88 kDa. This demonstrates that inulosucrase is successfully expressed and
purified.

![](https://static.igem.wiki/teams/5093/engineering/16.jpg)

Fig.16 Results of SDS-PAGE of supernatant, flowthrough,
wash, and elution parts for the protein purification of EC.2.4.1.9

Learn
2:

|  |  |
| --- | --- |
| Problem | Solution |
| How to determine the ratio of two enzymes | Further validation will be conducted via HPLC. |
| Why the results of SDS-PAGE were not clear | We may need to purify the protein by reconcentrating it. If the solution is too dilute, the results might not be precise. |

Other Test

We
utilized thin-layer chromatography to verify that this enzyme can degrade sucrose. The principle of
thin-layer chromatography is to use each component's different adsorption capacities on the same
adsorbent so that the solvent flows through the adsorbent process, continuous adsorption, and desorption
to achieve the mutual separation of the components. The reaction was carried out at 37°C for 1h in a
buffer system with the addition of 2% plantain sugar and the appropriate amount of recombinant enzyme
solution.

![](https://static.igem.wiki/teams/5093/engineering/17.jpg)

Fig.17 Results of
thin-layer chromatography. 1: The recombinant enzyme solution with a ratio of 1:1 between dextransucrase
and inulosucrase; 2: The recombinant enzyme solution with a ratio of 1:2 between dextransucrase and
inulosucrase; 3: The recombinant enzyme solution with a ratio of 2:1 between dextransucrase and
inulosucrase

The
results showed a reduction in sucrose and glucose production on the thin-layer chromatography plates
when only 2% sucrose substrate was added, and the reaction was carried out at 37°C for one hour. This
shows that this enzyme can break down sucrose to produce glucose and other substances in the reaction
system.

{% 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 %}

Experiments

[Preparation of LB Medium](#section1)
[Extraction of pET28a Plasmid](#section2)
[Heat Shock Transformation](#section3)
[Protein Expression and Purification](#section4)
[Thin Layer Chromatography (TLC)](#section5)

Preparation of LB Medium:

|  |  |
| --- | --- |
| Reagent | Concentration |
| Tryptone | 10g/L |
| Yeast Extract | 5g/L |
| NaCl | 10g/L |
| Agar Powder (for solid medium) | 1.5g/100mL |

Add distilled water to
a final volume of 1 L, then autoclave at 121°C for 15 minutes.

![/private/var/folders/ts/1z_35h513_d55mv_zqgz8l400000gn/T/com.kingsoft.wpsoffice.mac/picturecompress_20240831153955/output_1.pngoutput_1](https://static.igem.wiki/teams/5093/experiment/1.jpg)

·Activation of pET28a Strain

1. Prepare Kanamycin (Kana) solution at 100 mg/mL.
2. Inoculate the strain harbouring the pET28a plasmid into 5 mL of liquid LB medium at an inoculation
   ratio of 1%, and add kanamycin to a final concentration of 100 µg/mL.

3. Incubate at 37°C
and 220 rpm in a shaker for 12-16 hours.

4.After the prepared LB solid
medium plates cool to room
temperature, add kanamycin to a final 100 µg/mL concentration, mix thoroughly, and pour plates under
sterile conditions.

Extraction of pET28a Plasmid

1. For high-copy plasmids, take 2 mL of bacterial culture, centrifuge at 8000 x g for 2 minutes at
   room temperature to collect the cells, and discard the supernatant.

2. Add 250 μL of Buffer SP1 to the cell pellet and pipette to resuspend the cells thoroughly.
3. Add 250 μL of Buffer SP2, gently invert the tube 10 times to mix immediately, and let it stand at
   room temperature for 1 minute.

4. Add 350 μL of Buffer SP3 and gently invert the tube 10 times to mix immediately.
5. Centrifuge at 12000 x g for 10 minutes, transfer the supernatant to a spin column, and centrifuge
   at 9000 x g for 1 minute. Discard
   the flow-through and back the spin column into the
   same collection tube.

6. Add 500 μL of Buffer DW1 to the spin column and centrifuge at 9000 x g for 1 minute. Discard the
   flow-through and back
   the spin column into the same collection tube.

7. Add 500 μL of Wash Solution to the spin column and centrifuge at 9000 x g for 1 minute. Discard
   the flow-through and
   place the spin column back into the same collection tube.

8. Repeat step 7.
9. Centrifuge the empty spin column and collection tube at 9000 x g for 1 minute, then let it stand
   with the lid open for
   2 minutes to evaporate the ethanol.

10. Add 50 μL of Elution Buffer to the centre of the spin column membrane and let it stand at room
    temperature for 1 minute, and centrifuge at 9000 x g for 1 minute. Store the
    resulting plasmid DNA solution at -20°C.

![/private/var/folders/ts/1z_35h513_d55mv_zqgz8l400000gn/T/com.kingsoft.wpsoffice.mac/picturecompress_20240831153928/output_2.pngoutput_2](https://static.igem.wiki/teams/5093/experiment/2.jpg)

·Double
Digestion with NdeI/XhoI → Target
Gene/pET28a Plasmid

Preparation of the
Digestion System:

|  |  |
| --- | --- |
| Reagent | Volume |
| 10×CutSmart Buffer | 5 μL |
| pET28a | 5 μL |
| NdeI | 1 μL |
| XhoI | 1 μL |
| ddH2O | 38 μL |

1. Incubate the reaction mixture at 37°C for 20 minutes.
2. add 5 μL of 10✖ loading buffer
   after the reaction for staining.

·Agarose Gel Electrophoresis

|  |  |
| --- | --- |
| Component | Amount |
| Agarose | 0.5 g |
| 1×TAE Buffer | 50 mL |
| Nucleic Acid Stain | 3 μL |

Microwave on high for
2 minutes until completely melted.

2. Add 3 μL of nucleic
acid stain to the molten agarose, mix thoroughly, and pour into the mould. Allow to solidify.

3. Perform electrophoresis at 150V for 15 minutes.
4. Use a gel imaging system to photograph the gel and confirm the size of the target gene.

·Gel
Extraction of DNA Bands: pET28a/EC2.4.1.5/EC2.4.1.9

1. Separate the target DNA fragment from other fragments via agarose gel electrophoresis. Using a
   scalpel, excise the
   agarose gel slice containing the target DNA and place it into a 1.5 mL microcentrifuge tube. Weigh the
   gel slice.

2. Based on the weight and concentration of the gel slice, add Buffer B2 at a ratio of 300 μL per 100
   mg of agarose.
3. Incubate the tube in a 50°C water bath for 10 minutes, occasionally mixing until the gel slice is
   completely dissolved.

4. Transfer the dissolved solution into a spin column and centrifuge at 8,000 x g for 30 seconds.
   Discard the flow-Place the spin
   column back into the same collection tube.

5. Add 300 μL of Buffer B2 to the spin column and centrifuge at 8,000 x g for 30 seconds. Discard the
   flow-through and
   place the spin column back into the same collection tube.

6. Add 500 μL of Wash Solution to the spin column and centrifuge at 9,000 x g for 30 seconds. Discard
   the flow-Place the spin
   column back into the same collection tube.

7. Repeat step 6 once.
8. Centrifuge the empty spin column and collection tube at 9,000 x g for 1 minute to remove any
   residual wash buffer.

9. Add 15-40 μL of Elution Buffer to the centre of the spin column membrane, let it stand at room
   temperature for 1-2 minutes, and then centrifuge at 9,000 x g for 1 minute. Store the obtained DNA
   solution at -20°C or use it for subsequent experiments.

·T4 DNA
ligase-mediated ligation of
pET28a-2.4.1.5 / pET28a-2.4.1.9

|  |  |
| --- | --- |
| Component | Amount |
| Linearized pET28a plasmid | 0.02 μg |
| Insert (target gene) | 0.2 μg |
| T4 DNA ligase | 1 μL |
| 10× Ligase Buffer | 2 μL |
| ddH₂O | Y μL |
| total | 20 μL |

Mix thoroughly and
incubate at 37°C for 2 hours.

·Buffer
Preparation

1. IPTG (100 mM): Dissolve 2 g of IPTG in 8 mL of distilled water, then bring the volume to 10 mL
   with distilled water. Filter-sterilize using a 0.22 μm filter. Aliquot into 1 mL portions and store
   at -20°C.
2. His A Buffer (500 mL): Dissolve 1.42 g of Na₂HPO₄ (20 mM), 14.6 g of NaCl (500 mM), and 0.6808 g
   of imidazole (20 mM) in distilled water. Adjust pH to 7.4.
3. His B Buffer (250 mL): Dissolve 0.71 g of Na₂HPO₄ (20 mM), 7.3 g of NaCl (500 mM), and 8.5 g of
   imidazole (500 mM) in distilled water. Adjust pH to 7.4.
4. Citrate-Citric Acid Buffer (0.1 mM): Dissolve 21.01 g of citric acid monohydrate (C₆H₈O₇·H₂O) and
   29.41 g of sodium citrate dihydrate (Na₃C₆H₅O₇·2H₂O) in distilled water.

Heat Shock Transformation

Transformation of
Recombinant Product into DH5α Cells

1. Place the DH5α competent cells on ice to thaw. Add 10 μL of recombinant product to  100 μL of competent cells. Gently tap the
   tube to mix (do not vortex). Incubate on ice for 30
   minutes.

2. Perform heat shock by placing the tube in a 42°C water bath for 45 seconds, then immediately place
   the tube on ice
   to cool for 2-3 minutes.

3. Add 900 μL of LB liquid medium (without antibiotics) to the cells and incubate at 37°C with
   shaking for 1 hour (200-250 rpm).
   Centrifuge at 5,000 rpm (2,500 × g) for 5 minutes
   and discard the 900 μL supernatant. Resuspend the pellet in the remaining LB medium. Spread the
   resuspended cells onto LB-kanamycin plates using a sterile spreader.

4. Incubate the plates
at 37°C in an inverted position for 12-16 hours.

·PCR
identification of monoclonal/transformed colonies in DH5α

|  |  |
| --- | --- |
| Reagent | Volume |
| 2×Mix | 10 μL |
| T7-F | 1 μL |
| T7-R | 1 μL |
| ddH2O | 7 μL |

|  |  |
| --- | --- |
| 95℃ | 5min |
| 95℃ | 30s |
| 55℃ | 30s |
| 72℃ | 2min |
| 72℃ | 10min |
| 4℃ | ∞ |

According to the table above,
prepare the PCR
system. Pick a single colony from the plate using a 10 μL pipette tip and transfer it into the prepared
PCR mixture. Mix thoroughly by pipetting up and down, then place the mixture into the PCR machine for
amplification.

![/private/var/folders/ts/1z_35h513_d55mv_zqgz8l400000gn/T/com.kingsoft.wpsoffice.mac/picturecompress_20240831153928/output_3.pngoutput_3](https://static.igem.wiki/teams/5093/experiment/3.jpg)

·PCR
identification of monoclonal/transformed
colonies in BL21(DE3)

|  |  |
| --- | --- |
| Reagent | Volume |
| 2×Mix | 10 μL |
| T7-F | 1 μL |
| T7-R | 1 μL |
| ddH2 | 7 μL |

|  |  |
| --- | --- |
| 95℃ | 5min |
| 95℃ | 30s |
| 55℃ | 30s |
| 72℃ | 2min |
| 72℃ | 10min |
| 4℃ | ∞ |

According to the table
above, prepare the PCR system. Pick a single colony from the plate using a 10 μL pipette tip and
transfer it into the prepared PCR mixture. Mix thoroughly by pipetting up and down, then place the
mixture into the PCR machine for amplification.

·Agarose Gel Electrophoresis

|  |  |
| --- | --- |
| Component | Amount |
| Agarose | 0.5 g |
| 1×TAE Buffer | 50 mL |
| Nucleic Acid Stain | 3 μL |

1. Microwave on high for 2 minutes until completely melted.

2. Add 3 μL of nucleic
acid stain to the molten agarose, mix thoroughly, and pour into the mould. Allow to solidify.

3. Perform
electrophoresis at 150V for 15 minutes.

4. Use a gel imaging
system to photograph the gel and confirm the size of the target gene.

·Preparation of
seed culture fermentation
broth

1. Transfer a verified single colony into 3 mL of LB liquid medium. Add kanamycin antibiotic at a
   ratio of 1:1000 (3 μ

L)
incubate at 37°C with shaking at 220 rpm for 12-16 hours.

2. Inoculate the overnight culture of pET28a-2.4.1.5/2.4.1.9 into a sterile 50 mL centrifuge tube at
   a 1% inoculation

ratio. Add 40
mL of LB and 40 μL of kanamycin antibiotic at a
ratio of 1:100. Incubate at 37°C with shaking at

220
rpm for 3-4 hours until the OD600 reaches 0.6-0.8.

3. Add 0.2 mM IPTG to induce high-level protein expression and incubate at 16°C for over 20
   hours.

·SDS-PAGE
verification of protein
expression

SDS-PAGE Gel
Preparation

1. Assemble the gel casting apparatus and prepare the separating gel (1.50 mm thick mini-gel).
2. mix 4 mL of separating gel solution (2X) and 4 mL of separating gel buffer (2X) in a gel
   preparation cup.
3. Add 80 μL of modified catalyst to the mixture from step 2. Stir gently to mix thoroughly, avoiding
   bubble

formation.

4. Pour the mixed separating gel solution into the gel casting apparatus until the solution reaches
   1.5 cm below the

top
of the glass plate. Overlay the separating gel solution with a layer of water or alcohol to keep the gel
surface flat and free from bubbles.

5. Allow the gel to polymerise at room temperature (25°C) for 6-10 minutes. The gel is ready when a
   clear

boundary forms
between the separating gel and the overlay.

6. Prepare the stacking gel (1.50 mm thick mini-gel).
7. Carefully pour off the overlay. Mix 1.0 mL of stacking gel solution (2X) and 1.0 mL of stacking
   gel buffer (2X) in a

new
gel preparation cup.

8. Add 20 μL of modified catalyst to the mixture from step 7. Stir gently to mix thoroughly, avoiding
   bubble

formation.

9. Pour the stacking gel solution onto the polymerised separating gel until it reaches the top of the
   glass plate. Insert

The
comb slowly forms wells into the gel to avoid bubble formation.

10. Allow the stacking gel to polymerise for 10-15 minutes. Carefully remove the comb and rinse the
    wells with

electrophoresis
buffer using a pipette or syringe. The gel is now
ready for SDS-PAGE electrophoresis.

SDS-PAGE
Separating Gel Concentrations and
Optimal Separation Ranges:

|  |  |
| --- | --- |
| SDS-PAGE Gel Concentration | Optimal Separation Range |
| 6% PAGE gel | 70-300 kD |
| 8% PAGE gel | 30-200 kD |
| 10% PAGE gel | 20-80 kD |
| 12.5% PAGE gel | 15-60 kD |
| 15% PAGE gel | 10-45 kD |

Protein Expression and Purification

1. Centrifuge the overnight culture at 12,000 r/min for 10 minutes to collect the cell pellet.
   Resuspend the pellet in an
   appropriate amount of PBS buffer (pH 7.4).

2. Sonicate the cell suspension on ice with a power of 500 W (40%) in pulses of 3 seconds on and 3
   seconds off for 15 minutes. Centrifuge at 12,000 rpm for 30 minutes at 4°C, and collect the supernatant and pellet.

3. Nickel Affinity Purification (His-tag Protein Purification Kit)
4. Take 1 ml of well-mixed 50% His-tag Purification Resin, centrifuge at 4°C (1,000g for 10 seconds),
   and discard the
   storage buffer. Add 0.5 ml of non-denaturing lysis buffer to the resin, mix well to equilibrate,
   centrifuge at 4°C (1,000g for 10 seconds), and discard the buffer. Repeat the equilibration 1-2 times,
   discarding the buffer each time. Add 4 ml of bacterial lysate to the resin and incubate on a rotator at
   4°C for 60 minutes.

5. Transfer the mixture of lysate and His-tag Purification Resin into the empty affinity column
   provided in the kit.
6. Open the bottom cap of the purification column to allow the liquid to flow out by gravity. Collect
   20 μl of the flow-through for subsequent analysis.

7. Wash the column 5 times with 0.5-1 ml of non-denaturing wash buffer each time. Collect
   approximately 20 μl of the wash buffer for each wash for later analysis.

8. Elute the target His-tagged protein 6-10 times with 0.5 ml of non-denaturing elution buffer each
   time. Collect the eluates into
   different centrifuge tubes. The collected eluates are
   the purified His-tagged protein samples.

9. Mix the cell lysate supernatant with 5x SDS-PAGE loading buffer, boil at 100°C for 5-10 minutes,
   and centrifuge at
   12,000 r/min for 2 minutes. Perform 6% SDS-PAGE, ensuring equal protein loading. Stain the gel with Coomassie
   Brilliant Blue will visualise the protein bands.

![/private/var/folders/ts/1z_35h513_d55mv_zqgz8l400000gn/T/com.kingsoft.wpsoffice.mac/picturecompress_20240831153928/output_4.pngoutput_4](https://static.igem.wiki/teams/5093/experiment/4.jpg)

1.Thin Layer Chromatography (TLC)

1. Use sucrose as the substrate. In a buffer system containing 50 mM acetate-acetate sodium (pH 5.8)
   and 1 mM CaCl2, add 2%
   sucrose and an appropriate amount of recombinant
   enzyme. Conduct the reaction at 37°C to ensure the recombinant enzyme exhibits catalytic activity. The
   activity can be determined by analysing the reduction of sucrose and the formation of glucose, fructose,
   and oligosaccharides using thin-layer chromatography (TLC).

2. Analyze the hydrolysis products using Kieselgel 60 silica gel plates (Merck). The developing
   solvent is n-butanol acid
   (2:1:1, v/v/v). After centrifuging the samples at 10,000×g for 1 minute, spot 2 µL of the supernatant
   onto the silica gel plate. Develop the silica gel plate twice with the developing solvent; air dry it,
   then immerse it evenly in a colour development reagent composed of sulfuric acid (5:95, v/v). Finally,
   bake the plate at 130°C in an oven for 5 minutes to visualise the spots.

·Construction of Digestion Model
and Detection of
Sucrose Changes and Product Formation in Food Digestion Mixture

A
conical flask was used as the container. A food product rich in sucrose (or a mixture of a specific
amount of food and sucrose) was pulverised to prepare a simulated food suspension. Subsequently, a
particular amount of two recombinant enzyme solutions was added, and the mixture was incubated at 37°C
with shaking for 1 hour. The sucrose content and the amount of oligosaccharides produced in the reaction
products were quantitatively analysed using High-Performance Liquid Chromatography (HPLC).

|  |  |  |  |
| --- | --- | --- | --- |
| 2% Sucrose Treatment | | Trehalose Treatment | |
| Number | Sample Amount - Time | Number | Sample Amount - Time |
| 1 | 1-5 | 26 | 1-5 |
| 2 | 1-10 | 27 | 1-10 |
| 3 | 1-15 | 28 | 1-15 |
| 4 | 1-30 | 29 | 1-30 |
| 5 | 1-60 | 30 | 1-60 |
| 6 | 5-5 | 31 | 5-5 |
| 7 | 5-10 | 32 | 5-10 |
| 8 | 5-15 | 33 | 5-15 |
| 9 | 5-30 | 34 | 5-30 |
| 10 | 5-60 | 35 | 5-60 |
| 11 | 10-5 | 36 | 10-5 |
| 12 | 10-10 | 37 | 10-10 |
| 13 | 10-15 | 38 | 10-15 |
| 14 | 10-30 | 39 | 10-30 |
| 15 | 10-60 | 40 | 10-60 |
| 16 | 15-5 | 41 | 15-5 |
| 17 | 15-10 | 42 | 15-10 |
| 18 | 15-15 | 43 | 15-15 |
| 19 | 15-30 | 44 | 15-30 |
| 20 | 15-60 | 45 | 15-60 |
| 21 | 20-5 | 46 | 20-5 |
| 22 | 20-10 | 47 | 20-10 |
| 23 | 20-15 | 48 | 20-15 |
| 24 | 20-30 | 49 | 20-30 |
| 25 | 20-60 | 50 | 20-60 |

Chromatographic
Conditions:

Column: Waters
Sugar-Pak I column (6.5 × 300 mm)

Detector: Differential
Refractive Index (RI) detector

Detector Temperature:
40°C

Flow Rate: 0.5
mL·min⁻¹

Mobile Phase: 100%
deionised water

Injection Volume: 20
μL

Column Temperature:
80°C

![/private/var/folders/ts/1z_35h513_d55mv_zqgz8l400000gn/T/com.kingsoft.wpsoffice.mac/picturecompress_20240831153928/output_5.pngoutput_5](https://static.igem.wiki/teams/5093/experiment/5.jpg)

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

Results

[Overview](#section1)
[Construction of plasmids](#section2)
[Protein expression and purification](#section3)
[Functional verification of enzymes](#section4)
[Future plans](#section5)
[References](#section6)

Overview

In our project, we
developed a capsule containing two catalytic enzymesdextransucrase and
inulosucrase that are coded by genes
EC.2.4.1.5 and EC.2.4.1.9 to catalyse the
conversion from sucrose to two types of soluble dietary fibres
(SDFs) dextran and inulin respectively as shown in
Figure 1. Therefore, individuals may enjoy their diets without change in flavor while reducing the human
body's effective absorption of sugars in food and increasing fibre intake. With no change to the sugar
content of the food, this initiative intends to promote bowel movement, regulate gut bacteria, and lower
the quantity of sugar absorbed by the human body into the bloodstream.

![](https://static.igem.wiki/teams/5093/results/1.jpg)

Figure
1 The
overview flowchart of
our experiment

Construction of plasmids

We
designed 2
plasmids: pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9. The
DNA fragments EC.2.4.1.5 and EC.2.4.1.9 are amplified from the genome of Leuconostoc citreum[1]and
the
genome of Lactobacillus
johnsonii[2].

1. Identification and pre-modification of target genes

To construct our plasmids, we let
the company synthesise the
DNA fragments.To excise
two bands of interest (EC.2.4.1.5, EC.2.4.1.9), we used restriction endonuclease NdeⅠ and
XhoⅠ
to cutthe DNA fragments and the
pET28a vector.

Theoretically, the gene fragment
lengths for EC.2.4.1.5 and EC.2.4.1.9 are 4431bp and
2391bp, respectively.
After PCR
amplification, the TAE agarose gel electrophoresis results (Figure 2) proved the theoretical values, and
we could continue our experiment. After DNA extraction from the gel, T4 ligase was used to link the
vector and the target genes (EC.2.4.1.5 and EC.2.4.1.9) to produce the recombinant plasmid.

![](https://static.igem.wiki/teams/5093/results/2.png)

Figure
2.The identification of plasmid pET-28a, gene fragmentsEC.2.4.1.5 and EC.2.4.1.9 by restriction
enzymes digestion
and PCR amplification.

M: Maker;
EC.2.4.1.5:
Gene fragment EC.2.4.1.5 that digested
by Restriction
endonucleaseNdeⅠ and
XhoⅠ. EC.2.4.1.9: Gene fragment EC.2.4.1.9
that digested by
Restriction endonuclease NdeⅠ and XhoⅠ.

2. Plasmid profile

At the same time, Snapgene was used to create the profiles of the plasmids
pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9
so that parts of
the plasmids, including the target gene, restriction gene, and multiple cloning site, were
demonstrated
and understood.

![](https://static.igem.wiki/teams/5093/results/3.jpg)

Figure
3.
Plasmid profile
of pET28a-EC.2.4.1.5(left) and
pET28a-EC.2.4.1.9(right).

3. Plasmid transformation

In this part, we transformed the plasmids constructed
into
DH5α E.coli and replicated them. After extracting
and verifying the
plasmids, we transformed the plasmids into the bacteria again.

The
recombinant plasmid was transformedinto
E.coli (DH5α),
and we incubated them overnight at 37°C
after streak inoculating them on LB solid
medium plates that
included appropriate
antibiotics (LB-kana), as shown in
Figure 4. Afterward, we picked 3 individual colonies from each petri dish and
extracted their plasmids. A gel electrophoresis was run to confirm that the extracted plasmids were the
ones we required. As EC.2.4.1.5 and EC.2.4.1.9 have DNA fragment
lengths of 4431bp and 2391bp, our results show that we get the desired plasmids. The
DNA
sequencing diagram shows the desired lengths and sequencing for pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9
(Figure 5) indicates the plasmid construction of pET28a-EC.2.4.1.5 and pET28a-EC.2.4.1.9 are
successful.

![](https://static.igem.wiki/teams/5093/results/4.jpg)

Figure
4.
Incubate the
plasmids containing strains and gel
electrophoresis. A: Gel electrophoresis
was used to
confirm that the extracted plasmids were the ones we required. B:
pET28a-EC.2.4.1.5 containing strain. C:
pET28a-EC.2.4.1.9 containing
strain.

![](https://static.igem.wiki/teams/5093/results/5.jpg)

Figure
5.
The DNA sequencing
diagram for pET28a-EC.2.4.1.9

Protein expression and purification

1. Expression of protein

The recombinant
plasmid
was first transformed into E.coli BL21(DE3), incubated on LB solid medium plates (Kana+), and cultured at 37°C overnight. Four colonies of each
desired
plasmid were selected for PCR identification of transformants to confirm that the colonies have positive
results (shown in Figure 6) and then transferred into 1L
fresh LB
(Kana+) culture medium for the scale-up
cultivation. IPTG (0.2 mM) was used
to induce the
expression of genes EC.2.4.1.5 and EC.2.4.1.9
with OD600
around 0.6-0.8 and
cultured at 16℃ for 20h. The proteins were then extracted from the supernatant of the E.coli BL21(DE3) after ultrasonic cell disruption and
centrifugation.

![](https://static.igem.wiki/teams/5093/results/6.png)

Figure
6.DNA gel electrophoresis to verify the construction of
correct
recombinant plasmids fromE.coli BL21(DE3). A: DNA gel electrophoresis
verification of genes. B: E.coli BL21(DE3) that
contain EC.2.4.1.5 and EC.2.4.1.9

2. Protein purification

Nickel affinity chromatography is used to purify the
protein
sample we got for a clearer SDS-PAGE result with little interference by non-specifically bound
proteins. In Figure 7, the bands with sizes of167 kDa and 88 kDa for dextransucrase and inulosucrase demonstrate that the expected
proteins are expressed and
collected. The target proteins (dextransucrase and
inulosucrase) are successfully expressed and confirmed by the target bands
shown in the supernatant and flowthrough part. In the wash part, some target proteins are washed away
with other non-specific proteins, and in the elution part, a single and clear band illustrating the
target protein with the expected size for both dextransucrase and inulosucrase, which have sizes of
167 kDa and 88 kDa.

![](https://static.igem.wiki/teams/5093/results/7.jpg)

Figure 7 Results
of
SDS-PAGE of supernatant, flowthrough,
wash, and elution parts for the protein purification of dextransucrase and inulosucrase extracted
from EC.2.4.1.5 and EC.2.4.1.9 in E.coli
BL21(DE3)

Functional verification of enzymes

Both enzymes convert sucrose to 50% monosaccharides
and 50%
SDFs, while a lower proportion of monosaccharides
could be achieved by combining the two enzymes in an ideal
ratio. Thin-layer chromatography andhigh-performance liquid
chromatography (HPLC) were carried out to find the optimum ratio and
conditions.

1. Thin-layer chromatography

In a standard 2%
sucrose
solution, a certain amount of
tworecombinant enzyme
solutions with the ratio of 1:1, 1:2, and 2:1
between concentrations of dextransucrase and
inulosucrase was added, and the reaction was shaken at 37°C for 1h. After the reaction, thin-layer chromatography
detected
the sucrose changes in the reaction solution and the glucose content generated qualitatively. Figure 8
shows that the group with the least sucrose concentration undigested has a ratio of 2:1 between
dextransucrase and
inulosucrase.

![](https://static.igem.wiki/teams/5093/results/8.jpg)

Figure 8.Thin-layer
chromatography is used to identify the efficiency of different recombinant enzyme
solutions. 1: The recombinant enzyme
solution with a ratio of 1:1
between dextransucrase and inulosucrase. 2: The recombinant enzyme solution with a ratio of 1:2
between dextransucrase and inulosucrase. 3: The recombinant enzyme solution with a ratio of 2:1
between dextransucrase and inulosucrase.

2. High-performance liquid chromatography

In addition,
sugarcane
juice was used as a type of simulated food suspension to test enzyme function in real conditions. The
experiment took place under 37℃ to
simulate human body temperature. A 1:1 ratio of concentration between dextransucrase and inulosucraseis used with masses of 1mg,
5mg, 10mg, 15mg, and 20 mg of each
type of enzyme per 100 g sugarcane juice for 5 minutes, 10 mins, 15 mins, 30 mins, and 60 mins. Once the
reaction time is reached, the solution is immediately heated to 65℃ to
denature the enzymes. Afterward, HPLC is carried out to analyse the amount of sucrose and glucose
contained in the solution, the raw data are listed below (Table 1). We
used Origin Pro to
process data, forming 3D mappings aboutglucose and sucrose
concentrations in
various conditions (Figure 8). By analysing
the raw data and the 3D mapping, we could conclude that in general, with a longer reaction time,
catalysing tends to become more complete, which means higher glucose concentration while lower sucrose
concentration. In speaking of
each enzyme's sample size, except for the extremely high remaining sucrose with 1mg of
dextransucrase and inulosucrase, it is difficult to summarise the overall effects of the change in
glucose and sucrose concentrations. A low sucrose concentration (0g/100g) was left in the solution
when 20 mg of each enzyme was left in the solution and reacted for 60 mins, but a higher glucose
concentration was formed (2.9g/100g).

Table 1 The amount
of
sucrose and glucose contained in 100 g sugarcane solution with the existence of dextransucrase and
inulosucrase at 37℃.

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Sugarcane juice (100g) | | | Sugarcane juice (100g) | | |
| Sample size of each enzyme (mg) | Reaction time (min) | Sucrose remained (g) | Sample size of each enzyme (mg) | Reaction time (min) | Glucose produced (g) |
| 1 | 5 | 0.76 | 1 | 5 | 2.4 |
| 10 | 0.74 | 10 | 2.5 |
| 15 | 0.72 | 15 | 2.8 |
| 30 | 0.71 | 30 | 2.8 |
| 60 | 0.24 | 60 | 3.4 |
| 5 | 5 | 0.53 | 5 | 5 | 2.5 |
| 10 | 0.43 | 10 | 2.6 |
| 15 | 0.41 | 15 | 2.4 |
| 30 | 0.42 | 30 | 2.9 |
| 60 | 0.41 | 60 | 3.9 |
| 10 | 5 | 0.5 | 10 | 5 | 2.7 |
| 10 | 0.42 | 10 | 2.7 |
| 15 | 0.088 | 15 | 2.6 |
| 30 | 0.47 | 30 | 2.7 |
| 60 | 0.43 | 60 | 2.7 |
| 15 | 5 | 0.39 | 15 | 5 | 2.3 |
| 10 | 0.4 | 10 | 2.5 |
| 15 | 0.43 | 15 | 2.5 |
| 30 | 0.43 | 30 | 2.7 |
| 60 | 0.38 | 60 | 2.7 |
| 20 | 5 | 0.38 | 20 | 5 | 2.6 |
| 10 | 0.44 | 10 | 2.4 |
| 15 | 0.41 | 15 | 2.6 |
| 30 | 0.35 | 30 | 2.6 |
| 60 | 0 | 60 | 2.9 |

However, when the
sample
size is 10 mg for each enzyme, noticeably low remaining sucrose (0.088g/100g) and relatively low glucose
production (2.6g/100g) are observed when the reaction time is 15 mins. In conclusion, we determined that
allowing 10 mg of each enzyme to react in sugarcane juice for 15 mins is ideal to guarantee little
sucrose sustain and glucose synthesis at 37°C.

![](https://static.igem.wiki/teams/5093/results/9.jpg)

Figure
9 3D
mapping of
glucose(right) and sucrose(left) concentrations in various conditions under 37°C.

Future plans

Subsequently, we may
start the construction of the capsule to protect two protein-based enzymes from denaturation by the low
pH of gastric juice by adopting the microencapsulation technique, in which enzymes are coated in small
spheres. The capsule made of calcium chloride and sodium alginate provides high strength, elasticity,
and thermal stability to resist pepsin and hydrochloric acid in the stomach [3]. Moreover, the effect of
recombinant enzymes in the actual environment of the gastrointestinal tract should also be tested in the
future with distinct approaches, including the in vitro method, animal experiments,
and
human clinical
trials.

References

1. ‘dextransucrase [Leuconostoc citreum] - Protein - NCBI’. Accessed: Jul. 26, 2024.
   [Online].
2. ‘glycoside hydrolase family 68 protein [Lactobacillus johnsonii] - Protein - NCBI’.
   Accessed: Jul. 26, 2024. [Online]
3. YANG B. et al., ‘Preparation and
   properties of lipase microcapsules,’ China Dairy
   Industry, vol. 51, no. 9, pp. 10-14+64, 2023.

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