iGEM 2025 — Team subcat-hs

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}This serves as a chronological record of your team's progress throughout the season. It documents your
daily activities, experiments, discussions, and decisions.{% endblock %}
{% block extra\_css %}

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

# NOTEBOOK

![导航图片](https://static.igem.wiki/teams/5735/img/desc/p2-left-up-image.webp)

content

![圆点1](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot2.webp)
![圆点2](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot2.webp)

[Main Timeline](#main-timeline)
[Safety training is required before
the experiment begins](#safety-training)

### Main Timeline

**January：**

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |  |
| Theoretical knowledge learning and literature review |  |  |  |  |  |  |  |  |  |  |

**February：**

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Recruiting of new members |  |  |  |  |  |  |  |  |  |
| literature review |  |  |  |  |  |  |  |  |  |
| Project design |  |  |  |  |  |  |  |  |  |

**March：**

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Project design |  |  |  |  |  |  |  |  |  |  |
| Safety training |  |  |  |  |  |  |  |  |  |  |

**April：**

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Knowledge learning of synthetic biology |  |  |  |  |  |  |  |  |  |  |
| Writing of project plan |  |  |  |  |  |  |  |  |  |  |

**May：**

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Writing of project plan |  |  |  |  |  |  |  |  |  |  |
| Experimental operation and laboratory safety learning |  |  |  |  |  |  |  |  |  |  |

**June：**

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Enter the laboratory and start the experiment |  |  |  |  |  |  |  |  |  |

**July：**

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Synthetic biology experiment |  |  |  |  |  |  |  |  |  |  |
| Experimental data analysis |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |
| Expert Interview Guide |  |  |  |  |  |  |  |  |  |  |

**August：**

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Discussion and solution of experimental problems |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |

**September：**

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Data collation and analysis |  |  |  |  |  |  |  |  |  |  |
| Building the Team Wiki |  |  |  |  |  |  |  |  |  |  |
| Education |  |  |  |  |  |  |  |  |  |  |

**October：**

|  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Project arrangement and preparation |  |  |  |  |  |  |  |  |  |  |
| Drawing of experimental results |  |  |  |  |  |  |  |  |  |  |

### Safety training is required before the experiment begins

**Stage 1 experiment**
- Prepare LB for culturing competent cells
- Extract plasmid for target gene
- PCR extracted plasmid

**1. LB preparation**
- Tryptone 1 g
- Yeast extract 0.5 g
- NaCl 1 g
- Water 100 mL
Transfer to 2 conical flask
Add 1.5 g Agar in to one flask
Stored at 4 °C

**2. Plasmid extraction**
- UreB
- Nb-human
- Nb-6
- Nb-SCFV
- Centrifuge
- 1.5 ml centrifuge tube \*4
- Buffer S
- SP1
- SP2
- SP3
- DW1
- Wash solution
- Elution buffer

**3. PCR**
- PrimeSTAR Max Premix 25 μL
- Primer 1 2.5 μL
- Primer 2 2.5 μL
- Tamplate 1 μL
- Water 19 μL

**Stage 2 experiment**
- Digestion of plasmid and target DNA
- Ligation of plasmid and target DNA
- Agarose gel electrophoresis
- Transfer to competent cells

**1. Digestion**
- 10x restriction buffer 5 μL
- HindIII 1 μL
- NheI 1 μL
- Plasmid DNA and the PCR result 1 μL
- Water 40 μL

**2. Agarose gel electrophoresis**
- TAE buffer 100 mL
- agarose 1.2 g
- Samples(plasmid,PCR production) 20 μL
- Electrophoresis tank
- Marker 20 μL
Run for 30 min at 120V

**3. DNA gel extraction**
- Target band(1.7kb and5.4kb)
- Buffer B2
- Wash solution 500 μL
- Elution buffer 20 μL
Stored at -20 °C

**4. Ligation**
- 10× buffer and T4 DNA ligase 10 μL
- Insert (1710 bp) and purified vector (5369 bp) 8 μL
The vector:Insert=1:3
Incubate for 2 h at the room temperature

**5. Transfer to competent cells**
- DH5α(cloning) 50 μL
- BL21(express) 50 μL
- ECN1917(Probiotics) 50 μL
- Ligation production 2μL each
- LB agar 900 μL
- Kanamycin 100 μL

**Result:**

![Figure 1](https://static.igem.wiki/teams/5735/img/notebook/1.webp)

**Figure 1**

- Gel electrophoresis results showed bands at 1710 bp and 5369 bp after comparison with the marker, but
the
colours were faint, that means some plasmids and PCR products were successfully digested.

![Figure 2](https://static.igem.wiki/teams/5735/img/notebook/2.webp)

**Figure 2**

- The fact that competent cells can grow successfully on medium containing kanamycin indicates that the
constructed plasmid has been transferred into the cells, but further confirmation is needed to determine
whether the plasmid can be expressed.

**Stage 3 experiment**
- Monoclonal colony identification and selection
- Colony PCR
- Agarose gel electrophoresis
- Expand BL21 for expression
- IPTG-induced expression

**1. Colony PCR**
- 2× Taq Premix 10 μL
- Primer 1 μL
- Primer 2 1 μL
- Colony
- Water 7 μL
Cycle of heat

**2. Colony PCR verification**
- Agarose 1.2 g
- Water 100 mL

**3. Expansion of BL21**
- LB 100 mL
- Kanamycin 100 μL
Shake for 1 h at 37 °C

**4. Protein expression**
- IPTG 100 μL
- BL21
- LB 100 mL
- Kanamycin 100 μL
Shake for 1 night

**Result**
- The results of gel electrophoresis were not ideal. All bands were at the bottom. We speculated that
self-ligation may have occurred, or that the target gene was not inserted, or that some error occurred
during
the PCR process. However, after comparing our results with those of another group's repeated experiments,
we
speculated that the PCR failure may have been caused by enzyme denaturation.

![Figure 3](https://static.igem.wiki/teams/5735/img/notebook/3.webp)

**Figure 3**

We changed the experimental materials and conducted the experiment again. After optimizing the
experimental
scheme, we conducted the experiment again, and finally we got the correct PCR product.

![Figure 4](https://static.igem.wiki/teams/5735/img/notebook/4.webp)

**Figure 4**

Then, in order to obtain the protein we need, we added IPTG to the culture medium for protein.

![Figure 5](https://static.igem.wiki/teams/5735/img/notebook/5.webp)

**Figure 5**

**Stage 4 experiment**
- Ultrasonic disruption and protein extraction
- SDS-PAGE to verify protein

**1. Protein extraction**
- expanded BL21
- Centrifuge
- Ultrasonic crusher
- Lysozyme 50 μL
- BeyoGold His-tag 1000 mL
Repeat twice
- Lysis buffer 500 μL
- Elution buffer 500 μL
- Chromatography column

**2. SDS-PAGE**
- Agarose gel electrophoresis
- Lysis buffer 15 μL
- Marker(10-180KDa)
- Flow through
Wait 40 min under 120V
- Coomassie Brilliant Blue
Shake until protein stained

**Result:** The eluent is clearer than the flow-through liquid, indicating that impurity
proteins
have been filtered out.

![Figure 6](https://static.igem.wiki/teams/5735/img/notebook/6.webp)

**Figure 6**

**Stage 5 experiment**
- BCA protein concentration determination
- Western Blot

**1. BCA**
- BSA
- BCA regent A
- BCA regent B
Regent A:Regent B=50:1
- Extracted protein 20 μL each
Add lysis buffer until 20 μL
- Protein standard preparation solution 200 μL
- ELISA reader
Incubate at 37 °C for 30 min

**2. Western Blot**
- SDS-gel
- Splint
- Filter paper for transfer 2 sheets
- PDFV
- Methanol
- Sponge
- Transfer solution
- Electrospining tank
Add ice,powered on 150 mA for 80 min
- Blocking solution(5% BSA and BPST)
Add blocking solution in the PDFV for 30 min
- Mouse anti-his antibody
Wash extra antibody 3 times,10min per time
- Rabbit Anti-Mouse IgG H&L HRP secondary antibody
Incubating 1h at 4°C

**Result**

![Figure 7](https://static.igem.wiki/teams/5735/img/notebook/7.webp)

**Figure 7**

- We can estimate the relationship between protein concentration and absorbance based on the standard
protein
concentration. Then, we can calculate the protein concentration for each group of proteins using their
absorbance values. The results are shown in the table above.

![Figure 8](https://static.igem.wiki/teams/5735/img/notebook/8.webp)

**Figure 8**

![Figure 9](https://static.igem.wiki/teams/5735/img/notebook/9.webp)

**Figure 9**

![Figure 10](https://static.igem.wiki/teams/5735/img/notebook/10.webp)

**Figure 10**

![Figure 11](https://static.igem.wiki/teams/5735/img/notebook/11.webp)

**Figure 11**

- The sizes of each protein:human：34.1kDa UreB: 59.5kDa nb-6 27.5kDa scfv： 44.8KDa
Each group has bands at the corresponding protein size positions, indicating that the proteins in each
group
are expressed. There are many bands in the eluent, indicating that there are many impurity proteins in the
eluent. However, the protein bands in the first eluent are basically only close to the corresponding
protein
size, indicating that the proteins in the first eluent are relatively pure and that the target protein is
present, but the specificity needs to be further verified.

**Stage 6 experiment**
- immunodiffusion test
- Determination of the inhibition rate of Hp Urease activity by recombinant nanobodies

**1. Immunodiffusion test**
- Agarose 1 g
- Water 50 mL
- UreB 1 mL
- Nb-human,Nb-6,Nb-SCFV 100 μL each
- Culture medium
- Oxford cup
-CK 100 μL
Incubate overnight at 37 °C

**2. Determination of the inhibition rate of urease activity by recombinant nanobodies**
- UreB 100 μg/mL
- Nb-6,Nb-human,Nb-SCFV
Incubate overnight at 4 °C
- Lysis buffer
- Urea solution 50 μL
- 96-well plate
- Phenol red 50 μL
Incubate at 25 °C for 6 h

![vertical line](https://static.igem.wiki/teams/5735/img/desc/p2-right-vertical-line.webp)
![返回顶部](https://static.igem.wiki/teams/5735/img/desc/p2-right-mini.webp)

{% 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.{% endblock %}
{% block extra\_css %}

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

# ENGINEERING

![导航图片](https://static.igem.wiki/teams/5735/img/desc/p2-left-up-image.webp)

content

![圆点1](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点2](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点3](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点4](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)

[Overview](#overview)
[Cycle 1: Construction of
pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB](#cycle1)
[Cycle 2: Construction and verification of
pGEX4T-1-Nb-human](#cycle2)
[Reference](#reference)

### Overview

*Helicobacter pylori* (*H. pylori*) represent one of the most widespread bacterial
infections
globally. Current evidence indicates that nearly half of the global population is chronically infected
with
*H. pylori* [1, 2] and current clinical management relies primarily on antibiotic therapy.
Nevertheless, the escalating antibiotic resistance resulting from overuse has led to an increasing rate of
treatment failure. Urease is a critical factor for the colonization and pathogenicity of *H.
pylori*
in the stomach and *H. pylori* possesses one of the highest levels of urease content and enzymatic
activity among all known bacteria[3]. Urease is formed as a heterodimer composed of two distinct subunits,
UreA and UreB, with the UreB subunit containing the catalytic active site of *H. pylori*[4]. UreB,
as
a key subunit of urease, facilitates the efficient incorporation of Ni²⁺ into the enzyme's active site and
stabilizes the site against metal chelation[5]. In this study, we constructed a genetically engineered
expression system for a recombinant nanobody specifically targeting the urease subunit (UreB) of *H.
pylori*. However, due to the limitation of biological safety, we can't use Ureb of *Helicobacter
pylori* for experiments, so we use Ureb of Bacillus subtilis instead in this experiment. First, the
recombinant nanobody was produced using an *Escherichia coli* expression system. The binding effect
of our antibody to Bacillus subtilis was explored through experiments, and then the binding effect of
antibody to *Helicobacter pylori* Ureb was simulated by computer. Finally, we investigated the
expression of the recombinant nanobody in food-grade probiotic bacteria, aiming to utilize food-grade
probiotics as delivery vehicles to administer the antibody in the form of live bacteria.
      **Our design consists of two main cycles:**
      **Cycle 1: Construction of pET28a-Sumo-Nb-human([BBa\_25BHCZON](https://registry.igem.org/parts/bba-25bhczon)),
pET28a-Sumo-Nb-SCFV([BBa\_25IL2D7N](https://registry.igem.org/parts/bba-25il2d7n)), pET28a-Sumo-UreB-Nb6([BBa\_25O8SZ84](https://registry.igem.org/parts/bba-25o8sz84)), pET28a-UreB([BBa\_25WB0RT4](https://registry.igem.org/parts/bba-25wb0rt4))**
      **Cycle 2: Construction and verification of
pGEX4T-1-Nb-human****([BBa\_25D33Y5Sz](https://registry.igem.org/parts/bba-25d33y5s))**
![Figure 1. Overall experimental design concept](https://static.igem.wiki/teams/5735/engineering/figure-1.webp)
**Figure 1. Overall experimental design concept**

### Cycle 1: Construction of pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB

**Design:**
Based on literatures, the three genes (Nb-human, UreB-Nb6, and Nb-scFv) targeting the urease subunit B
(UreB) of *H. pylori* were chosen to be constructed the recombined nano-antibody. This program
employed the pET28a expression system to construct four optimized plasmids: pET28a-SUMO-Nb-human,
pET28a-SUMO-Nb-scFv, pET28a-SUMO-UreB-Nb6, and pET28a-UreB, aiming to aquire the recombinant protein and
urease unit UreB. In the plasmid design, all target genes were fused with a SUMO tag (6×His tag) to
enhance
protein solubility and expression efficiency. Additionally, a 6×His tag on both the N-terminal and
C-terminal can be used to facilitate subsequent protein purification via nickel-affinity chromatography.
The
LacI gene on the plasmid provides an inducible and controllable switch for gene expression through a
dynamic
regulatory mechanism of "repression-release". We can use IPTG to induce the expression of the protein.
Furthermore, a signal peptide sequence (MKYLLPTAAAGLLLLAAQPA) was integrated into the vector to optimize
the
production yield of antibody proteins(Nb-human, UreB-Nb6, and Nb-scFv) by regulating their secretion
pathway, thereby improving their therapeutic potential. This plasmid system fully leverages the strong T7
promoter and prokaryotic expression advantages of the pET28a vector, while combining the synergistic
effects
of the SUMO tag, His tag, and signal peptide, thereby establishing a robust foundation for the efficient
expression and study of nanobodies (Figure 2).
![Figure 2. The design of pET28a-SUMO-Nb-human, pET28a-SUMO-Nb-scFv, pET28a-SUMO-UreB-Nb6, and pET28a-UreB](https://static.igem.wiki/teams/5735/engineering/figure-2.webp)
**Figure 2. The design of pET28a-SUMO-Nb-human,
pET28a-SUMO-Nb-scFv, pET28a-SUMO-UreB-Nb6, and pET28a-UreB**
**Build:**
![Figure 3. The overview of the process of the plasmid construction](https://static.igem.wiki/teams/5735/engineering/figure-3.webp)
**Figure 3. The overview of the process of the plasmid
construction**
**Acquisition of target gene fragment**
      Based on the literature, we choose the genes (Nb-human, UreB-Nb6, and
Nb-scFv) as the target genes. The target gene sequence was synthesized by the biotech company. The
amplified
target fragment using the synthesized target gene fragment as the template and the vector (pET28a-1) were
identified by agarose gel electrophoresis after enzymatic digestion by NheI and HindⅢ, respectively. The
Gene fragment lengths of Sumo-Nb-SCFV, Sumo-UreB-Nb6, Sumo-Nb-human and UreB were about 807bp, 655bp,
936bp,375bp, respectively (Figure 4A). Electrophoresis results showed that the band size was consistent
with
the expected size. The size of plasmid skeleton after digestion (pET28a-Sumo-Nb-human,
pET28a-Sumo-Nb-SCFV,
pET28a-Sumo-UreB-Nb6, pET28a-UreB) was consistent with the expectation (Figure 4B). It indicates that the
target gene has been successfully amplified. Subsequently, we used T4 DNA ligase to link the linearized
plasmids to the target gene fragment.
![Figure 4. Agarose gel electrophoresis of the target gene fragment and Skeleton](https://static.igem.wiki/teams/5735/engineering/figure-4.webp)
**Figure 4. Agarose gel electrophoresis of the target
gene
fragment and Skeleton. A.Target gene. B.Skeleton.**
**Construction of pET28a-Sumo-Nb-human([BBa\_25BHCZON](https://registry.igem.org/parts/bba-25bhczon))**
      Through antibiotic screening, colony PCR, and DNA sequencing
verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-human in *E.Coil* DH5α and
*E.Coil* BL21). Figure 5A depict the growth of transformed colonies on Kanamycin-resistant plates
post-transformation. Bacteria growing on the plate are the strains that may be successfully constructed.
The
colonies selected from the plate served as PCR templates for colony PCR. Agarose gel electrophoresis was
used to verify the correctness of PCR products and the results show that the range of the target size is
consistent with the expectation (Figure 5B). Subsequently, the target-sized amplicons were sent for
sequencing. According to the sequence results shown in Figure 5C, the target gene was successfully ligated
with the vector without obvious mutations, confirming the successful construction of the
pET28a-Sumo-Nb-human plasmid.
![Figure 5. The validation and sequencing of the plasmid pET28a-Sumo-Nb-human](https://static.igem.wiki/teams/5735/engineering/figure-5.webp)
**Figure 5. The validation and sequencing of the plasmid
pET28a-Sumo-Nb-human
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis map; C. Sequencing comparison
results.**
**Construction of pET28a-Sumo-Nb-SCFV([BBa\_25IL2D7N](https://registry.igem.org/parts/bba-25il2d7n))**
      Through antibiotic screening, colony PCR, and DNA sequencing
verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-SCFV in *E.Coil* DH5α and
*E.Coil* BL21). Figure 6A depicts the growth of transformed colonies on Kanamycin-resistant plates
post-transformation. Bacteria growing on the plate are the strains that may be successfully constructed.
The
colonies selected from the plate served as PCR templates for colony PCR. Agarose gel electrophoresis was
used to verify the correctness of PCR products and the results show that the range of the target size is
consistent with the expectation (Figure 6B). Subsequently, the target-sized amplicons were sent for
sequencing. According to the sequence results shown in Figure 6C, the target gene was successfully ligated
with the vector without obvious mutations, confirming the successful construction of the
pET28a-Sumo-Nb-SCFV
plasmid.
![Figure 6. The validation and sequencing of the plasmid pET28a-Sumo-Nb-SCFV](https://static.igem.wiki/teams/5735/engineering/figure-6.webp)
**Figure 6. The validation and sequencing of the plasmid
pET28a-Sumo-Nb-SCFV
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis map; C. Sequencing comparison
results.**
**Construction of pET28a-Sumo-Nb-6([BBa\_25O8SZ84](https://registry.igem.org/parts/bba-25o8sz84))**
      Through antibiotic screening, colony PCR, and DNA sequencing
verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-6 in *E.Coil* DH5α and
*E.Coil* BL21). Figure 7A depicts the growth of transformed colonies on Kanamycin-resistant plates
post-transformation. Bacteria growing on the plate are the strains that may be successfully constructed.
The
colonies selected from the plate served as PCR templates for colony PCR. Agarose gel electrophoresis was
used to verify the correctness of PCR products, and the results show that the range of the target size is
consistent with the expectation (Figure 7B). Subsequently, the target-sized amplicons were sent for
sequencing. According to the sequence results shown in the Figure 7C, the target gene was successfully
ligated with the vector without obvious mutations, confirming the successful construction of the
pET28a-Sumo-Nb-6 plasmid.
![Figure 7. The validation and sequencing of the plasmid pET28a-Sumo -Nb6](https://static.igem.wiki/teams/5735/engineering/figure-7.webp)
**Figure 7. The validation and sequencing of the plasmid
pET28a-Sumo -Nb6
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis map; C. Sequencing comparison
results.**
**Construction of pET28a-Sumo-UreB([BBa\_25WB0RT4](https://registry.igem.org/parts/bba-25wb0rt4))**
      Through antibiotic screening and DNA sequencing verification, we
verified the recombinant plasmids (pET28a-Sumo-UreB in *E.Coil* DH5α and *E.Coil* BL21).
Figure 8A depicts the growth of transformed colonies on Kanamycin-resistant plates post-transformation.
Bacteria growing on the plate are the strains that may be successfully constructed. Subsequently, We
selected Single clony for sequencing. According to the sequence results shown in Figure 8B, the target
gene
was successfully ligated with the vector without obvious mutations, confirming the successful construction
of the pET28a-Sumo-UreB.
![Figure 8. The validation and sequencing of the plasmid pET28a-Sumo-UreB](https://static.igem.wiki/teams/5735/engineering/figure-8.webp)
**Figure 8. The validation and sequencing of the plasmid
pET28a-Sumo-UreB.
A. Cloned strains on resistant plates; B. Sequencing comparison results.**
**Test:**
![Figure 9. The specific process of the test](https://static.igem.wiki/teams/5735/engineering/figure-9.webp)
**Figure 9. The specific process of the
test**
**Protein expression and validation**
      **SDS-PAGE was used to verify the protein expression
level**
      After confirming that the strain was constructed correctly, we used
IPTG
as inducer to induce the expression of the target protein in the next step. After protein induction, we
broke the cell by physical and chemical methods, then extracted、separated and purified the protein inside
the cell.
      Finally, we verified whether the target protein was successfully
expressed by SDS-PAGE. The molecular weights of the corresponding target proteins (Nb-6, Nb-human Nb-scfv,
UreB) are 27.5 kDa, 34.1 kDa, 44.8 kDa and 14 kDa, respectively. After coomassie brilliant blue staining,
we
found that the results of SDS-PAGE were completely consistent with expectations. All four target
proteins(Nb-6(Figure 10A), Nb-human (Figure 10B), Nb-scfv(Figure 10C), UreB(Figure 10D)) were successfully
induced to express.
![Figure 10. SDS-PAGE was used to verify the protein expression](https://static.igem.wiki/teams/5735/engineering/figure-10.webp)
**Figure 10. SDS-PAGE was used to verify the protein
expression. A. The SDS-PAGE result of Nb-6;B. The SDS-PAGE result of Nb-human; C. The SDS-PAGE result of
Nb-scfv; D. The SDS-PAGE result of UreB.**
**Protein concentration was determined by BCA method**
      Using the BCA (Bicinchoninic Acid Assay) method, we determined the
concentration of the purified protein mentioned above. We applied the microplate reader to measure the
absorbance at a wavelength of 562 nm during this process and calculated the protein concentration of the
sample based on the standard curve. (Figure 11)
![Figure 11. The BCA result of the target proteins](https://static.igem.wiki/teams/5735/engineering/figure-11.webp)
**Figure 11. The BCA result of the target
proteins.**
      By substituting the measured absorbance values of the samples (y)
using
this fitting equation, the concentrations of each protein (x) were calculated as follows:

![Table 1. The concentration of the target protein](https://static.igem.wiki/teams/5735/engineering/table-1.webp)
**Table 1. The concentration of the target
protein**
**Western blot was used to verify whether the protein was expressed**
      This SDS-PAGE result shows the protein which has the same weight as
the
target protein, but we still can't be sure the corresponding strip is the target protein. Our target
protein
has his tag; therefore, we conducted western blot experiments using anti-his antibodies to further verify
to
confirm. Through western blot we found that the bands were indeed the target proteins and not any other
non-specific expression product (Figure 12). Experiments have proved that our antibody protein has been
successfully expressed. In this experiment, in order to improve the solubility of Ureb protein, we
introduced
several of his tags into Ureb protein, so we did not verify the protein expression of Ureb by WB. However,
based on the previous SDS-PAGE experiment, we basically confirmed that the Ureb protein was successfully
expressed.
![Figure 12. The Western blot result of Nb-human, UreB-Nb6, Nb-SCFV](https://static.igem.wiki/teams/5735/engineering/figure-12.webp)
**Figure 12. The Western blot result of Nb-human,
UreB-Nb6,
Nb-SCFV.**
**Immunodiffusion assay of three recombinant nanobodies**
      We used the Oxford cup diffusion experiment to qualitatively evaluate
the
binding specificity of the expressed antibody to the homologous antigen. As shown in Figure.13 (A, B, C),
after 36 hours of incubation, a slight white precipitation ring (protein stability may be impaired) was
formed
around the well plates loaded with three antibody preparations (Nb-6, Nb-SCFV, Nb-human), indicating that
there was an interaction between urease and antibody. However, it can be found that the range of our
precipitation ring is very narrow, which also means that the binding effect of our antibody against
Bacillus
subtilis Ureb is not very significant. This may be because the antibody we designed is specific to Ureb of
Helicobacter pylori, and the homology of Ureb protein between Helicobacter pylori and Bacillus subtilis is
not
particularly high, so our antibody may not have a particularly good binding effect on Ureb of Bacillus
subtilis.
![Figure 13. Oxford Cup test results](https://static.igem.wiki/teams/5735/engineering/figure-13.webp)
**Figure 13. Oxford Cup test results. A: Nb-6; B:
Nb-SCFV;C:
Nb-human.
(The number means control and different concentration of the recombinant nanobody：1：Blank 2：Negative
control
3：25μg/ml antibody 4：200μg/mL antibody)**
**Nanobody-mediated urease inhibition assay of three recombinant nanobodies**
      We assessed the inhibitory potency of recombinant nanobodies against
urease. After incubation, urea and phenol red were added, and the extent of urease inhibition was
quantified
colorimetrically.
      The experimental results show that the rate at which urea is
decomposed
by
urease in the experimental group with antibody is almost the same as that in the control group. This shows
that our antibody binding effect on Ureb of Bacillus subtilis is poor. Consistent with the above analysis,
our
antibody is specific to Helicobacter pylori, so its inhibitory effect on Bacillus subtilis is not good.
This
means that if our antibody is used to target Ureb of Helicobacter pylori, it may have a better
experimental
effect. Unfortunately, due to the limitation of biological safety, we can only simulate the binding of our
antibodies to Helicobacter pylori by computer.
![Figure 14. Colorimetric changes and quantitative analysis of urease-recombinant nanobody interaction](https://static.igem.wiki/teams/5735/engineering/engineering-14.webp)
**Figure 14. Colorimetric changes and quantitative
analysis
of urease-recombinant nanobody interaction**
**The mimic antibody binds to Helicobacter pylori Ureb by molecular docking**
      In this experiment, the model is a very important part of our
experiment.
Due to the consideration of biological safety, we can't use Ureb of Helicobacter pylori in practical
experiments, so we tested the binding effect of nano-antibody and Ureb of Helicobacter pylori by molecular
docking. Through computer simulation, we deeply analyzed the potential inhibitory effect of our
nano-antibody
on Helicobacter pylori and established a good model foundation for subsequent experiments.
      The 3D position of Ureb protein after interaction with three
antibodies
is
shown in the figure. Among them, the green protein is UreB protein, the blue protein is Nb6 protein
(Figure15A), the pink protein is Nb-Human protein (Figure15B) and the purple protein is Nb-ScFv protein
(Figure15C), and the bonding situation among the residues has been marked in the figure, in which the red
dotted line represents hydrogen bond interaction, the yellow dotted line represents salt bridge
interaction,
and the number near the dotted line of the interaction bond is the bond length of the interaction bond in
Amy.
Through the comprehensive analysis of these interactions, we can screen out the nano-antibody protein with
the
strongest binding ability.
![Figure 15 Interaction between Ureb protein and nano-antibody protein](https://static.igem.wiki/teams/5735/engineering/figure-15.webp)
**Figure 15 Interaction between Ureb protein and
nano-antibody protein. A. Schematic diagram of the interaction between UreB protein and Nb-6 protein, in
which UreB protein is shown in green and NB6 protein is shown in blue. B. Schematic diagram of
interaction
between UreB protein and Nb-Human protein, in which UreB protein is shown in green and NB-human protein
is
shown in pink. C. Schematic diagram of interaction between UreB protein and Nb-ScFv protein, in which
UreB
protein is shown in green and Nb-ScFv protein is shown in purple.**
      Using the Interface analyzer module of FoldX, the binding energies of
UreB
protein with three proteins are shown in the following table(Table 2), among which UreB protein has a
strong
binding tendency with Nb-SvFc protein and Nb-Human protein (protein-protein binding energy is less than
-25
kcal/mol, which means that there is a strong binding tendency). The interaction of three pairs of proteins
can
be further observed in combination with subsequent molecular dynamics simulation.
**Table 2 The binding energies of UreB protein with
three
proteins**
![Table 2 The binding energies of UreB protein with three proteins](https://static.igem.wiki/teams/5735/engineering/table-2.webp)
**The mimic antibody binds to Helicobacter pylori UreB by molecule dynamics**
      RMSD curve shows the changes of roots mean square displacement of
three
proteins relative to UreB protein in the simulation process and is used to evaluate the stability of
binding
between proteins in this study. From the graph analysis, it can be seen that the RMSD curve of Nb6 protein
relative to UreB protein shows a certain fluctuation trend in the first 80ns of the simulation. After 80
ns,
the RMSD curve gradually converges and vibrates around 0.8nm until the end of the simulation. The RMSD
curve
of Nb-ScFv protein relative to UreB protein experienced a certain fluctuation trend in the first 60ns of
the
simulation, and gradually converged after 60ns, vibrating around 0.5nm until the end of the simulation.
The
RMSD curve of Nb-Human protein relative to UreB protein showed a rapid increase in the first 20ns of the
simulation, and then gradually converged, vibrating around 1.2nm until the end of the simulation.
![Figure 16 RMSD Curve of Ureb and Three Proteins in Simulated Docking Process](https://static.igem.wiki/teams/5735/engineering/figure-16.webp)
**Figure 16 RMSD Curve of Ureb and Three Proteins in
Simulated Docking Process.**
      Nb-6 protein is composed of more flexible random curls at the
interaction
interface in the first 80ns of simulation, and the interface is pulled by UreB protein in the process of
dynamic simulation, so the protein interaction interface maintains a certain conformational adjustment in
the
first 80 ns of simulation, which leads to a certain fluctuation trend of RMSD curve. After 80 ns, with the
interaction between proteins gradually stabilized, and there was no obvious conformational change again,
the
skeleton structure of the two proteins gradually stabilized, only maintaining a certain change in the
mutual
orientation of side chains. Therefore, after 80 ns, the RMSD curve gradually converged and vibrated around
0.8
nm until the end of the simulation. It can be considered that Nb6 protein and UreB protein formed a stable
complex after 100ns molecular dynamics simulation.
![Figure 17 Schematic Diagram of Conformation Rendering of UreB-Nb6 Protein Complex at 0 or 100ns](https://static.igem.wiki/teams/5735/results/result-14.webp)
**Figure 17 Schematic Diagram of Conformation Rendering
of
UreB-Nb6 Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and blue shows Nb6
protein.**
      Nb-ScFv protein changed little relative to UreB protein in the whole
simulation process, so the RMSD value was low in the whole kinetic simulation process. In the first 60 ns
of
the simulation, the RMSD curve kept a certain fluctuation trend, which was due to the fact that although
the
skeleton atoms of the two proteins did not change obviously at the interaction interface, the orientation
of
the side chains changed to some extent, so the RMSD curve still showed a certain fluctuation trend. After
60ns, as the interaction mode between the two proteins tends to be stable, the RMSD curve also tends to
converge and vibrates around 0.5nm until the end of the simulation.
![Figure 18 Schematic Diagram of Conformation Rendering of UreB-Nb-ScFv Protein Complex at 0 or 100ns](https://static.igem.wiki/teams/5735/results/result-15.webp)
**Figure 18 Schematic Diagram of Conformation Rendering
of
UreB-Nb-ScFv Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and purple shows
Nb-ScFv
protein.**
      In the first 20 ns of the simulation, Nb-Human protein appears to be
close
to UreB protein, which is most obviously marked in the above figure. In the first 20ns of the simulation,
Nb-Human protein tends to twist and fold spontaneously under the action of UreB protein, and this
conformation
is maintained until the end of the simulation. After 20ns, the interaction between the two proteins tends
to
be stable, and the skeleton atoms do not change obviously again, so the RMSD curve tends to converge
gradually. During this period, the vibration of the RMSD curve still comes from the mutual orientation
change
of the side chains of the two proteins, and the whole two proteins tend to form stable complexes.
![Figure 19 Schematic Diagram of Conformation Rendering of UreB-Nb-Human Protein Complex at 0 or 100ns](https://static.igem.wiki/teams/5735/results/result-16.webp)
**Figure 19 Schematic Diagram of Conformation Rendering
of
UreB-Nb-Human Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and pink shows
Nb-Human
protein.**
      Combined with the analysis of the movement trajectory of the protein
complex, it can be seen that the three proteins have no obvious displacement relative to the UreB protein
during the whole simulation process, so the RMSD value is relatively stable. Similarly, in the early stage
of
the simulation, the structure of the interaction between the three proteins and UreB protein has changed
to
some extent, so the RMSD curve shows a certain fluctuation trend in the first certain period of the
simulation, and then gradually converges with the dynamic simulation. On the whole, after 100ns molecular
dynamics simulation, all three proteins formed a stable complex with UreB protein, which can be combined
with
subsequent analysis to further observe the binding behavior of the three proteins.
      RMSF analysis showed the structural adaptability of each residue in
protein, which was used in this study to analyze the flexibility and movement intensity of amino acid
residues
in protein during the whole simulation process. It can be seen from the figure that the overall RMSF of
the
protein remained at a low level during the whole simulation process. The reason why the RMSF at the
N-terminal
and C-terminal of the protein is large is that this residue is at the head end of the protein and is less
constrained by other domains in the protein, which leads to greater flexibility and has a certain impact
on
its own stability. In addition, the reason why the rest of the protein RMSF is large may be that there is
some
disturbance due to the combination of small molecules, or because the peptide itself is flexible and has
some
disturbance during the simulation.
      By comparing the RMSF curves of three protein complexes, it can be
seen
that the RMSF value of Nb-ScFv protein and Nb-Human protein is higher than that of Nb-6 protein after
forming
complexes with Ureb protein. Which may be due to the stronger interaction between Nb-ScFv protein and
Nb-Human
protein and UreB protein, so the flexible residues at the interaction interface are more affected, which
leads
to the more obvious flexible jitter of different residues of Ureb protein. At the same time, it was
observed
that the RMSF value of N-terminal of Nb-Human protein increased obviously. Combined with the above
analysis,
it can be seen that this is because the Nb-Human protein moved towards UreB protein in the first 20ns of
simulation, so the RMSF value of this random curly segment increased, which is consistent with the above
analysis.
      On the whole, the RMSF values of the three proteins are low in the
simulation process, so it can be considered that the proteins have little jitter in the simulation
process,
keep stable vibration in the solution environment, and the sampling and analysis are reliable.
![Figure 20 RMSF curve of each protein in the simulation process](https://static.igem.wiki/teams/5735/engineering/engineering-20.webp)
**Figure 20 RMSF curve of each protein in the simulation
process. A. The RMSF curve of Ureb. B. The RMSF curve of Nb-6. C. The RMSF curve of Nb-ScFv. D. The RMSF
curve of Nb-Human.**
      The Radius of Gyration (Rg) can be used to characterize the
compactness
of
protein structure, and it can also be used to characterize the change of protein's peptide chain looseness
in
the simulation process. Combined with the above analysis and Rg diagram analysis, it can be seen that the
Rg
curves of the complexes formed by Nb6 protein and Nb\_ScFv protein with UreB protein respectively remained
stable throughout the simulation process and changed within 2.5nm and 2.8nm respectively until the end of
the
simulation. After the complex of Nb\_Human protein and UreB protein was formed, the Rg curve showed an
upward
trend in the first 20ns of the simulation, and then it tended to converge, maintaining the vibration
around
2.75nm until the end of the simulation.
      Combined with the above analysis, it can be seen that after Nb6
protein
and Nb-ScFv protein form complexes with UreB protein, the protein complexes remain relatively stable
during
the whole dynamic simulation process. As mentioned in the aforementioned RMSD analysis, only the mutual
orientation of the residues at the interaction interface of the two proteins changes during the simulation
process, so the RMSD curve remains in a low state, showing a certain fluctuation trend. As the skeleton
atoms
of the proteins do not change obviously as a whole, the Rg curve does not change significantly, but only
keeps
a certain vibration until the end of the simulation. After the complex of Nb-Human protein and UreB
protein
was formed, the random curl of Nb-Human protein was close to UreB protein in the first 20 ns of
simulation,
and the overall compactness became larger, so the Rg curve increased in the first 20 ns of simulation.
After
20ns, with the convergence of the two protein complexes after exercise, the Rg curve also tends to be
stable,
vibrating around 2.75 nm until the end of the simulation.
      On the whole, the Rg curve of protein is stable in the simulation
process
and the sampling is reliable.
![Figure 21 Radius of Gyration](https://static.igem.wiki/teams/5735/engineering/figure-21.webp)
**Figure 21 Radius of Gyration**
      We analyzed the changes of the number of hydrogen bonds formed between
proteins with the simulation time and systematically investigated the stability of protein binding from
the
perspective of interaction. From the graph analysis, it can be seen that the number of hydrogen bonds
formed
between Nb-6 protein and Nb-ScFv protein and UreB protein respectively remained relatively stable and had
a
certain amplitude during the whole simulation process. The number of hydrogen bonds formed between
Nb-Human
protein and UreB protein showed a certain fluctuation trend in the first 40ns of the simulation, and
gradually
increased as a whole, and stabilized after 40ns until the end of the simulation.
      Based on the above analysis and the analysis of the protein complex's
trajectory, it can be seen that although Nb-6 protein and Nb-ScFv protein have no obvious displacement
relative to UreB protein during the whole simulation process, due to the conformational adjustment between
the
residues at the interaction interface of the two proteins, accompanied by the residue change and flexible
jitter of the protein flexible binding cavity, this movement leads to the continuous formation and
destruction
of hydrogen bonds between the residues at the protein interaction interface, which leads to a certain
vibration of the hydrogen bond number curve with the change of simulation time, and the whole is
relatively
stable; Nb-Human protein appeared close to UreB protein in the first 20ns of the simulation, and
maintained
this conformation until the end of the simulation, so the number of hydrogen bonds formed with UreB
protein
showed a certain fluctuation trend in the first 40ns of the simulation, and the whole showed an upward
trend
due to the increase of protein interaction interface. After 40ns, with the gradual convergence of the
residues
at the interaction interface between the two proteins, the number curve of hydrogen bonds tends to be
stable
until the end of the simulation.
      In addition, it can be noted that although the number curve of
hydrogen
bonds changes obviously with the simulation, there are hydrogen bonds between the two proteins at all
times,
which explains that the binding between proteins is stable from the perspective of force.
![Figure 22 The change of the number of hydrogen bonds formed between proteins with the simulation time](https://static.igem.wiki/teams/5735/engineering/figure-22.webp)
**Figure 22 The change of the number of hydrogen bonds
formed between proteins with the simulation time.**

      After eliminating the cycle of the simulated trajectory, combined with
the
aforementioned RMSD analysis, the movement trajectory of protein complex in the period of 90ns-100ns was
extracted for binding free energy (MM/GBSA) analysis, and the energy term was decomposed.
      The results showed that the binding energy between protein and UreB
was
mainly van der Waals energy (Nb6 protein was -88.53 kcal/mol, Nb-ScFv protein was -109.01 kcal/mol,
Nb-Human
protein was -139 kcal/mol), and the electrostatic energy of Nb-6 protein was -26.58 kcal/mol,Nb-ScFv
protein
is -40.13 kcal/moland Nb-Human protein is -43.19 kcal/mol), which indicates that electrostatic interaction
is
beneficial to protein binding, and proteins interact mainly through van der Waals interaction. The
aforementioned hydrogen bond analysis and force analysis show that there are certain van der Waals
interactions and hydrophobic interactions between different amino acids of each protein, so van der Waals
can
significantly facilitate the binding between proteins and occupy a dominant position; At the same time,
there
is a certain electrical property at the protein-protein interaction interface, so there is a certain
electrostatic interaction at the protein-protein interaction interface, which leads to the contribution of
electrostatic energy to the binding. In addition, we noticed that the solvation energies of Nb6 protein,
Nb-ScFv protein and Nb-Human protein were 26.53 kcal/mol, 41.46 kcal/mol and 42.60 kcal/mol respectively
in
this simulation, suggesting that the addition of solvents was not conducive to the binding between
proteins.
      The overall binding energy of Nb-6 protein is -88.59 kcal/mol, Nb-ScFv
protein is -107.68 kcal/mol, and Nb-Human protein is -139.59 kcal/mol), which indicates that the binding
tendency of proteins is relatively large, and the relative order of binding tendency is Nb-Human > Nb-ScFv
>
Nb-6, which further explains the binding between proteins from the perspective of energy.
      Based on the above computer simulation analysis, our nano-antibody has
a
good binding effect on Ureb protein of Helicobacter pylori in theory, among which Nb-Human has the best
binding effect. Unfortunately, due to the limitation of biological safety, we can't directly verify the
inhibitory effect of antibodies on Helicobacter pylori. Later, we may consider cooperating with more
professional research institutions to verify the actual inhibitory effect of our antibodies on
Helicobacter
pylori.
![Figure 23 Analysis of binding free energy (MM/GBSA)](https://static.igem.wiki/teams/5735/engineering/figure-23.webp)
**Figure 23 Analysis of binding free energy
(MM/GBSA)**

**Table 3 Energy analysis in simulation
process**
![Table 3 Energy analysis in simulation process](https://static.igem.wiki/teams/5735/engineering/engineering-table3.webp)
**Learn:**
      Expression of the antibody gene in *E. Coli* BL21(DE3) revealed
that the Nb-human gene encodes a protein exhibiting potent urease inhibitory activity. To enable the
expression of this recombinant nanobody in food-grade probiotic strains, it is necessary to verify whether
the
Nb-human gene can be successfully delivered using the current vector system, express, and whether the
resulting antibody retains significant urease-inhibitory function. Therefore, we propose testing the
expression of the Nb-human gene in probiotic bacteria to establish a foundation for the subsequent oral
delivery of functional antibodies via live bacterial carriers.

### Cycle 2: Construction and verification of pGEX4T-1-Nb-human

**Design:**
      For antibody expression validation in probiotics, the pET28a vector
was
unsuitable due to its dependence on T7 RNA polymerase—an enzyme absent in *Escherichia coli* Nissle
1917(EcN). *Escherichia coli* Nissle 1917 (EcN) is one of the most extensively studied probiotic
strains. It has been demonstrated to be a suitable probiotic for a wide range of gastrointestinal
disorders
and represents a promising and safe candidate for the treatment of gastrointestinal diseases. Currently,
EcN
has been successfully employed as one of the safest carriers for therapeutic molecules in both *in
vivo* and *in vitro* applications so we choose it as recombinant nanobody delivery strain. We
employed the pGEX-4T-1 plasmid as an alternative expression vector to clone and express the target gene
Nb-human. The LacI gene, SUMO tag, signal peptide sequence and kanamycin resistance gene fragment are
similar to the construction of the plasmid pET28a-Sumo-Nb-human.
![Figure 24. The map of the plasmid pGEX4T-1-Nb-human](https://static.igem.wiki/teams/5735/engineering/figure-24.webp)
**Figure 24. The map of the plasmid
pGEX4T-1-Nb-human**
**Build:**
![Figure 25. The overview of the construction of pGEX4T-1-Nb-human](https://static.igem.wiki/teams/5735/engineering/figure-25.webp)
**Figure 25. The overview of the construction of
pGEX4T-1-Nb-human**
      Following the same procedure as the construction of
pET28a-Sumo-Nb-human, we digested the target gene amplification product and the pGEX-4T-1 plasmid with
restriction enzymes, followed by ligation using T4 DNA ligase. The ligation product was then transfered
into
DH5α competent cells, and through colony PCR and DNA sequencing，we verified the success of the
construction
of the pGEX4T-1-Nb-human plasmid(Figure 26).
![Figure 26. The validation and sequencing of the plasmid pGEX4T-1-Nb-human](https://static.igem.wiki/teams/5735/engineering/figure-26.webp)
**Figure 26. The validation and sequencing of the
plasmid
pGEX4T-1-Nb-human. A: The transformed strain growing on the resistant plate; B: The comparison results
of
the target genes of the positive cloned strains after sequencing.**
**Test:**
      **Protein expression and validation(EcN1917)**
      We transfered the pGEX4T-1-Nb-human expression plasmid into
*Escherichia coli* Nissle 1917 competent cells, induced the expression of the target protein by 1mM
IPTG and purified the acquired Nb-human protein using nickel affinity purification. SDS-PAGE results
(Figure
27) indicated that the antibody protein (34.1 kDa) encoded by Nb-human was successfully expressed in the
EcN
1917.
![Figure 27. SDS-PAGE electrophoresis gel of Nb-human protein expressed in EcN 1917](https://static.igem.wiki/teams/5735/engineering/figure-27.webp)
**Figure 27. SDS-PAGE electrophoresis gel of Nb-human
protein expressed in EcN 1917.**
**Learn:**
1. In this study, we employed a basic assay to evaluate the activity of recombinant nanobodies. Under the
premise of biological safety, to further validate their functionality, additional experiments are required
to determine whether these nanobodies can inhibit *Helicobacter pylori* growth, proliferation, or
in
vivo colonization. Subsequent studies should include in vitro pharmacodynamic assays and in vivo animal
validation experiments.
2. Although the host strain used in this study is generally recognized as safe food-grade microorganism,
the
expression vector carries an antibiotic resistance gene. If administered orally, this could pose potential
risks due to horizontal gene transfer. Therefore, for future therapeutic applications, alternative vectors
without antibiotic resistance markers should be considered to ensure biosafety.

### Reference

[1] Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. Global Prevalence of Helicobacter
pylori Infection: Systematic Review and Meta-Analysis. Gastroenterology. 2017;153:420-9.
[2] Malfertheiner P, Camargo MC, El-Omar E, Liou J-M, Peek R, Schulz C, et al. Helicobacter pylori
infection. Nature Reviews Disease Primers. 2023;9.
[3] Eaton KA, Brooks CL, Morgan DR, Krakowka S. Essential role of urease in pathogenesis of gastritis
induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun. 1991;59:2470-5.
[4] Almarmouri C, El-Gamal MI, Haider M, Hamad M, Qumar S, Sebastian M, et al. Anti-urease therapy: a
targeted approach to mitigating antibiotic resistance in Helicobacter pylori while preserving the gut
microflora. Gut Pathogens. 2025;17.
[5] Mobley HL. The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic
ulceration. Aliment Pharmacol Ther. 1996;10 Suppl 1:57-64.

![vertical line](https://static.igem.wiki/teams/5735/img/desc/p2-right-vertical-line.webp)
![返回顶部](https://static.igem.wiki/teams/5735/img/desc/p2-right-mini.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide
sufficient information for other teams to replicate our work.{% endblock %}
{% block extra\_css %}

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

# EXPERIMENTS

![导航图片](https://static.igem.wiki/teams/5735/img/desc/p2-left-up-image.webp)

content

![圆点1](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot2.webp)
![圆点2](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot2.webp)
![圆点3](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点4](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot4.webp)

[Preparation of LB Culture Base](#preparation)
[Construction of plasmid](#construction)
[Protein expression](#expression)
[Function test](#function-test)

### Preparation of LB Culture Base

**Experimental objective：**
To formulate a functional and nutritive liquid and solid culture medium for bacterial growth. Liquid media
serve various purposes, including microbial propagation and testing.

**Equipment list：**
1)Tryptone
2)Yeast extract
3)Sodium chloride
4)Distilled water
5)Sterilized conical flask, cap
6)Autoclave
7)Lab refrigerator
8)Measuring cylinder
9)Weighing scale

**Method:**
1)Weigh out 2.50 g of premix LB broth powder by weighing scale (including 1.00 g tryptone, 0.50 g yeast
extract and 1.00 g sodium chloride)
2)Transfer the LB broth powder into 250 mL conical flask
3)Add distilled water up to total volume of 100 mL by 100 mL measuring cylinder
4)Close the cap and autoclave it for 20 min at least
5)Wait until it cools

### Construction of plasmid

**2.1 pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB**

**Extraction of plasmid DNA**
**Experimental objective：**
To extract and purify plasmid DNA from cultured bacteria for use in downstream applications such as
PCR.the
target gene fragments.

**Equipment list:**
1)Benchtop microcentrifuge (capable of 12,000× g)
2)Centrifuge tubes (1.5 mL)
3)Collection tubes for spin columns
4)Adjustable-volume micropipettes
5)Sterilized pipette tips
6)Silica membrane-based spin columns
7)Waste collection tubes
8)Buffer SP1 (with RNase A)
9)Buffer SP2
10)Buffer SP3
11)Wash Solution
12)Elution Buffer
13)Buffer DW1
14)Overnight bacterial culture
15)centrifuge tube racks

**Method:**
1)Verification:
Confirm RNase A has been added to Buffer SP1
Verify absolute ethanol has been added to Wash Solution
Check Buffers SP2 and SP3 for precipitation; warm to 37 °C if precipitated
2)Column equilibration:
Place spin column in a collection tube
Add 500 μL Buffer S to the column
Centrifuge at 12,000× g for 1 min
Discard flow-through and reassemble column-collection tube assembly
3)Bacterial Pellet Harvesting:
Transfer 1.5 mL bacterial culture to a microcentrifuge tube
Centrifuge at 8,000× g for 2 min
Discard supernatant completely
4)Cell Lysis and Neutralization
Resuspend pellet in 250 μL Buffer SP1 by vigorous pipetting
Add 250 μL Buffer SP2 and immediately mix by inverting tube 5–10 times
Incubate at room temperature for 2–4 min
Add 350 μL Buffer SP3 and immediately mix by inverting 5–10 times
Centrifuge at 12,000× g for 5–10 min.
5)DNA Binding and Washing
Transfer supernatant to the equilibrated spin column
Centrifuge at 8,000× g for 30 sec. Discard flow-through
Add 500 μL Buffer DW1, centrifuge at 9,000× g for 30 sec. Discard flow-through
Add 500 μL Wash Solution, centrifuge at 9,000× g for 30 sec. Discard flow-through
Repeat Wash Solution step
Centrifuge empty column at 9,000× g for 1 min to remove resting washing solution
6) DNA Elution
Transfer spin column to a clean 1.5 mL microcentrifuge tube
Add 50 μL Elution Buffer to the center of the membrane
Incubate at room temperature for 1 min
Centrifuge at 9,000× g for 1 min
Repeat elution buffer step
Conserve the DNA solution in tube

**Obtaining target gene fragments by PCR**
**Experimental objective：**
To amplify the target gene fragments (Nb-human, UrB-Nb6, UreB and Nb-SCFV ) for later recombination
materials.

**Equipment list:**
1) 2×PrimeSTAR Max Premix
2) Sumo-Nb-human, Sumo-UrB-Nb6, UreB and Sumo-Nb-SCFV gene template
3) double distilled water
4) Primer-R
5) Primer-F
6) Centrifuge tube
7) PCR thermal cycler

**Method:**
1)Add 25 µL 2×PrimeSTAR Max Premix, pNb-human template 1µL, primer-R 1µL, and primer-F 1 µL to centrifuge
tube. Add double distilled water to the centrifuge tube until reaching 50 µL. If bubbles are observed in
the
mixture, centrifuge the mixture for a few seconds to remove the bubbles.
2) Insert mixture into a PCR thermal cycler. Denature the fragments at 95 ˚C for 3 min
3) Perform 30 cycles of 30 sec 95 ˚C denaturing, 30 sec 55 ˚C annealing, and 90 sec extension 72 ˚C
extension.
4) Extend the fragments for 1 min at 72 ˚C
5) If not use immediately, preserve the fragments at 4 ˚C
6) Repeat step 1-5 for Somo-UreB-Nb6, Sumo-UreB and Sumo-Nb-SCFV.

**Vector linearization**
**Experimental objective：**
To linearize the vector via double restriction enzyme digestion, generating sticky ends for downstream
ligation.

**Equipment list：**
1)Ice bucket
2)Dry bath incubator
3)Adjustable micropipettes (0.5-10 μL, 10-100 μL)
4)centrifuge tubes
5)Waste collection tubes
6)Restriction enzyme: HindIII
7)Restriction enzyme: NheI
8)Compatible 10× reaction buffer
9)Plasmid extract (from previous step)
10)Double distilled water

**Method:**
1)Pre-cooling:
Thaw HindIII and NheI enzymes on ice
Chill 1.5 mL microcentrifuge tubes in ice bucket
2)Add 3 μL double distilled water into the centrifuge tube
3)Add 5 μL 10× Reaction Buffer into the centrifuge tube
4)Add 1.5 μg Plasmid extract into the centrifuge tube
5)Add 1 μL HindIII into the centrifuge tube
6)Add 1 μL NheI into the centrifuge tube
7)Incubate at 37 ˚C for 1 h in a calibrated dry bath incubator

**Digestion of the target gene fragments**
**Experimental objective：**
To digest the target genes by double restriction digestion, yielding compatible sticky ends for downstream
ligation.

**Equipment list：**
1)Ice bucket
2)Dry bath incubator
3)Adjustable micropipettes (0.5-10 μL, 10-100 μL)
4)centrifuge tubes
5)Waste collection tubes
6)Restriction enzyme: HindIII
7)Restriction enzyme: NheI
8)Compatible 10× reaction buffer
9)PCR product (from previous step)
10)Double distilled water

**Method:**
1)Pre-cooling:
Thaw HindIII and NheI enzymes on ice
Chill 1.5 mL centrifuge tubes in ice bucket
2)Add 40.5 μL double distilled water into the centrifuge tube
3)Add 5 μL 10× Reaction Buffer into the centrifuge tube
4)Add 1.5 μg amplication of target genes extract into the centrifuge tube
5)Add 1 μL HindIII into the centrifuge tube
6)Add 1 μL NheI into the centrifuge tube
7)Incubate at 37 ˚C for 1 h in a calibrated dry bath incubator

**Agarose gel preparation**
**Experimental objective:**
To produce a functional and well-shaped gel for electrophoresis of DNA

**Equipment list:**
1)Casting Tray
2)Weighing scale
3)Measuring cylinder
4)Well comb
5)Microwave
6)1x TAE
7)Agarose powder
8)1000x Nucleic acid gel stain
9)Conical flask

**Method:**
1)Weigh out 0.8g Agarose powder by weighing scale
2)Transfer the Agarose powder into 250 mL conical flask
3)Add TAE (Running buffer) up to total volume of 100 mL by 100 mL measuring cylinder
4)Heat the material with microwave
5)Melt the mixture at 30 sec intervals
6)See whether bubbles are forming, if did successfully mix
7)Add 100 μL nucleic acid gel stain (1000x)
8)Let it cool for 5~10 min
9)Pour the agarose mixture into the casting tray
10)Place the appropriate well comb to create the wells
11)Wait to solidify at least 30 min
12)Remove the comb and place the gel in the gel box

**Agarose gel electrophoresis**
**Experimental objective:**
To verify the restriction digestion of the target gene fragments and vector, followed by gel purification
of
the digested products.

**Equipment list：**
1)sample
2)Marker (DNA ladder)
3)Prepared gel (from previous step)
4)10x Loading buffer (dye)
5)Horizontal gel electrophoresis system
6)Adjustable micropipettes

**Method:**
1)Extract 50 µL of every sample, add 5 µL of loading buffer (10x) to each
2)Place the gel in the horizontal electrophoresis system
3)Extract 50 µL of the mixed solution containing the sample, mix them till their colors are almost
uniform
4)Add the marker to the first well and load 50 µL of each sample into the wells
5)The procedure runs at 120 V for 25 min
6)Afterwards, take out the gel from the device and proceed to following steps

**Gel Extraction and Purification of DNA**
**Experimental objective:**
To verify the DNA bands of digested target genes fragments and plasmids and thus preparing for the next
step
of gel extraction to extract the bands that are of the correct length.

**Equipment list:**
7)UV transilluminator
8)Clean scalpel
9)Weighing scale
10)Water bath (50 °C calibrated)
11)Microcentrifuge tube rack
12)Silica-membrane spin columns
13)Collection tubes (2 mL)
14)Nuclease-free 1.5 mL microcentrifuge tubes
15)Buffer B2 (Gel solubilization buffer)
16)Wash Solution
17)Elution Buffer
18)Agarose gel
19)pipettes and sterilized pipette heads
20)centrifuge

**Method:**
1)Pre-Extraction Preparation
Check Buffer B2 for precipitation
Preheat water bath to 50 °C
2)Excise target band under UV light, Minimize UV exposure (<30 sec)
3)Weigh gel slice and record the mass
4)Add Buffer B2 (3× gel weight)
5)Incubate at 50 °C for 5 min to melt the gel
6)Transfer lysate to spin column, centrifuge at 8,000× g for 30 sec
7)Discard flow-through
8)Add 500 μL Wash Solution centrifuge at 9,000× g for 30 sec
9)Repeat wash step centrifuge at 9,000× g for 30 sec
10)Dry column (empty) centrifuge at 9,000 ×g for 1 min
11)Transfer column to clean 1.5 mL tube
12)Add 20 μL Elution Buffer to membrane center, place at room temp for 1 min
13)Centrifuge to collect DNA at 9,000 ×g for 1 min
14)Store eluate

**Ligation of the target gene and vector for plasmid construction**
**Experimental objective:**
Ligate the plasmid fragment after enzymatic digestion with the target gene fragment, constructing the
target gene-containing plasmid for subsequent protein expression and purification.

**Equipment list:**
1)T4 DNA ligase
2)T4 DNA ligase buffer
3)PCR products of target genes
4)vector（plasmid）
5)nanodrop
6)Adjustable micropipettes

**Method:**
1)Test the concentration of vector and target genes using Nanodrop
2)Prepare the system (20 μL)
Add 1 μL T4 DNA ligase
Add 2 μL T4 DNA ligase buffer
vector 2 μL
target DNA fragment 50 ng
Place the system at in a 16°C metal bath for 2 hours to react.

**Transformation of competent cells and plating via heatshock**
**Experimental objective:**
To transfer the target gene-carrying plasmid into competent cells for plasmid amplification (for
subsequent verification) or recombinant protein expression.

**Equipment list:**
1)Ligated samples
2)LB Agar plates and liquid solution
3)Laminar flow cabinet
4)Inoculation loop
5)Shaking incubator
6)Antibiotic (kanamycin)
7)Component cells E.coli DH5α, E.coli BL21(DE3)

**Method:**
1)Ligated samples are added to component cells
2)Placed on ice for 30 min
3)Heat at 42 °C for 45 sec, then immediately put on ice for 2 min
4)Add 900 μL LB solution, incubate for 30 min
5)Shake at 220 rpm for 50 min at 37 °C using shaking incubator
6)Apply end product to agar plates with antibiotic (kanamycin), incubate for 12-16 h at 37 °C

**Positive clone verification**
**Experimental objective:**
To confirm the successful transformation of the target gene into competent cells, PCR amplification
followed by gel electrophoresis was performed on bacterial colonies.

**Equipment list:**
1)Adjustable micropipettes
2)LB Agar plates with bacterial colonies
3)Centrifuge
4)Measuring cylinder
5)Centrifuge tube
6)2×PrimeSTAR Max Premix
7)Double distilled water
8)Primer-R
9)Primer-F
10)PCR thermal cycler
11)Marker (DNA ladder)
12)Prepared gel (from previous step)
13)10x Loading buffer (dye)
14)Horizontal gel electrophoresis system

**Method:**
1)Preparation of a 20 μL system
Add 10 µL 2×PrimeSTAR Max Premix, primer-R 1.0 µL, and primer-F 1.0 µL to centrifuge tube.
Add double distilled water to the centrifuge tube until reaching 20 µL.
If bubbles are observed in the mixture, centrifuge the mixture for a few seconds to remove the
bubbles
2)Within sterilized fume hood workstation, remove a colony of preprepared bacteria from the agar plate
and
add to 1.5mL centrifuge tube
(Note: choose larger colonies but also be aware that colonies near the sides of the petri dish are most
likely to be contaminated or are undesired bacteria and are less ideal)
3)Insert mixture into a PCR thermal cycler. Denature the fragments at 95 ˚C for 3 min
4)Perform 30 cycles of 30 sec 95 ˚C denaturing, 30 sec 55 ˚C annealing, and 90 sec extension 72 ˚C
extension.
5)Extend the fragments for 1 min at 72 ˚C
6)Extract 50 µL of every sample
7)Place the gel in the horizontal electrophoresis system
8)Extract 50 µL of the mixed solution containing the sample, mix them till their colors are almost
uniform
9)Add the marker to the first well and load 10 µL of each sample into the wells
10)The procedure runs at 120 V for 25 min
11)Afterwards, take out the gel from the device and observe the position of the bands using a gel
imager

**2.2 Construction of pGEX4T-1-Nb-human**
The construction of the pGEX4T-1-Nb-human is same as the construction of pET28a-Sumo-Nb-human,
pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6, pET28a-UreB. And there were some difference.
1. First, we used Nb-human as the target gene instead of all three target genes for expression and
purification when the plasmid was constructed.
2. The vector was replaced by pGEX4T-1.
3. We transferred the constructed plasmid into Escherichia coli Nissle 1917 for expression and
purification, and we got the desired vector carrying the target gene.

### Protein expression

**3.1 Protein expression of pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6,
pET28a-UreB**

**Protein induction and expression in E.coli BL21**
**Experimental objective:**
To scale up the cultivation of genetically verified bacterial clones for subsequent target protein
induction, expression, and functional validation.

**Equipment list：**
1)Adjustable micropipettes
2)Culture tube
3)Spectrophotometer
4)Shaking incubator
5)Antibiotic (kanamycin)
6)Laminar flow cabinet
7)Conical flask
8)LB liquid solution
9)Colonies samples
10)IPTG (isopropyl β-D-1-thiogalactopyranoside)

**Method:**
1）Add 4 mL of LB liquid solution into a sterile culture tube
2）Based on the agarose gel electrophoresis results (from the previous step), select and transfer colonies
confirmed to carry the target genes into the culture tube
3）Incubate the culture tube in a shaking incubator at 37 °C with 220 rpm shaking for 4 h
4）Add 100 mL of LB liquid medium with 50 μg/mL kanamycin into a sterile conical flask
5）Transfer the entire bacterial suspension from the culture tube into the conical flask
6）Place the flask in a shaking incubator and culture at 37 °C, 220rpm.
7）Measure the optical density (OD₆₀₀) using a spectrophotometer until the reading reaches 0.4–0.6
8）Add 100 µL of IPTG (isopropyl β-D-1-thiogalactopyranoside) to induce protein expression at 25°C or 16
°C,
20h

**Protein extraction and purification**
**Experimental objective：**
To lyse bacterial cells and extract proteins for subsequent purification and concentration
measurement

**Equipment list：**
1)Centrifuge
2)LB solution (with sample)
3)50 mL centrifuge tubes
4)1.5 mL centrifuge tubes
5)Adjustable-volume micropipettes
6)Ultrasonic cell disruptor with microtip probe
7)Ice bath
8)Column stands with clamps
9)4 °C cold room or refrigerated cabinet
10)-20 °C freezer (for enzyme stock storage)
11)100 mg/mL Lysozyme
12)Non-denaturing lysis buffer with protease inhibitor cocktail
13)50% BeyoGold™ His-tag Purification Resin
14)Lateral shaker
15)Affinity chromatography column
16)Wash buffer
17)Elution buffer

**Method：**
1)Transfer the LB solution (with sample) into 50 mL centrifuge tubes
2)Centrifuge induced culture at 12,000 rpm for 4 min, discard supernatant completely
3)Add 4 mL non-denaturing lysis buffer with protease inhibitors per gram wet pellet, fully resuspend the
bacterial pellet
4)Add lysozyme from 100 mg/mL stock to final 1 mg/mL, incubate on ice for 30 min
5)Sonicate on ice: 3 sec ON / 3 sec OFF, at 40% power for 15 min
6)Centrifuge to obtain the bacterial lysate supernatant
7)Transfer 1 mL of a well-mixed 50% slurry of BeyoGold™ His-tag Purification Resin into a microcentrifuge
tube, centrifuge at 4 °C and discard the supernatant.
8)Add 0.5 mL of non-denaturing lysis buffer to the resin pellet, mix thoroughly to equilibrate the
resin.
9)Add 4 ml of bacterial lysate supernatant to it and gently shake it at 4 ºC for 60 min by lateral
shaker
10)Transfer the mixture into an empty affinity chromatography column
Remove the bottom cap of the purification column and allow the liquid to flow through by gravity. Collect
20
µL of the flow-through for subsequent analysis
11)Repeat the flow-through step a total of 2 times
12)Apply 0.5 mL of non-denaturing wash buffer, each time collecting 20 µL of the wash-through for
subsequent
analytical detection
Proceed with column washing and the subsequent elution step.
13)Repeat the washing step a total of 3 times
14)Apply 0.5 mL of non-denaturing elution buffer to elute the bound proteins, collecting each fraction in
separate microcentrifuge tubes.
15)Repeat the elution step a total of 4 times. The collected eluates constitute the purified His-tagged
protein samples

**Dialysis for salt removal**
**Experimental objective：**
The main purpose of dialysis and desalination of the target protein is to to remove the excess NaCl and
imidazole ions in the protein solution, ensuring the purity and stability of the protein

**Equipment list：**
1) Dialysis bag
2) 20 mM Tris-HCl（pH = 8.0）
3) Beaker
4) Magnetic stirrer
5) Clip

**Method:**
1) The purified recombinant protein solution was placed into a dialysis bag with a molecular weight
cut-off
of 10 kDa.
2) The two ends of the dialysis bag were clamped into a beaker containing 20 mM Tris-HCl (pH = 8.0)
dialysate
3) Placed the dialysis bag at 4℃, and dialyzed overnight with continuous stirring by a magnetic
stirrers.

**Concentrate the protein after dialysis**
**Experimental objective：**
It can increase the concentration and reduce the volume in an efficient and gentle way, laying the
foundation for further research or application of proteins.

**Equipment list：**
1) 10 kDa ultrafiltration tube
2) Cone shaped bottle
3) High-speed centrifuge
4)50 mL centrifuge tubes
5) 1.5 mL centrifuge tubes

**Method:**
The protein after dialysis was concentrated by ultrafiltration with a 10 kDa ultrafiltration tube,
packaged
and stored at -80℃

**SDS-PAGE**
**A. PAGE gel preparation**
**Experimental objective：**
To produce a functional and well-shaped gel for electrophoresis of proteins

**Equipment list:**
1)Casting tray
2)15 mL centrifuge tubes
3)Adjustable-volume micropipettes
4)Well comb
5)Resolving gel solution
6)Resolving gel buffer
7)Stacking gel solution
8)Colored stacking gel buffer
9)Polymerization accelerator

**Method:**
1)Add 4 mL of resolving gel solution and 4 mL of resolving gel buffer and mix thoroughly
2)Add 1 mL of stacking gel solution and 1 mL of colored stacking gel buffer and mix thoroughly
3)Add 80 μL of polymerization accelerator to the resolving gel mixture and mix gently
4)Immediately pour the solution into the gel-casting assembly, ensuring the liquid level remains 0.5 cm
below the top edge of the short glass plate
5)After resolving gel solidification, add 20 μL of polymerization accelerator to the stacking gel mixture
and mix gently
6)Carefully layer the stacking gel solution on top of the polymerized resolving gel, insert the
well-forming
comb, avoiding bubble formation
7)Allow gel polymerization, remove the comb carefully and rinse wells with running buffer before
electrophoresis.
8)Wait for 40minutes
The percentage of the gel is 15% based on the target protein size.

**SDS-PAGE gel electrophoresis**
**Experimental objective:**
To verify successful expression and purification of the target protein by SDS-PAGE analysis of induced
lysates and elution fractions.

**Equipment list：**
1)Sample
2)Marker
3)Prepared gel (from previous step)
4)Loading buffer (dye)
5)Vertical gel electrophoresis system
7)Adjustable micropipettes

**Method:**
1)Extract 20 µL of every sample, add 5 µL of loading buffer (5x) to each
2)Place the gel in the vertical electrophoresis system
3)Add the marker to the first well and load 15 µL of each sample into the wells
1)The procedure runs at 180V for 40 minutes.
2)Afterwards, take out the gel from the device
3)Stain with Coomassie Brilliant Blue R-250 for 15min and proceed to following steps

**Target protein concentration determination**
**Experimental objective：**
To quantify the target protein concentration and evaluate its expression using the Bicinchoninic Acid
(BCA)
assay.

**Equipment list:**
1)Adjustable micropipettes
2)Protein standard
3)Protein standard diluent
4)BCA Reagent A
5)BCA Reagent B
6)96-well plate
7)Protein samples
8)Incubator
9)Microplate reader

**Method:**
1)Add 0.8 mL of protein standard diluent to one vial of protein standard (20 mg BSA). After thorough
dissolution, prepare a 25 mg/mL protein standard solution, then dilute\* to a final concentration of 0.5
mg/mL.
\*The protein samples should be in the same diluent as that used for the standard preparation
2)Prepare 7.65 mL of BCA working solution by mixing 7.5 mL of BCA Reagent A with 0.15 mL of BCA Reagent B,
followed by thorough vortexing
3)Add 0, 1, 2, 4, 8, 12, 16, and 20 μL of the standard solution to the designated wells of a 96-well
plate,
adjusting the final volume in each well to 20 μL with standard diluent. The resulting standard
concentrations are 0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL, respectively
4)Add 20 μL of protein samples to the designated sample wells of the 96-well plate
5)Add 200 μL of BCA working solution to each well and incubate at 37°C for 20–30 minutes
6)Measure the absorbance at 562 nm using a microplate reader
7)Calculate the protein concentration of the samples based on the standard curve and the sample volume
used.

**Western Blot (WB)**
**A.SDS-PAGE gel electrophoresis and Membrane Transfer**
The SDS-PAGE operation is consistent with the above.

**Experimental objective:**
To transfer the extracted proteins from the PAGE gel to a PVDF membrane for subsequent analysis

**Equipment list：**
1)Gel sample (from previous step)
2)Adjustable micropipettes
3)Ice bath
4)PVDF membrane
5)Anhydrous methanol
6)Transfer cassette
7)Filter paper
8)Filter pad
9)Transfer tank
10)Pre-chilled transfer buffer

**Method:**
1)Pre-activate the PVDF membrane by soaking in methanol.
2)Assemble the transfer sandwich in the following order (from anode to cathode): transfer cassette, filter
pad, filter paper, gel, PVDF membrane, filter paper, filter pad, and transfer cassette
Ensure all components are fully saturated with transfer buffer
3)Secure the assembled cassette and place it into the transfer tank filled with pre-chilled transfer
buffer
4)Perform electrophoretic transfer at 150mA for 2h under ice-cooled conditions to prevent
overheating

**B. Non-specific binding blockade**
**Experimental objective：**
To block non-specific binding sites

**Equipment list：**
1)Lateral shaker
2)1x PBST buffer
3)5% BSA-PBST blocking buffer

**Method：**
1)Remove the PVDF membrane from the transfer tank
2)Wash the PVDF membrane 3–5 times with PBST buffer
3)Block the PVDF membrane with 50 mL of blocking solution(5% BSA-PBST) and incubate with gentle shaking on
a
lateral shaker for 30 min

**C. Antibody Incubation**
**Experimental objective：**
To specifically identify the protein of interest, the membrane was incubated with a primary antibody, then
with an HRP-labeled secondary antibody, and finally subjected to TMB for visualization

**Equipment list：**
1)Adjustable micropipettes
2)Lateral shaker
3)1x PBST buffer
4)Anti-His tag primary antibody
5)Rabbit Anti-Mouse IgG H&L HRP secondary antibody

**Method：**
1)Remove the PVDF membrane from the blocking buffer
2)Wash the PVDF membrane 3–5 times with PBST buffer
3)Incubate the membrane with anti-His tag primary antibody for 2 h at 4 °C temperature under gentle
shaking
4)Wash the PVDF membrane 3–5 times with PBST buffer
5)Incubate the membrane with Rabbit Anti-Mouse IgG H&L HRP secondary antibody for 1 h at room temperature
under gentle shaking

**D. Detection**
**Experimental objective：**
To validate the specificity of the target protein, the membrane was developed with TMB substrate,
resulting
in visible colorimetric signals at the sites of antibody binding

**Equipment list：**
1)Adjustable micropipettes
2)1x PBST buffer
3)TMB color-developing solution

**Method：**
1)Wash the PVDF membrane 3–5 times with PBST buffer
2)Uniformly drop 100 µL of TMB color-developing solution onto the membrane, covering the area on the
membrane
3)Incubate in the dark for 5 to 30 min until the desired result is achieved

**3.2 Protein expression of pGEX4T-1-Nb-human**
The protein expression of Nb-human on the pGEX4T-1 is similar to the above. The induction condition was
set
to induce at 25℃ for 20 hours under the action of 1mM IPTG.

### Function test

**4.1 Function test of pET28a-Sumo-Nb-human, pET28a-Sumo-Nb-SCFV, pET28a-Sumo-UreB-Nb6,
pET28a-UreB**

**Immunodiffusion Assay**
**Experimental objective：**
To qualitatively verify whether the expressed antibody protein effectively binds to its target
antigen

**Equipment list：**
1)Double distilled water
2)Weighing scale
3)Conical flask
4)Agarose powder
5)Measuring cylinder
6)Microwave
7)Ureb sample
8)Antibody sample (Nb-human, Nb6 and SCFV)
9)Culture dish
10)Incubator
11)Oxford cups
12)Adjustable micropipettes

**Method：**
1)Weigh out 1.00 g Agarose powder by weighing scale
2)Transfer the Agarose powder into 250 mL conical flask
3)Add double distilled water up to total volume of 100 mL by 100 mL measuring cylinder
4)Heat the material with microwave, melt the mixture at 30 sec intervals
5)Add 1 mL UREB sample into the conical flask
6)Transfer the mixture into a culture dish
7)After solidification of the culture medium, wells were created using Oxford cups, with clear labeling of
antibody names and control groups
8)Add 100 μL antibody sample into each well
9)Incubate at 37 °C for 24-36h.

**Nanobody-Mediated Urease Inhibition Assay**
**Experimental objective：**
To quantitatively measure the inhibitory activity of recombinant nanobodies against urease

**Equipment list:**
1)Adjustable micropipettes
2)Diluent
3)Ureb sample
4)Antibody sample (Nb-human, Nb6 and SCFV)
5)96-well plate
6)Incubator
7)Microplate reader
8)Urea

**Method：**
1)Dilute Ureb sample to achieve a final concentration of 100 μg/mL
2)Perform two-fold serial dilutions of each antibody (Nb-human, Nb6 and SCFV) in assay buffer to create
concentrations of 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0 μg/mL
3)Aliquot 100 μL of UreB solution (100 μg/mL) into each well of microtiter plates
4)Transfer 50 μL of diluted antibody solutions to corresponding antigen-coated wells.
5)Incubate at 4 °C overnight
6)Preparation of 1 M Urea Solution
7)Aliquot 50 μL of 1 M Urea solution into each well of microtiter plates
8)Incubation at 25 °C for 6-10 h
8)Measure the absorbance at 550 nm using a microplate reader and calculate inhibition ratio.

![vertical line](https://static.igem.wiki/teams/5735/img/desc/p2-right-vertical-line.webp)
![返回顶部](https://static.igem.wiki/teams/5735/img/desc/p2-right-mini.webp)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}Present the results of your project, along with a detailed analysis and discussion of their
significance. Also outline future plans and reflections on the impact of your project.{% endblock %}
{% block extra\_css %}

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

# RESULTS

![导航图片](https://static.igem.wiki/teams/5735/img/desc/p2-left-up-image.webp)

content

![圆点1](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点2](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点3](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点4](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点5](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点6](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)
![圆点7](https://static.igem.wiki/teams/5735/img/desc/p2-left-dot3.webp)

[Construction of the plasmid](#construction)
[Protein expression and
validation](#protein-expression)
[Functional Test](#functional-test)
[Computer simulation of
antibody
binding to Helicobacter pylori UreB](#computer-simulation)
[Transforming plasmid into
probiotics](#transforming-plasmid)
[Future plan](#future-plan)
[Reference](#reference)

### Construction of the plasmid

Helicobacter pylori (H. pylori) represent one of the most widespread bacterial infections globally.
Current
evidence indicates that nearly half of the global population is chronically infected with H. pylori [1, 2]
and current clinical management relies primarily on antibiotic therapy. Nevertheless, the escalating
antibiotic resistance resulting from overuse has led to an increasing rate of treatment failure. Urease is
a
critical factor for the colonization and pathogenicity of H. pylori in the stomach and H. pylori possesses
one of the highest levels of urease content and enzymatic activity among all known bacteria[3]. Urease is
formed as a heterodimer composed of two distinct subunits, UreA and UreB, with the UreB subunit containing
the catalytic active site of H. pylori[4]. UreB, as a key subunit of urease, facilitates the efficient
incorporation of Ni²⁺ into the enzyme's active site and stabilizes the site against metal chelation[5]. In
this study, we constructed a genetically engineered expression system for a recombinant nanobody
specifically targeting the urease subunit (UreB) of H. pylori. However, due to the limitation of
biological
safety, we can't use Ureb of Helicobacter pylori for experiments, so we use Ureb of Bacillus subtilis
instead in this experiment. First, the recombinant nanobody was produced using an Escherichia coli
expression system. The binding effect of our antibody to Bacillus subtilis was explored through
experiments,
and then the binding effect of antibody to Helicobacter pylori Ureb was simulated by computer. Finally, we
investigated the expression of the recombinant nanobody in food-grade probiotic bacteria, aiming to
utilize
food-grade probiotics as delivery vehicles to administer the antibody in the form of live bacteria.
![Figure 1. Overall experimental design concept](https://static.igem.wiki/teams/5735/results/figure-1.webp)
***Figure 1. Overall experimental design
concept***
**1.1. Acquisition of target gene fragment**
      Based on the literature, we choose the genes (Nb-human, UreB-Nb6, and
Nb-scFv) as the target genes. The target gene sequence was synthesized by the biotech company. The
amplified
target fragment using the synthesized target gene fragment as the template and the vector (pET28a-1) were
identified by agarose gel electrophoresis after enzymatic digestion by NheI and HindⅢ, respectively. The
Gene fragment lengths of Sumo-Nb-SCFV, Sumo-UreB-Nb6, Sumo-Nb-human and UreB were about 807bp, 655bp,
936bp,375bp, respectively (Figure 2A). Electrophoresis results showed that the band size was consistent
with
the expected size. The size of plasmid skeleton after digestion (pET28a-Sumo-Nb-human,
pET28a-Sumo-Nb-SCFV,
pET28a-Sumo-UreB-Nb6, pET28a-UreB) was consistent with the expectation (Figure 2B). It indicates that the
target gene has been successfully amplified. Subsequently, we used T4 DNA ligase to link the linearized
plasmids to the target gene fragment.
![Figure 2. Agarose gel electrophoresis of the target gene fragment and Skeleton](https://static.igem.wiki/teams/5735/results/result-2.webp)
***Figure 2. Agarose gel electrophoresis of the
target
gene fragment and Skeleton. A.Target gene. B.Skeleton.***
**1.2. Construction of pET28a-Sumo-Nb-human**
      Through antibiotic screening, colony PCR, and DNA sequencing
verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-human in E.Coil DH5α and E.Coil BL21).
Figure 3A depict the growth of transformed colonies on Kanamycin-resistant plates post-transformation.
Bacteria growing on the plate are the strains that may be successfully constructed. The colonies selected
from the plate served as PCR templates for colony PCR. Agarose gel electrophoresis was used to verify the
correctness of PCR products and the results show that the range of the target size is consistent with the
expectation (Figure 3B). Subsequently, the target-sized amplicons were sent for sequencing. According to
the
sequence results shown in Figure 3C, the target gene was successfully ligated with the vector without
obvious mutations, confirming the successful construction of the pET28a-Sumo-Nb-human plasmid.

![Figure 3. The validation and sequencing of the plasmid pET28a-Sumo-Nb-human](https://static.igem.wiki/teams/5735/results/figure-3.webp)
***Figure 3. The validation and sequencing of the
plasmid pET28a-Sumo-Nb-human
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis
map; C. Sequencing comparison results.***
**1.3. Construction of pET28a-Sumo-Nb-SCFV**
      Through antibiotic screening, colony PCR, and DNA sequencing
verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-SCFV in E.Coil DH5α and E.Coil BL21).
Figure 4A depicts the growth of transformed colonies on Kanamycin-resistant plates post-transformation.
Bacteria growing on the plate are the strains that may be successfully constructed. The colonies selected
from the plate served as PCR templates for colony PCR. Agarose gel electrophoresis was used to verify the
correctness of PCR products and the results show that the range of the target size is consistent with the
expectation (Figure 4B). Subsequently, the target-sized amplicons were sent for sequencing. According to
the
sequence results shown in Figure 4C, the target gene was successfully ligated with the vector without
obvious mutations, confirming the successful construction of the pET28a-Sumo-Nb-SCFV plasmid.
![Figure 4. The validation and sequencing of the plasmid pET28a-Sumo-Nb-SCFV](https://static.igem.wiki/teams/5735/results/figure-4.webp)
***Figure 4. The validation and sequencing of the
plasmid pET28a-Sumo-Nb-SCFV
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis
map;
C. Sequencing comparison results.***
**1.4. Construction of pET28a-Sumo-Nb-6**
      Through antibiotic screening, colony PCR, and DNA sequencing
verification, we verified the recombinant plasmids (pET28a-Sumo-Nb-6 in E.Coil DH5α and E.Coil BL21).
Figure
5A depicts the growth of transformed colonies on Kanamycin-resistant plates post-transformation. Bacteria
growing on the plate are the strains that may be successfully constructed. The colonies selected from the
plate served as PCR templates for colony PCR. Agarose gel electrophoresis was used to verify the
correctness
of PCR products, and the results show that the range of the target size is consistent with the expectation
(Figure 5B). Subsequently, the target-sized amplicons were sent for sequencing. According to the sequence
results shown in the Figure 5C, the target gene was successfully ligated with the vector without obvious
mutations, confirming the successful construction of the pET28a-Sumo-Nb-6 plasmid.
![Figure 5. The validation and sequencing of the plasmid pET28a-Sumo -Nb6](https://static.igem.wiki/teams/5735/results/figure-5.webp)
***Figure 5. The validation and sequencing of the
plasmid pET28a-Sumo -Nb6
A. Cloned strains on resistant plates; B. Colony PCR electrophoresis map;
C.
Sequencing comparison results.***
**1.5. Construction of pET28a-Sumo-UreB**
      Through antibiotic screening and DNA sequencing verification, we
verified the recombinant plasmids (pET28a-Sumo-UreB in E.Coil DH5α and E.Coil BL21). Figure 6A depicts the
growth of transformed colonies on Kanamycin-resistant plates post-transformation. Bacteria growing on the
plate are the strains that may be successfully constructed. Subsequently, We selected Single clony for
sequencing. According to the sequence results shown in Figure 6B, the target gene was successfully ligated
with the vector without obvious mutations, confirming the successful construction of the
pET28a-Sumo-UreB.
![Figure 6. The validation and sequencing of the plasmid pET28a-Sumo-UreB](https://static.igem.wiki/teams/5735/results/figure-6.webp)
***Figure 6. The validation and sequencing of the
plasmid pET28a-Sumo-UreB.
A. Cloned strains on resistant plates; B. Sequencing comparison
results.***

### Protein expression and validation

**2.1. SDS-PAGE was used to verify the protein expression level**
      After confirming that the strain was constructed correctly, we used
IPTG
as inducer to induce the expression of the target protein in the next step. After protein induction, we
broke the cell by physical and chemical methods, then extracted、separated and purified the protein inside
the cell.
      Finally, we verified whether the target protein was successfully
expressed by SDS-PAGE. The molecular weights of the corresponding target proteins (Nb-6, Nb-human Nb-scfv,
UreB) are 27.5 kDa, 34.1 kDa, 44.8 kDa and 14 kDa, respectively. After coomassie brilliant blue staining,
we
found that the results of SDS-PAGE were completely consistent with expectations. All four target
proteins(Nb-6(Figure 7A), Nb-human (Figure 7B), Nb-scfv(Figure 7C), UreB(Figure 7D)) were successfully
induced to express.
![Figure 7. SDS-PAGE was used to verify the protein expression](https://static.igem.wiki/teams/5735/results/figure-7.webp)
***Figure 7. SDS-PAGE was used to verify the protein
expression. A. The SDS-PAGE result of Nb-6;
B. The SDS-PAGE result of Nb-human; C. The SDS-PAGE
result of Nb-scfv; D. The SDS-PAGE result of UreB.***
**2.2. Protein concentration was determined by BCA method**
      Using the BCA (Bicinchoninic Acid Assay ) method, we determined the
concentration of the purified protein mentioned above. We applied the microplate reader to measure the
absorbance at a wavelength of 562 nm during this process and calculated the protein concentration of the
sample based on the standard curve. (Figure 8)
![Figure 8. The BCA result of the target proteins](https://static.igem.wiki/teams/5735/results/figure-8.webp)
***Figure 8. The BCA result of the target
proteins***
      By substituting the measured absorbance values of the samples (y)
using
this fitting equation, the concentrations of each protein (x) were calculated as follows:
**Table 1. The concentration of the target
protein**
![Table 1. The concentration of the target protein](https://static.igem.wiki/teams/5735/results/table-1.webp)
**2.3. Western blot was used to verify whether the protein was expressed**
      This SDS-PAGE result shows the protein which has the same weight as
the
target protein, but we still can't be sure the corresponding strip is the target protein. Our target
protein
has his tag; therefore we conducted western blot experiments using anti-his antibodies to further verify
to
confirm. Through western blot we found that the bands were indeed the target proteins and not any other
non-specific expression product (Figure 9). Experiments have proved that our antibody protein has been
successfully expressed. In this experiment, in order to improve the solubility of Ureb protein, we
introduced several of his tags into Ureb protein, so we did not verify the protein expression of Ureb by
WB.
However, based on the previous SDS-PAGE experiment, we basically confirmed that the Ureb protein was
successfully expressed.
![Figure 9. The Western blot result of Nb-human, Nb6, Nb-SCFV](https://static.igem.wiki/teams/5735/results/figure-9.webp)
***Figure 9. The Western blot result of Nb-human,
Nb6,
Nb-SCFV.***

### Functional Test

**3.1 Immunodiffusion assay of three recombinant nanobodies**
      We used the Oxford cup diffusion experiment to qualitatively evaluate
the binding specificity of the expressed antibody to the homologous antigen. As shown in Figure.10 (A, B,
C), after 36 hours of incubation, a slight white precipitation ring (protein stability may be impaired)
was
formed around the well plates loaded with three antibody preparations (Nb-6, Nb-SCFV, Nb-human),
indicating
that there was an interaction between urease and antibody. However, it can be found that the range of our
precipitation ring is very narrow, which also means that the binding effect of our antibody against
Bacillus
subtilis Ureb is not very significant. This may be because the antibody we designed is specific to Ureb of
Helicobacter pylori, and the homology of Ureb protein between Helicobacter pylori and Bacillus subtilis is
not particularly high, so our antibody may not have a particularly good binding effect on Ureb of Bacillus
subtilis.
![Figure 10. Oxford Cup test results](https://static.igem.wiki/teams/5735/results/figure-10.webp)
***Figure 10. Oxford Cup test results. A: Nb-6; B:
Nb-SCFV;C: Nb-human.
(The number means control and different concentration of the recombinant
nanobody：1：Blank 2：Negative control 3：25μg/ml antibody 4：200μg/mL antibody)***

**3.2 Nanobody-mediated urease inhibition assay of three recombinant nanobodies**
      We assessed the inhibitory potency of recombinant nanobodies against
urease. After incubation, urea and phenol red were added, and the extent of urease inhibition was
quantified
colorimetrically.
      The experimental results show that the rate at which urea is
decomposed
by urease in the experimental group with antibody is almost the same as that in the control group. This
shows that our antibody binding effect on Ureb of Bacillus subtilis is poor. Consistent with the above
analysis, our antibody is specific to Helicobacter pylori, so its inhibitory effect on Bacillus subtilis
is
not good. This means that if our antibody is used to target Ureb of Helicobacter pylori, it may have a
better experimental effect. Unfortunately, due to the limitation of biological safety, we can only
simulate
the binding of our antibodies to Helicobacter pylori by computer.
![Figure 11. Colorimetric changes and quantitative analysis of urease-recombinant nanobody interaction](https://static.igem.wiki/teams/5735/results/result-11.webp)
***Figure 11. Colorimetric changes and quantitative
analysis of urease-recombinant nanobody interaction***

### Computer simulation of antibody binding to Helicobacter pylori UreB

**4.1. The mimic antibody binds to Helicobacter pylori Ureb by molecular docking**
      In this experiment, the model is a very important part of our
experiment. Due to the consideration of biological safety, we can't use Ureb of Helicobacter pylori in
practical experiments, so we tested the binding effect of nano-antibody and Ureb of Helicobacter pylori by
molecular docking. Through computer simulation, we deeply analyzed the potential inhibitory effect of our
nano-antibody on Helicobacter pylori and established a good model foundation for subsequent
experiments.
      The 3D position of Ureb protein after interaction with three
antibodies
is shown in the figure. Among them, the green protein is UreB protein, the blue protein is Nb6 protein
(Figure12A), the pink protein is Nb-Human protein (Figure12B) and the purple protein is Nb-ScFv protein
(Figure12C), and the bonding situation among the residues has been marked in the figure, in which the red
dotted line represents hydrogen bond interaction, the yellow dotted line represents salt bridge
interaction,
and the number near the dotted line of the interaction bond is the bond length of the interaction bond in
Amy. Through the comprehensive analysis of these interactions, we can screen out the nano-antibody protein
with the strongest binding ability.
![Figure 12 Interaction between Ureb protein and nano-antibody protein](https://static.igem.wiki/teams/5735/results/figure-12.webp)
***Figure 12 Interaction between Ureb protein and
nano-antibody protein. A. Schematic diagram of the interaction between UreB protein and Nb-6 protein,
in
which UreB protein is shown in green and NB6 protein is shown in blue. B. Schematic diagram of
interaction between UreB protein and Nb-Human protein, in which UreB protein is shown in green and
NB-human protein is shown in pink. C. Schematic diagram of interaction between UreB protein and
Nb-ScFv
protein, in which UreB protein is shown in green and Nb-ScFv protein is shown in
purple.***
      Using the Interface analyzer module of FoldX, the binding energies of
UreB protein with three proteins are shown in the following table(Table 2), among which UreB protein has a
strong binding tendency with Nb-SvFc protein and Nb-Human protein (protein-protein binding energy is less
than -25 kcal/mol, which means that there is a strong binding tendency). The interaction of three pairs of
proteins can be further observed in combination with subsequent molecular dynamics simulation.
**Table 2 The binding energies of UreB protein with
three
proteins**
![Table 2 The binding energies of UreB protein with three proteins](https://static.igem.wiki/teams/5735/results/table-2.webp)

**4.2. The mimic antibody binds to Helicobacter pylori Ureb by molecule dynamics**
      RMSD curve shows the changes of roots mean square displacement of
three
proteins relative to UreB protein in the simulation process and is used to evaluate the stability of
binding
between proteins in this study. From the graph analysis, it can be seen that the RMSD curve of Nb6 protein
relative to UreB protein shows a certain fluctuation trend in the first 80ns of the simulation. After 80
ns,
the RMSD curve gradually converges and vibrates around 0.8nm until the end of the simulation. The RMSD
curve
of Nb-ScFv protein relative to UreB protein experienced a certain fluctuation trend in the first 60ns of
the
simulation, and gradually converged after 60ns, vibrating around 0.5nm until the end of the simulation.
The
RMSD curve of Nb-Human protein relative to UreB protein showed a rapid increase in the first 20ns of the
simulation, and then gradually converged, vibrating around 1.2nm until the end of the simulation.
![Figure 13 RMSD Curve of Ureb and Three Proteins in Simulated Docking Process](https://static.igem.wiki/teams/5735/results/figure-13.webp)
***Figure 13 RMSD Curve of Ureb and Three Proteins in
Simulated Docking Process.***

      Nb-6 protein is composed of more flexible random curls at the
interaction interface in the first 80ns of simulation, and the interface is pulled by UreB protein in the
process of dynamic simulation, so the protein interaction interface maintains a certain conformational
adjustment in the first 80 ns of simulation, which leads to a certain fluctuation trend of RMSD curve.
After
80 ns, with the interaction between proteins gradually stabilized, and there was no obvious conformational
change again, the skeleton structure of the two proteins gradually stabilized, only maintaining a certain
change in the mutual orientation of side chains. Therefore, after 80 ns, the RMSD curve gradually
converged
and vibrated around 0.8 nm until the end of the simulation. It can be considered that Nb6 protein and UreB
protein formed a stable complex after 100ns molecular dynamics simulation.
![Figure 14 Schematic Diagram of Conformation Rendering of UreB-Nb6 Protein Complex](https://static.igem.wiki/teams/5735/results/result-14.webp)
***Figure 14 Schematic Diagram of Conformation
Rendering of UreB-Nb6 Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and blue
shows Nb6 protein.***
      Nb-ScFv protein changed little relative to UreB protein in the whole
simulation process, so the RMSD value was low in the whole kinetic simulation process. In the first 60 ns
of
the simulation, the RMSD curve kept a certain fluctuation trend, which was due to the fact that although
the
skeleton atoms of the two proteins did not change obviously at the interaction interface, the orientation
of
the side chains changed to some extent, so the RMSD curve still showed a certain fluctuation trend. After
60ns, as the interaction mode between the two proteins tends to be stable, the RMSD curve also tends to
converge and vibrates around 0.5nm until the end of the simulation.
![Figure 15 Schematic Diagram of Conformation Rendering of UreB-Nb-ScFv Protein Complex](https://static.igem.wiki/teams/5735/results/result-15.webp)
***Figure 15 Schematic Diagram of Conformation
Rendering of UreB-Nb-ScFv Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and
purple shows Nb-ScFv protein.***
      In the first 20 ns of the simulation, Nb-Human protein appears to be
close to UreB protein, which is most obviously marked in the above figure. In the first 20ns of the
simulation, Nb-Human protein tends to twist and fold spontaneously under the action of UreB protein, and
this conformation is maintained until the end of the simulation. After 20ns, the interaction between the
two
proteins tends to be stable, and the skeleton atoms do not change obviously again, so the RMSD curve tends
to converge gradually. During this period, the vibration of the RMSD curve still comes from the mutual
orientation change of the side chains of the two proteins, and the whole two proteins tend to form stable
complexes.
![Figure 16 Schematic Diagram of Conformation Rendering of UreB-Nb-Human Protein Complex](https://static.igem.wiki/teams/5735/results/result-16.webp)
***Figure 16 Schematic Diagram of Conformation
Rendering of UreB-Nb-Human Protein Complex at 0 or 100ns. Among them, green shows UreB protein, and
pink
shows Nb-Human protein.***
      Combined with the analysis of the movement trajectory of the protein
complex, it can be seen that the three proteins have no obvious displacement relative to the UreB protein
during the whole simulation process, so the RMSD value is relatively stable. Similarly, in the early stage
of the simulation, the structure of the interaction between the three proteins and UreB protein has
changed
to some extent, so the RMSD curve shows a certain fluctuation trend in the first certain period of the
simulation, and then gradually converges with the dynamic simulation. On the whole, after 100ns molecular
dynamics simulation, all three proteins formed a stable complex with UreB protein, which can be combined
with subsequent analysis to further observe the binding behavior of the three proteins.
      RMSF analysis showed the structural adaptability of each residue in
protein, which was used in this study to analyze the flexibility and movement intensity of amino acid
residues in protein during the whole simulation process. It can be seen from the figure that the overall
RMSF of the protein remained at a low level during the whole simulation process. The reason why the RMSF
at
the N-terminal and C-terminal of the protein is large is that this residue is at the head end of the
protein
and is less constrained by other domains in the protein, which leads to greater flexibility and has a
certain impact on its own stability. In addition, the reason why the rest of the protein RMSF is large may
be that there is some disturbance due to the combination of small molecules, or because the peptide itself
is flexible and has some disturbance during the simulation.
      By comparing the RMSF curves of three protein complexes, it can be
seen
that the RMSF value of Nb-ScFv protein and Nb-Human protein is higher than that of Nb-6 protein after
forming complexes with Ureb protein. Which may be due to the stronger interaction between Nb-ScFv protein
and Nb-Human protein and UreB protein, so the flexible residues at the interaction interface are more
affected, which leads to the more obvious flexible jitter of different residues of Ureb protein. At the
same
time, it was observed that the RMSF value of N-terminal of Nb-Human protein increased obviously. Combined
with the above analysis, it can be seen that this is because the Nb-Human protein moved towards UreB
protein
in the first 20ns of simulation, so the RMSF value of this random curly segment increased, which is
consistent with the above analysis.
      On the whole, the RMSF values of the three proteins are low in the
simulation process, so it can be considered that the proteins have little jitter in the simulation
process,
keep stable vibration in the solution environment, and the sampling and analysis are reliable.

![Figure 17 RMSF curve of each protein in the simulation process](https://static.igem.wiki/teams/5735/results/result-17.webp)
***Figure 17 RMSF curve of each protein in the
simulation process. A. The RMSF curve of Ureb. B. The RMSF curve of Nb-6. C. The RMSF curve of
Nb-ScFv.
D. The RMSF curve of Nb-Human.***
      The Radius of Gyration (Rg) can be used to characterize the
compactness
of protein structure, and it can also be used to characterize the change of protein's peptide chain
looseness in the simulation process. Combined with the above analysis and Rg diagram analysis, it can be
seen that the Rg curves of the complexes formed by Nb6 protein and Nb\_ScFv protein with UreB protein
respectively remained stable throughout the simulation process and changed within 2.5nm and 2.8nm
respectively until the end of the simulation. After the complex of Nb\_Human protein and UreB protein was
formed, the Rg curve showed an upward trend in the first 20ns of the simulation, and then it tended to
converge, maintaining the vibration around 2.75nm until the end of the simulation.
      Combined with the above analysis, it can be seen that after Nb6
protein
and Nb-ScFv protein form complexes with UreB protein, the protein complexes remain relatively stable
during
the whole dynamic simulation process. As mentioned in the aforementioned RMSD analysis, only the mutual
orientation of the residues at the interaction interface of the two proteins changes during the simulation
process, so the RMSD curve remains in a low state, showing a certain fluctuation trend. As the skeleton
atoms of the proteins do not change obviously as a whole, the Rg curve does not change significantly, but
only keeps a certain vibration until the end of the simulation. After the complex of Nb-Human protein and
UreB protein was formed, the random curl of Nb-Human protein was close to UreB protein in the first 20 ns
of
simulation, and the overall compactness became larger, so the Rg curve increased in the first 20 ns of
simulation. After 20ns, with the convergence of the two protein complexes after exercise, the Rg curve
also
tends to be stable, vibrating around 2.75 nm until the end of the simulation.
      On the whole, the Rg curve of protein is stable in the simulation
process and the sampling is reliable.
![Figure 18 Radius of Gyration](https://static.igem.wiki/teams/5735/results/figure-18.webp)
***Figure 18 Radius of Gyration***

      We analyzed the changes of the number of hydrogen bonds formed between
proteins with the simulation time and systematically investigated the stability of protein binding from
the
perspective of interaction. From the graph analysis, it can be seen that the number of hydrogen bonds
formed
between Nb-6 protein and Nb-ScFv protein and UreB protein respectively remained relatively stable and had
a
certain amplitude during the whole simulation process. The number of hydrogen bonds formed between
Nb-Human
protein and UreB protein showed a certain fluctuation trend in the first 40ns of the simulation, and
gradually increased as a whole, and stabilized after 40ns until the end of the simulation.

      Based on the above analysis and the analysis of the protein complex's
trajectory, it can be seen that although Nb-6 protein and Nb-ScFv protein have no obvious displacement
relative to UreB protein during the whole simulation process, due to the conformational adjustment between
the residues at the interaction interface of the two proteins, accompanied by the residue change and
flexible jitter of the protein flexible binding cavity, this movement leads to the continuous formation
and
destruction of hydrogen bonds between the residues at the protein interaction interface, which leads to a
certain vibration of the hydrogen bond number curve with the change of simulation time, and the whole is
relatively stable; Nb-Human protein appeared close to UreB protein in the first 20ns of the simulation,
and
maintained this conformation until the end of the simulation, so the number of hydrogen bonds formed with
UreB protein showed a certain fluctuation trend in the first 40ns of the simulation, and the whole showed
an
upward trend due to the increase of protein interaction interface. After 40ns, with the gradual
convergence
of the residues at the interaction interface between the two proteins, the number curve of hydrogen bonds
tends to be stable until the end of the simulation.
      In addition, it can be noted that although the number curve of
hydrogen
bonds changes obviously with the simulation, there are hydrogen bonds between the two proteins at all
times,
which explains that the binding between proteins is stable from the perspective of force.

![Figure 19 The change of the number of hydrogen bonds formed between proteins with the simulation time](https://static.igem.wiki/teams/5735/results/figure-19.webp)
***Figure 19 The change of the number of hydrogen
bonds
formed between proteins with the simulation time.***

      After eliminating the cycle of the simulated trajectory, combined with
the aforementioned RMSD analysis, the movement trajectory of protein complex in the period of 90ns-100ns
was
extracted for binding free energy (MM/GBSA) analysis, and the energy term was decomposed.
      The results showed that the binding energy between protein and UreB
was
mainly van der Waals energy (Nb6 protein was -88.53 kcal/mol, Nb-ScFv protein was -109.01 kcal/mol,
Nb-Human
protein was -139 kcal/mol), and the electrostatic energy of Nb-6 protein was -26.58 kcal/mol, Nb-ScFv
protein is -40.13 kcal/mol and Nb-Human protein is -43.19 kcal/mol), which indicates that electrostatic
interaction is beneficial to protein binding, and proteins interact mainly through van der Waals
interaction. The aforementioned hydrogen bond analysis and force analysis show that there are certain van
der Waals interactions and hydrophobic interactions between different amino acids of each protein, so van
der Waals can significantly facilitate the binding between proteins and occupy a dominant position; At the
same time, there is a certain electrical property at the protein-protein interaction interface, so there
is
a certain electrostatic interaction at the protein-protein interaction interface, which leads to the
contribution of electrostatic energy to the binding. In addition, we noticed that the solvation energies
of
Nb6 protein, Nb-ScFv protein and Nb-Human protein were 26.53 kcal/mol, 41.46 kcal/mol and 42.60 kcal/mol
respectively in this simulation, suggesting that the addition of solvents was not conducive to the binding
between proteins.
      The overall binding energy of Nb-6 protein is -88.59 kcal/mol, Nb-ScFv
protein is -107.68 kcal/mol, and Nb-Human protein is -139.59 kcal/mol), which indicates that the binding
tendency of proteins is relatively large, and the relative order of binding tendency is Nb-Human > Nb-ScFv
>
Nb-6, which further explains the binding between proteins from the perspective of energy.

      Based on the above computer simulation analysis, our nano-antibody has
a
good binding effect on Ureb protein of Helicobacter pylori in theory, among which Nb-Human has the best
binding effect. Unfortunately, due to the limitation of biological safety, we can't directly verify the
inhibitory effect of antibodies on Helicobacter pylori. Later, we may consider cooperating with more
professional research institutions to verify the actual inhibitory effect of our antibodies on
Helicobacter
pylori.

![Figure 20 Analysis of binding free energy (MM/GBSA)](https://static.igem.wiki/teams/5735/results/figure-20.webp)
***Figure 20 Analysis of binding free energy
(MM/GBSA)***

***Table 3 Energy analysis in simulation
process***
![Table 3 Energy analysis in simulation process](https://static.igem.wiki/teams/5735/model/model-table2.webp)

### Transforming plasmid into probiotics

**5.1. Construction of the pGEX4T-1-Nb-human plasmid**
      Based on preliminary experimental results, among the three recombinant
nanobodies, the antibody encoded by the Nb-human gene exhibited the strongest urease inhibitory activity.
Given this finding, we selected this gene for cloning into the pGEX4T-1 expression vector to achieve
optimal
expression in Escherichia coli Nissle 1917(probiotic).

      Following the same procedure as the construction of
pET28a-Sumo-Nb-human, we digested the target gene amplification product and the pGEX-4T-1 plasmid with
restriction enzymes, followed by ligation using T4 DNA ligase. The ligation product was then transfered
into
DH5α competent cells, and through DNA sequencing，we verified the success of the construction of the
pGEX4T-1-Nb-human plasmid(Figure 21).

![Figure 21. The validation and sequencing of the plasmid pGEX4T-1-Nb-human](https://static.igem.wiki/teams/5735/results/figure-21.webp)
***Figure 21. The validation and sequencing of the
plasmid pGEX4T-1-Nb-human. A: The transformed strain growing on the resistant plate; B: The comparison
results of the target genes of the positive cloned strains after sequencing.***

**5.2. Protein expression in Escherichia coli Nissle 1917**
      Then, we transferred the pGEX4T-1-Nb-human expression plasmid into
Escherichia coli Nissle 1917 competent cells, induced the expression of the target protein by 1mM IPTG and
purified the acquired Nb-human protein using nickel affinity purification. SDS-PAGE results (Figure 22)
indicated that the antibody protein (34.1 kDa) encoded by Nb-human was successfully expressed in the EcN
1917.

![Figure 22. SDS-PAGE electrophoresis gel of Nb-human protein expressed in EcN 1917](https://static.igem.wiki/teams/5735/results/figure-22.webp)
***Figure 22. SDS-PAGE electrophoresis gel of
Nb-human
protein expressed in EcN 1917.***

### Future plan

1. In this study, recombinant antibodies were induced for expression with IPTG at an appropriate
temperature. However, the obtained recombinant nanobodies exhibited lower purity and concentration than
expected. Although the urease inhibitory activity of these nanobodies was confirmed, the suboptimal yield
may affect subsequent antibody potency assessments. To obtain high-quality antibodies, a systematic
purification strategy must be implemented.
2. In the experiment, due to the limitation of biological safety, we can't use Ureb of Helicobacter pylori
for the experiment, so we use Ureb of Bacillus subtilis as a substitute. However, because our antibody is
specific to Helicobacter pylori, even in computer simulation, it has a good binding effect on Ureb of
Helicobacter pylori. But in order to draw an accurate conclusion, we may consider cooperating with more
professional research institutions to verify the actual inhibitory effect of our antibodies on
Helicobacter
pylori. In this experiment, we utilized purified UreB as the substrate to assess the activity of the
recombinant nanobodies. Subsequent studies should apply these nanobodies directly to Helicobacter pylori
to
evaluate their ability to inhibit bacterial growth and proliferation, thereby strengthening the
credibility
of our findings.

### Reference

[1] Hooi JKY, Lai WY, Ng WK, Suen MMY, Underwood FE, Tanyingoh D, et al. Global Prevalence of Helicobacter
pylori Infection: Systematic Review and Meta-Analysis. Gastroenterology. 2017;153:420-9.
[2] Malfertheiner P, Camargo MC, El-Omar E, Liou J-M, Peek R, Schulz C, et al. Helicobacter pylori
infection. Nature Reviews Disease Primers. 2023;9.
[3] Eaton KA, Brooks CL, Morgan DR, Krakowka S. Essential role of urease in pathogenesis of gastritis
induced by Helicobacter pylori in gnotobiotic piglets. Infect Immun. 1991;59:2470-5.
[4] Almarmouri C, El-Gamal MI, Haider M, Hamad M, Qumar S, Sebastian M, et al. Anti-urease therapy: a
targeted approach to mitigating antibiotic resistance in Helicobacter pylori while preserving the gut
microflora. Gut Pathogens. 2025;17.
[5] Mobley HL. The role of Helicobacter pylori urease in the pathogenesis of gastritis and peptic
ulceration. Aliment Pharmacol Ther. 1996;10 Suppl 1:57-64.

![vertical line](https://static.igem.wiki/teams/5735/img/desc/p2-right-vertical-line.webp)
![返回顶部](https://static.igem.wiki/teams/5735/img/desc/p2-right-mini.webp)

{% endblock %}