iGEM 2024 — Team testdaily-i-beijing

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# 20240408-20240502: Molecular Biology Experiments

| Molecular biology experiments | |
| --- | --- |
| Overview | Specific content |
| Resuscitation strains | Resuscitation of pet28a-BMP-4-DH5, pet28a-VEGF DH5 |
| Extract plasmid, prepare target gene fragment by PCR, cut pET28a plasmid by nco1&xho1, connect | The gel concentration after enzyme digestion of plasmid is extremely low |
| PCR obtained FTD, pet28a backbone, BMP2 sequence, and electrophoresis verification | The PCR template for the fragment used for seamless cloning is FTD and BMP-4 produced by the primers P above. |
| PCR was used to prepare FTD and BMP-4 templates, and PCR was used again to prepare SM FTD and BMP-4 sequences. | ![](https://static.igem.wiki/teams/5374/notebook/fig1-1.png) BMP-4 SMBMP-4 M (from bottom to top: 100 250 500) FTDD SMFTD |
| PCR preparation of pet28a template | ![](https://static.igem.wiki/teams/5374/notebook/fig1-2.png) Gradient PCR determines the annealing temperature of vector preparation. As the temperature rises, large fragments increase and small fragments decrease. |
| Preparation of pet28a backbone, SM-FTD, SM-BMP-4, gel excision recovery/PCR product purification and seamless cloning | ![](https://static.igem.wiki/teams/5374/notebook/fig1-3.png) |
| There are a lot of colonies on the plates for seamless cloning BMP-4 and FTD, but there are also colonies on the negative control plate, but they are very few in comparison. |  |
| Colony PCR verification | ![](https://static.igem.wiki/teams/5374/notebook/fig1-4.png) 1~7: BMP-4 No. 1~7; 8~12: FTD No. 1~5; 13: marker; 14~15: FTD No. 6~7; 16~19: seamless cloning negative pair; 20~21: PCR negative pair (I don’t know why the 16~19 seamless cloning negative pair also exploded, but it can be obtained by sequencing the connection product obtained earlier, so it’s not a big problem) |
| Top 10 strains of B-pet28a and FTD-pet28a were stored and plasmids were extracted | Transform FTD-pet28a and BMP2-pet28a |
| into six expression competent cells (BL21 rosetta shuffleT7, B&k12 AE, BL21Plyss) | FTD's have all grown out, B's BL21plyss and rosetta have not grown out, waiting to be re-done ![](https://static.igem.wiki/teams/5374/notebook/fig1-5.png) |
| Re-transfer of BL21plyss and rosetta to BMP-4 | All the re-transplanted ones have grown out, pick a single colony |
| Colony PCR | ![](https://static.igem.wiki/teams/5374/notebook/fig1-6.png) T7 primer amplified BMP-4 fragment: 531bp; T7 primer amplified FTD fragment: 882bp; T7 primer amplified sfGFP fragment: 880bp  (marker: Sangon Biotech 2K)  From left to right in the above picture: AE, BL21, R, P, ShuffleT7B, ShuffleT7K12, BL21 is the FTD gene, and the rest are BMP-4 genes  ![](https://static.igem.wiki/teams/5374/notebook/fig1-7.png) T7 primer amplified BMP-4 fragment: 531bp; T7 primer amplified FTD fragment: 882bp  (marker: Sangon Biotech 2K) ShuffleT7K12 |
| Fermentation test to see who can express soluble protein better | Collect bacteria, crush |

# 20240520-20240615: Protein Fermentation and Purification

![](https://static.igem.wiki/teams/5374/notebook/fig2-1.png)

The cultured bacteria were collected, ultrasonically broken one by one, and centrifuged at 17000xg. The samples
were whole bacteria, supernatant, and precipitate.

## Solarbio marker

The lanes on the right of the marker are divided into four groups, one of which is BL21, AE, ORIGAMI, and
SHUFFLEYT7. The four groups are whole bacteria, supernatant, and precipitate. Except for the supernatant group of
shuffleT7, which was not diluted, the samples in the other 11 lanes were diluted twice.

I found that BL21 could not express and the yield of shuffleT7 was too low. Both rosetta and origami had proteins
in the supernatant, but considering the growth rate, I chose AE because rosetta grows faster.

![](https://static.igem.wiki/teams/5374/notebook/fig2-2.png)

## Concentration (ug/ml)

275.9038 FTDBMP4 concentrate

10.65392 50KDA concentrate↑

# 20240701-20240910: Cell Characterization Experiments

![](https://static.igem.wiki/teams/5374/notebook/fig2-3.png)

See the figure for the plating design and related calculations

The lyophilized powder was dissolved in 1 mL of tris-hcl (pH 8.0)

#### 1. Biocompatibility:

Live and dead fluorescence staining d1

Cell proliferation (such as CCK8) d1d3d7

#### 2. Cell attachment:

Cytoskeleton and cell nucleus immunofluorescence staining d1

Observe cell morphology using SEM after gradient dehydration of cells d1

#### 3. Osteogenic differentiation:

Alkaline phosphatase expression images d1d7d14

Alizarin red staining d1d7d14

![](https://static.igem.wiki/teams/5374/notebook/fig2-4.png)

## Cell culture, digestion and cryopreservation

(1) Growth medium: α-MEM +10%FBS+1%PS

Osteogenic induction medium (chemical): Growth medium plus 10 mM sodium glycerophosphate, 60 mg/L ascorbic acid,
dexamethasone 100 nM (valid for 1 month, see next page for specific amounts)

(2) Cell digestion: remove the culture medium, add 5-10ml PBS to wash once, add 3ml pancreatin to the T75 bottle,
digest in the incubator for 3min, stop digestion with 5ml growth medium, and centrifuge at 1000rpm for 5min.

(3) Cryopreservation solution preparation DMSO:FBS:culture medium = 1:2:7, cell volume 1 x106/ml cryopreservation
solution,

First, mix 2 parts of serum, 2 parts of culture medium and 1 part of DMSO evenly, and then slowly add the mixture
to the cell-culture medium system to achieve a slow increase in DMSO concentration to reduce DMSO damage to
cells.

Use gradient cryopreservation boxes for freezing. Gradient cryopreservation boxes need to be restored to room
temperature before use. After being placed in the cryopreservation box for at least 7 hours, they can be transferred
to liquid nitrogen for storage.

## 2. Recommended cell inoculation volume

Cell culture plate specifications Cell inoculation volume (/well) Culture medium volume Related experiments

96-well 1x104 200 μl CCK 8. Live-death staining

24-well 5x104 1ml Differentiation (staining)

6-well 2x105 1.5ml-2ml Differentiation (mRNA)

T75 bottle 1 x106 (rats grow 3x106; humans grow 1.8 x106) 12-15ml Cell culture

White cell culture plate packaging paper is an adherent culture plate, and green is an anti-adhesive plate!!

## 3. Osteogenesis-related detection

Observe trend changes (first experiment), set time points for 1, 4, 7, 14, 21, and 28 days for detection

If there is no need to observe trends and mature experimental systems, 14 days can be selected as the detection
time point (except Alizarin Red)

Detection Recommended reagent brand Time point (optional)

ALP quantitative detection Beyotime P0321S 14 days<

Calcium content detection Nanjing Jiancheng C004-2-1 21 days/28 days

dsDNA (normalized Yisheng 12641ES03 Same as ALP/calcium content detection

ALP staining Beyotime C3206 14 days

Alizarin red staining Solarbio 21 days/28 days

Immunofluorescence RUN-2, BMP 2, Collagen I, Abcam 14 days

OCN, OPN

50mL osteogenic culture medium requires:

108mg sodium β-glycerophosphate

3mg ascorbic acid

1.96ug dexamethasone

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# 1 Overview of DBTL Cycles in Our Project

Our project is focused on the development of an innovative dual-cell-factor release system designed for bone repair. This system is engineered to address the shortcomings of existing bone repair materials, including issues such as unregulated cell factor release and inadequate biocompatibility. To achieve a functional and optimized system, we have conducted three iterative cycles of the **Design-Build-Test-Learn (DBTL) process**. This approach has enabled us to develop a system that facilitates the sequential and controlled release of Vascular Endothelial Growth Factor (VEGF) and Bone Morphogenetic Protein-4 (BMP-4). This mimics the body's natural healing cascade, where angiogenesis precedes osteogenesis

![](https://static.igem.wiki/teams/5374/enginsuc/fig-1-1.png)

Fig 1.1 Schematic diagram of this project

Synthetic Biology applies engineering principles to the design of biological systems, endowing organisms with tailored functions. It involves the systematic redesign, reconstruction, and even de novo synthesis of biological components. The field's development is characterized by an iterative approach that adheres to the Design-Build-Test-Learn (DBTL) cycle, ensuring a continuous refinement of the engineered systems.

Our project consists of three iterative Design-Build-Test-Learn (DBTL) cycles aimed at developing a dual cell factor release system for bone repair:

**DBTL Cycle 1:** We screened and identified suitable collagen-binding domains (CBDs) for VEGF and BMP-4.

**DBTL Cycle 2:**We developed a dual cell factor release system for sequential cell factor delivery using the selected CBDs.

**DBTL Cycle 3:** We created a low-endotoxin collagen hydrogel using E. coli ClearColi, ensuring biocompatibility and controlled release.

In our project, **Cycle 2** and **Cycle 3** are executed concurrently, each targeting the development of a distinct core component of our bone repair material at different scales. **Cycle 2** is dedicated to the **microscale**, with the primary objective of designing the engineering cell factors that stimulate angiogenesis and osteogenesis. Concurrently, **Cycle 3** is concentrated on the **macroscale**, where the focus is on developing a collagen hydrogel. This hydrogel not only fills the bone defect site but also acts as a carrier for the delivery of the engineered cell factors. Collectively, these cycles are designed to synergize, with Cycle 2 enhancing the bioactivity of the agents and Cycle 3 perfecting the scaffold for their delivery, culminating in an integrated bone repair system.

# 2 DBTL Cycle 1: Screening Collagen-Binding Domains (CBDs)

## 2.1 Design

The goal of the first cycle was to identify suitable **collagen-binding domains (CBDs)** for the sequential release of **VEGF** and **BMP-4**. Different CBDs were fused with Enhanced Green Fluorescent Protein (EGFP) to screen for those that minimally impact the bioactivity of the fusion proteins and can control the binding to collagen. We chose pET28a plasmid as the vector and Escherichia coli BL21 as the host cell. Below is the collagen binding module and plasmid design we selected.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-2-1.png)

Fig 2.1 Plasmid design

**SPARC/OD-EGFP:**
SPARC (Secreted Protein Acidic and Rich in Cysteine) and OD (Osteonectin Domain): This domain is involved in regulating the interaction between cells and the extracellular matrix (ECM), particularly binding to collagen and influencing matrix remodeling. It plays a role in tissue repair and bone mineralization.

**CBD MMPs-EGFP:**
CBD MMPs (Collagen-Binding Domain from Matrix Metalloproteinases): MMPs are enzymes responsible for the breakdown of ECM components like collagen. This domain has a strong affinity for collagen, making it suitable for applications requiring high binding affinity.

**DB-EGFP:**
DB (Discoidin Domain): Found in various ECM proteins, this domain is known for its collagen-binding properties. It’s important in cell-matrix interactions and tissue organization.

**DDR-EGFP:**
DDR (Discoidin Domain Receptor): DDRs are receptor tyrosine kinases activated by collagen. They are involved in regulating ECM remodeling and cell adhesion, with strong collagen-binding capabilities.

**AGR-EGFP:**
AGR (Agrin): A multi-domain proteoglycan involved in the organization of the ECM and synapse formation. It binds to collagen and influences cellular interactions with the ECM.

**COMP-EGFP:**
COMP (Cartilage Oligomeric Matrix Protein): COMP is a key ECM protein that binds collagen and stabilizes matrix structures. It plays a role in tissue integrity, especially in cartilage and tendons.

**VWF-A Domain-EGFP:**
VWF-A (von Willebrand Factor A Domain): This domain is known for mediating collagen binding, particularly in blood clotting and platelet adhesion. It is essential for collagen interaction in the vascular system.

**FTD-EGFP:**
FTD (Fibrinogen-like Domain): FTD is commonly found in proteins involved in coagulation and ECM remodeling, where it plays a role in binding collagen and other matrix components.

## 2.2 Build

We constructed several **CBD-EGFP fusion proteins expressing plasmids** and expressed them in bacterial systems to analyze their activity.

We first commissioned a company to synthesize the gene sequences of each CBD and then used seamless cloning to connect the gene sequences of each CBD with the EGFP sequence already in the laboratory.

In this **stage**, the colony PCR results show successful amplification of the eight different **CBD-EGFP fusion proteins**, confirming the correct construction of the engineered strains. The bands visible in the gel correspond to the expected sizes of the fusion proteins:

* **SPARC/OD**, **DB**, and **DDR** are shorter sequences. After seamless cloning with **EGFP**, their expected PCR product size is approximately **800 bp**.
* The remaining five CBDs—**CBD MMPs, AGR, COMP, VWF-A, and FTD**—are longer tags. After seamless cloning with **EGFP**, their expected PCR product size is around **1200 bp**.

The PCR bands in the gel align well with these predicted sizes, demonstrating that each fusion construct was correctly assembled, which is crucial for proceeding with protein expression and purification.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-2-2.png)

Fig 2.2 Plasmid design

We then used these E. coli to express proteins and measured the fluorescence value of each protein host bacteria at the same bacterial concentration. Some of the fusion proteins were then purified by Ni-NTA for further analysis.

## 2.3 Test

The fluorescence activity of each fusion protein was tested to evaluate whether the CBDs affected EGFP activity. The relative fluorescence values of the eight **CBD-EGFP fusion proteins** were measured to assess the impact of the collagen-binding domains (CBDs) on EGFP activity. As shown in the graph:

* **SPARC/OD-EGFP, CBD MMPs-EGFP, DB-EGFP, VWF-A Domain-EGFP**, and **FTD-EGFP** exhibited fluorescence values close to or higher than that of the control **EGFP**, indicating that these CBDs did not significantly affect the bioactivity of EGFP.
* **DDR-EGFP, AGR-EGFP,** and **COMP-EGFP** showed slightly lower fluorescence values, suggesting some impact on EGFP activity, but still maintaining measurable fluorescence.

Overall, the majority of CBDs retained sufficient EGFP activity, which confirms that they can be used in further experiments for controlled release without significantly impairing the functionality of the fusion proteins. These results support the selection of CBDs that balance collagen binding with maintaining protein bioactivity.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-2-3.png)

Fig 2.3 The fluorescence activity of each fusion protein

Binding affinity tests (via microscale thermophoresis) were also conducted to determine which CBDs had high binding affinity (for BMP-4) and low binding affinity (for VEGF). Based on the binding experiment shown in the graph, the results illustrate the relative binding affinities of different **CBD-EGFP fusion proteins** to collagen, with the binding affinity represented by the dissociation constant **(Kd)**. The **midpoint of each curve** on the x-axis corresponds to the Kd value, indicating how tightly the CBDs bind to collagen.

* **FTD-EGFP** shows the strongest binding to collagen, with the lowest Kd value. From the graph, the FTD curve's midpoint is approximately **8.3\*10^-7 M**, indicating a high binding affinity.
* **CBD MMPs-EGFP** exhibits the weakest binding to collagen, with the highest Kd value. The midpoint of the CBD MMPs curve is approximately at **6.7\*10^-6 M**, indicating a lower binding affinity compared to the other CBDs.

These Kd values highlight that **FTD** is the most suitable for applications requiring strong binding, while **CBD MMPs** may be used in situations where weaker, more transient binding is needed.

## 2.4 Learn

From the binding results, FTD was chosen for BMP-4 due to its high affinity for collagen, ensuring a more sustained release. Meanwhile, CBD MMPs were selected for VEGF because of their low affinity, allowing for a faster and more transient release. These selections laid the foundation for the next cycle, where these domains were fused with the respective cell factors to achieve controlled and sequential release, mimicking natural bone healing processes.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-2-4.png)

Fig 2.4 What we learned in Cycle 1

# 3 DBTL Cycle 2: Developing the Dual Cell Factor Release System

## 3.1 Design

Based on the selections from Cycle 1, the second cycle aimed to design a dual cell factor release system. The goal was to ensure that VEGF, responsible for promoting angiogenesis, is released first with a low-affinity collagen-binding domain (CBD MMPs), followed by the controlled, delayed release of BMP-4 for osteogenesis, using the high-affinity binding domain (FTD). This sequential release would better mimic natural bone healing processes.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-3-1.png)

Fig 3.1 Fusion Cell factor expression plasmid

## 3.2 Build

In the **Build stage** of **Cycle 2**, the primary objective was to successfully express and purify two functional fusion proteins: **CBD MMPs-VEGF** and **FTD-BMP-4**, which are critical for the controlled release system. The key to achieving the desired gradient release lies in the intrinsic binding affinity of the **collagen-binding domains (CBDs)** to the collagen matrix. Since the release timing depends on the CBDs’ strength of interaction with collagen, we could focus solely on the expression of the cell factors without needing to alter the hydrogel’s crosslinking properties.

The success of this cycle hinged on producing ,**soluble, bioactive proteins**. For **CBD MMPs-VEGF**, expression in **E. coli BL21 (DE3)** yielded high concentrations of soluble protein under standard conditions, confirming that this fusion protein could be expressed easily and efficiently.

In contrast, **FTD-BMP-4** proved more challenging. The presence of multiple disulfide bonds in BMP-4 led to the formation of inclusion bodies when expressed in BL21, which made the protein insoluble and inactive. To overcome this, we explored various optimized bacterial strains designed to facilitate the proper folding of disulfide bond-containing proteins.

Several strains were tested:

* **E. coli Origami (DE3)** improved disulfide bond formation but still resulted in considerable protein aggregation.
* **E. coli Shuffle T7-B** enhanced soluble expression, but its slow growth rate limited its practicality for large-scale production.
* Finally, **ArcticExpress (DE3) pRARE** was selected as the most suitable expression host, balancing both high protein yield and improved solubility of **FTD-BMP-4**.

The culmination of this stage was obtaining both fusion proteins in their active, soluble forms, laying the foundation for further testing of their controlled release and biological activity in subsequent phases. In Fig3.2, lanes 1 to 4, 5 to 8, and 9 to 12 are whole bacteria, supernatant after disruption, and precipitate after disruption, respectively. Each group consists of four lanes, and the order within each group is BL21, ArcticExpress (DE3) pRARE, Shuffle T7-B, Origami (DE3)

![](https://static.igem.wiki/teams/5374/enginsuc/fig-3-2.png)

Fig 3.2 Results of expression system optimization a) SDS-PAGE; b) semi-quantitative analysis (Lanes 1 to 4, 5 to 8, and 9 to 12 are whole bacteria, supernatant after disruption, and precipitate after disruption, respectively. Each group consists of four lanes, and the order within each group is BL21, ArcticExpress (DE3) pRARE, Shuffle T7-B, Origami (DE3))

According to Fig3.2, **ArcticExpress (DE3) pRARE** stands out for its high proportion of soluble protein expression.The reasons for ArcticExpress's success in producing a high level of **soluble protein** are as follows:

**Chaperone Support:**
ArcticExpress is engineered to co-express two cold-adapted chaperones, **Cpn60** and **Cpn10**, which facilitate the proper folding of proteins, especially those with complex structures like **FTD-BMP-4**, which contains multiple disulfide bonds. These chaperones help reduce the formation of misfolded proteins and inclusion bodies, promoting correct folding at lower temperatures, which is ideal for proteins prone to aggregation.

**pRARE Plasmid:**
The **pRARE plasmid** in ArcticExpress provides additional tRNAs for rare codons that are underrepresented in standard E. coli strains. This allows for more efficient and accurate translation of eukaryotic or complex proteins like BMP-4, which may otherwise experience stalling during translation in standard hosts due to rare codon usage.

**Cold-Adapted System:**
ArcticExpress is designed to perform well at lower temperatures, which can slow down the protein synthesis process, giving more time for complex proteins with disulfide bonds to fold correctly and avoid aggregation.

In the gel (left) and the bar chart (right), ArcticExpress shows significantly improved soluble protein expression compared to other strains like BL21, which primarily produces inclusion bodies. This makes ArcticExpress the optimal choice for expressing soluble and active **FTD-BMP-4**, ensuring that a higher percentage of the total protein is in the desired soluble form.

## 3.3 Test

The system was tested through a series of in vitro cell assays to evaluate the controlled release of the two cell factors:
VEGF release was monitored to ensure rapid availability for angiogenesis.
BMP-4 release was tested to confirm delayed but sustained release for bone formation.
ALP (Alkaline Phosphatase) staining and other bioassays were conducted to assess the biological activity of the cell factors in promoting angiogenesis and osteogenesis.

### 3.3.1 Sequential release

This experiment monitored the release profiles of CBD MMPs-VEGF and FTD-BMP-4 from collagen hydrogels over time. The two fusion proteins were incorporated into collagen hydrogels, and their release was quantified using ELISA assays. The hydrogels were incubated in PBS at 37°C, with supernatant samples collected at regular intervals to measure the amount of protein released. After each sampling, fresh PBS was added to the hydrogels to maintain consistent conditions throughout the experiment.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-3-3.png)

Fig 3.3 Time-Dependent Release Profiles of CBD MMPs-VEGF and FTD-BMP-4 from Collagen Hydrogel

The results reveal a significant difference in the release kinetics between the two cell factors. **CBD MMPs-VEGF** showed a rapid and early release pattern, starting with an accelerated increase in the release rate during the first 10 days. By day 10, more than 50% of the VEGF had already been released from the hydrogel. This sharp increase continued, reaching approximately 75% release by day 20. Following this, the release rate gradually slowed, with the curve plateauing as nearly 100% of the VEGF was released by day 30. The release curve of VEGF shows an initial rapid release phase, followed by a stabilization period where the release rate gradually levels off. This fast-release behavior can be attributed to the lower binding affinity of the CBD MMPs domain, allowing VEGF to dissociate from the collagen matrix more quickly.

In contrast, **FTD-BMP-4** exhibited a much slower and more controlled release. During the first 10 days, less than 20% of BMP-4 was released, indicating a more gradual release process. The release steadily increased, with the curve following a linear trend. By day 20, around 30% of BMP-4 had been released, and by day 30, approximately 50% was released. The release continued steadily, reaching close to 100% around day 40. This extended release profile reflects the higher binding affinity of the FTD domain to collagen, which ensures a slower and sustained release of BMP-4 throughout the experiment.

These contrasting release profiles demonstrate the effectiveness of using different collagen-binding domains to control the timing and rate of cell factor release, with CBD MMPs facilitating a faster release for VEGF and FTD ensuring a prolonged release for BMP-4.These results confirm that the controlled, sequential release of the two cell factors can be achieved by leveraging the different binding affinities of the CBDs, with **CBD MMPs-VEGF** releasing faster to initiate angiogenesis, followed by the slower release of **FTD-BMP-4** to promote osteogenesis.

### 3.3.2 In vitro biological activity(VEGF)

The **tube formation** is a widely used in vitro method for assessing the ability of endothelial cells, such as **HUVECs** (Human Umbilical Vein Endothelial Cells), to form capillary-like structures, mimicking the process of angiogenesis. This assay is particularly useful for evaluating the angiogenic potential of **cell factors**, small molecules, or other treatments.

In the assay, endothelial cells are seeded on a **Matrigel** matrix, which provides an extracellular matrix environment that encourages the cells to migrate, align, and connect with each other, forming **tubular networks**. When angiogenic **cell factors**, such as **VEGF**, are present, these processes are enhanced, leading to more extensive and faster formation of tube-like structures.

The process of **cell connection and tube formation** involves several key cellular mechanisms:

1. **Migration:** Endothelial cells move toward each other, driven by chemotactic signals, such as VEGF, which promote cell motility. VEGF binds to its receptors on endothelial cells, activating intracellular signaling pathways that guide this migration.
2. **Adhesion:** As cells come into contact, they adhere to each other via cell-cell adhesion molecules such as **VE-cadherin**. These molecules facilitate stable connections between neighboring cells, which are critical for forming continuous structures.
3. **Cell Structure Rearrangement:** The internal framework of endothelial cells undergoes dynamic changes, allowing the cells to elongate and align with each other. This is crucial for shaping the cells into tube-like formations.
4. **Lumen Formation:** Once the cells are aligned and connected, they start forming hollow tubes that resemble capillaries. This involves the coordinated action of intracellular vacuoles and the fusion of these vacuoles between neighboring cells.

Overall, the tube formation assay provides insights into the angiogenic potential of treatments like **CBD MMPs-VEGF**, as seen in our experiment. The results suggest that **CBD MMPs-VEGF** promotes cell migration, adhesion, and network formation, confirming its ability to stimulate angiogenesis through effective **cell factor** release.

In the tube formation assay, human umbilical vein endothelial cells (HUVECs) were seeded onto a Matrigel-coated plate to facilitate tube formation. The experimental group was treated with CBD MMPs-VEGF at a final concentration of 100 ng/ml, while the control group received no VEGF. The cells were incubated at 37°C with 5% CO₂ for 4-6hours, during which tube formation was observed. The formation of capillary-like structures was monitored under a microscope to assess angiogenesis. The extent of tube formation between the experimental and control groups was compared to determine the impact of CBD MMPs-VEGF on promoting angiogenesis.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-3-4.png)

Fig 3.4 Comparison of Tube Formation in HUVECs a) Control; b)CBD MMPs-VEGF Treatment

Image a (Without CBD MMPs-VEGF): In the control group, where no CBD MMPs-VEGF was added, the HUVECs exhibited weak angiogenic activity. Only a few scattered cell connections were observed, with minimal evidence of network formation. Quantitatively, the tube formation rate (measured by parameters such as total tube length, number of branch points, and closed loops or tube closure rate) was significantly reduced. The tube closure rate, which measures the percentage of formed capillary-like loops, was close to zero, reflecting the lack of organized tube structures. This result confirms that without the presence of VEGF, the cells are unable to initiate or sustain effective angiogenesis.
Image b (CBD MMPs-VEGF at 100 ng/ml): In the experimental group treated with 100 ng/ml CBD MMPs-VEGF, the HUVECs showed robust tube formation, with clearly defined and well-organized capillary-like structures. Quantitative analysis revealed a dramatic increase in several angiogenesis indicators. The tube closure rate increased to approximately 70-80%, indicating a high number of complete tube loops. Additionally, the total tube length and number of branch points were significantly higher compared to the control, demonstrating enhanced network complexity and cell connectivity. These findings suggest that the addition of VEGF significantly promoted angiogenesis, with the cells forming a dense and interconnected tubular network, characteristic of active capillary formation.
This comparison highlights the essential role of CBD MMPs-VEGF in promoting angiogenesis, as quantified by standard tube formation metrics. The increased tube closure rate, total tube length, and branch points in the experimental group strongly indicate that the VEGF produced in the system retains its biological function and effectively stimulates endothelial cell organization and network formation. The comparison between these two images confirms that the CBD MMPs-VEGF produced in this study retains biological activity and effectively stimulates tube formation, validating its functional ability to promote angiogenesis.

### 3.3.3 In vitro biological activity(BMP-4)

In our osteogenic differentiation experiments, **MC3T3-E1 cells**, a well-established **pre-osteoblast cell line** derived from mouse calvaria, were used. These cells are commonly employed in bone research because they have the inherent capacity to differentiate into mature osteoblasts when cultured under osteogenic conditions. During the early stages of osteogenic differentiation, **MC3T3-E1 cells** express **alkaline phosphatase (ALP)**, which is critical for mineralization. ALP staining can be used to measure the early osteogenic activity in these cells. As differentiation progresses, **MC3T3-E1** cells begin to deposit calcium in the extracellular matrix, which is a key indicator of bone formation. This can be detected using **Alizarin Red staining**, which binds specifically to the calcium deposits and allows for the visualization of mineralization. Thus, by using both **ALP staining** and **Alizarin Red staining**, the osteogenic differentiation process of **MC3T3-E1 cells** can be effectively tracked from early osteoblast activity (ALP expression) to late-stage bone formation (calcium deposition).
So, ALP (Alkaline Phosphatase) Staining and Alizarin Red Staining are two key methods used to assess different stages of osteogenic differentiation in cells, providing insights into the bone formation process.

**ALP Staining Mechanism:**
ALP is an early marker of osteogenic differentiation, highly expressed by pre-osteoblasts and osteoblasts during the early stages of bone formation. ALP plays a crucial role in the **mineralization** process by hydrolyzing phosphate-containing compounds, and releasing inorganic phosphate, which contributes to the formation of hydroxyapatite crystals. When cells are induced toward osteogenic differentiation, an increase in ALP activity is observed. During **ALP staining**, a substrate (e.g., BCIP/NBT) reacts with ALP to produce a blue-purple color, indicating active ALP expression. By measuring this staining, you can assess how efficiently the cells are entering the osteogenic pathway and preparing for mineral deposition.

**Alizarin Red Staining Mechanism:**
Alizarin Red S staining detects **calcium deposits**, a hallmark of late-stage osteogenic differentiation. As osteoblasts mature, they begin to produce and deposit extracellular matrix, which undergoes **mineralization** as calcium and phosphate ions crystallize into hydroxyapatite, the main mineral component of bone. Alizarin Red S binds specifically to calcium ions, forming an orange-red complex that can be observed microscopically. By assessing the intensity and extent of red staining, you can determine the extent of mineralization, indicating that the cells have progressed to the later stages of osteoblast differentiation and are actively contributing to bone matrix formation.

Together, **ALP staining** and **Alizarin Red staining** offer a comprehensive view of osteogenic differentiation. ALP staining identifies early differentiation events, while Alizarin Red staining confirms matrix mineralization, showing that the cells are functionally mature osteoblasts capable of bone formation.

For the ALP staining, cells were cultured for 7 and 14 days, then washed with PBS and fixed with 4% paraformaldehyde for 10-15 minutes at room temperature. After washing with PBS, the cells were incubated with ALP staining solution (e.g., BCIP/NBT) for 30-60 minutes at 37°C. ALP activity, indicating early osteogenic differentiation, was visualized as blue-purple staining under a microscope. For the Alizarin Red staining, cells were cultured for 21 and 28 days, washed with PBS, and fixed with 4% paraformaldehyde for 10-15 minutes. After washing with distilled water, Alizarin Red S solution (pH 4.1-4.3) was applied for 20-30 minutes to detect calcium deposits. The cells were then rinsed with distilled water, and red staining was observed to indicate matrix mineralization, a marker of mature osteoblast activity.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-3-5.png)

Fig 3.5 ALP and Alizarin Red Staining of MC3T3-E1 Cells at Different Time Points During Osteogenic Differentiation

**ALP Staining Results (Day 7 and Day 14):** From the images provided, on **Day 7** (top-left images), the ALP staining shows minimal blue-purple staining, suggesting that osteogenic differentiation had only just begun. By **Day 14** (bottom-left images), there is a noticeable increase in blue-purple staining, indicating elevated ALP activity, which correlates with increased osteoblast differentiation. The stronger ALP activity seen on Day 14 demonstrates that the cells are undergoing early osteogenic differentiation, which is being effectively stimulated by the BMP-4 cell factor, confirming its biological activity in promoting bone formation.

**Alizarin Red Staining Results (Day 21 and Day 28):** In the **Day 21** images (top-right), there are initial signs of mineralization with scattered red deposits, indicating the onset of calcium deposition. By **Day 28** (bottom-right), the staining intensity has significantly increased, with larger and more densely stained red areas, showing substantial mineralization of the extracellular matrix. This confirms the later-stage osteogenic activity, as BMP-4 continues to drive the maturation of osteoblasts and the deposition of calcium, which is a hallmark of bone tissue formation.

These results confirm that the BMP-4 produced in our system retains its biological activity, as it successfully stimulates both early (ALP activity) and late (mineralization) osteogenic differentiation.

## 3.4 Learn

From the results, we confirmed that the gradient release of the two cell factors was achieved due to the differences in the collagen-binding domains (CBDs) fused to each factor, rather than any adjustments to the crosslinking density or hydrogel composition. VEGF was released first due to the lower affinity of CBD MMPs to collagen, while BMP-4 exhibited a more sustained release profile thanks to the high-affinity binding of FTD. These findings successfully demonstrated the functionality of the dual cell factor release system, which not only provided controlled, gradient release but also maintained excellent biological activity of both factors. This system proves effective for further applications and sets the stage for future optimization.

# 4 DBTL Cycle 3: Development and Validation of the Collagen Hydrogel System

## 4.1 Design

In the third cycle, the goal was to create a collagen hydrogel system capable of delivering two cell factors—VEGF and BMP-4—in a controlled and sequential manner. Collagen was selected as the base material for the hydrogel due to its numerous advantages in bone repair. As a naturally occurring protein, collagen is biocompatible, biodegradable, and has excellent bioactivity, making it an ideal scaffold for supporting cell adhesion, proliferation, and differentiation. Furthermore, its structural similarity to the extracellular matrix provides an optimal environment for promoting tissue regeneration. Collagen’s ability to be easily crosslinked and modified allows for the incorporation of cell factors and controlled release mechanisms, making it a versatile material for bone defect filling while simultaneously delivering bioactive molecules. Based on these advantages, we designed the collagen hydrogel to act as both a physical scaffold for bone tissue regeneration and a carrier for sequentially releasing VEGF and BMP-4. Therefore, we chose to express **Type I collagen** using E. coli, as Type I collagen is more robust and widely distributed in bone tissue.
For our project, we are adopting a similar approach in the selection and design of a recombinant Type I collagen protein for bone repair applications
**Sequence Selection:** The foundation of our design is based on the human Type I collagen sequence, chosen for its natural role in supporting bone structure and promoting cell adhesion. The selected sequence retains the key functional regions of native collagen to ensure that our product effectively mimics the biological properties of natural bone tissue.
**Codon Optimization:** To ensure efficient production in E. coli, we will optimize the codon usage of the collagen gene. This adjustment improves the yield of the recombinant protein while ensuring proper folding and stability during expression in bacterial systems, allowing for scalable production of the collagen hydrogel.
**Enhancement of Bioactivity:** To further enhance the cell adhesion properties, we will incorporate specific peptide sequences at the N-terminal of the collagen. These sequences are designed to improve interaction with cells, thereby increasing the efficacy of collagen in promoting osteogenic activity and bone regeneration.
**Structural Modifications:** Similar to the patented approach, we will introduce targeted modifications to the collagen sequence. These changes will ensure the protein maintains high structural stability and over 90% sequence homology to native collagen while enhancing its performance in tissue repair applications. The modifications may involve selective addition, deletion, or substitution of amino acids to optimize both biological function and structural integrity.

This design methodology will allow us to create a recombinant Type I collagen that is highly effective in bone repair, ensuring both the strength and bioactivity required for successful tissue regeneration.

We have designed the pET32a-Collagen I plasmid to enable the recombinant expression of Type I collagen in E. coli. Utilizing the pET32a backbone, the plasmid incorporates a strong T7 promoter to drive high-level transcription of the collagen gene.

![](https://static.igem.wiki/teams/5374/enginsuc/fig-4-1.png)

Fig 4.1 Schematic of pET32a-Collagen I Expression Plasmid for Recombinant Collagen Production

## 4.2 Build

Using E. coli ClearColi, we expressed low-endotoxin recombinant collagen, which was then incorporated into the hydrogel system. The collagen hydrogel was prepared by embedding the previously engineered fusion proteins—CBD MMPs-VEGF and FTD-BMP-4—into the matrix. The hydrogel was designed to ensure VEGF would be released quickly to stimulate angiogenesis, followed by a slower, sustained release of BMP-4 to support osteogenesis.

## 4.3 Test

The release profiles of the two cell factors were monitored over time using **ELISA**. The results showed that **VEGF** was rapidly released during the first 20 days due to the lower binding affinity of **CBD MMPs** to collagen, while **BMP-4** was released more gradually over 40 days, reflecting the stronger collagen-binding capacity of **FTD**. Both cell factors retained their bioactivity, as confirmed by **tube formation assays** for VEGF and **ALP** and **Alizarin Red staining** for BMP-4, demonstrating successful angiogenesis and osteogenesis, respectively.

## 4.4 Learn

The third cycle confirmed that the **dual cell factor release system** achieved both **gradient release** and maintained **strong biological activity** of the embedded cell factors. VEGF was released first to promote angiogenesis, followed by BMP-4 for bone formation. This hydrogel system showed excellent potential for tissue engineering applications without the need for further modification to the matrix. These insights validated the design and performance of the release system and set the foundation for future applications in regenerative medicine.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
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# 1 Preparation of Experimental Reagents and Media

Each group will prepare the corresponding volume/quantity of reagents/media.

## 1.1 Experimental Objective

To prepare reagents needed for subsequent experiments.

#### Genetic Engineering:

* LB Liquid Medium: 20 ml/tube \* 5
* LB Liquid Medium: 20 ml/bottle \* 10
* CaCl2 0.1M: 50 ml
* LB Solid Medium (1.5% Agar): 200 ml

#### Protein Engineering:

* Solution A
* Solution B
* Pure Water
* 20% Ethanol

## 1.2 Experimental Procedure: Preparation of Reagents for Genetic Engineering

### 1.2.1 Preparation and Sterilization of Solutions

#### LB Medium

A total of 300 ml is needed, but 400 ml will be prepared to ensure sufficient volume during aliquoting.

Preparation Method: [Tryptone: 4g, Yeast Extract: 2g, NaCl: 4g] + [400 ml pure water], stir well in a beaker, aliquot 20 ml into 10 conical flasks, and the remainder into a blue-cap bottle, and autoclave (loosen the cap for the blue-cap bottle).

#### LB Solid Medium (1.5%)

[Tryptone: 2g, Yeast Extract: 1g, NaCl: 4g] + [200 ml pure water] + [Agar powder: 3g], prepare directly in a 500 ml conical flask, do not stir, cover, and autoclave (loosen the cap for the blue-cap bottle).

#### Calcium Chloride (for competent cell preparation)

[0.55g CaCl2] + [50 ml pure water], prepare directly in a 100 ml blue-cap bottle, and autoclave.

#### Plastic Consumables

1.5 ml centrifuge tubes, 20 pcs, packaged in a plastic bag, and autoclaved.

### 1.2.2 Pouring Plates

Add Ampicillin (1 ml + 1 μl, final concentration 50 μg/ml) to the sterilized and cooled LB solid medium below 60°C, mix well, and pour plates into 90 mm plastic petri dishes, approximately 10-15 ml per plate, at least 10 plates needed.

## 1.3 Experimental Procedure: Preparation of Reagents for Protein Engineering

Prepare solutions according to the formulas for Solution A and Solution B, adjust pH, filter through a membrane, and degas by ultrasound. Each group prepares 1L of Solution A and 500 ml of Solution B.

Filter and degas 1L of deionized water by ultrasound, prepare 1L per group.

Prepare 20% ethanol solution, filter through a membrane, and degas by ultrasound.

# 2 Inoculation (Preparation for Plasmid Extraction)

Add 15 ml of autoclaved LB medium from 1.2.1 to a sterile 50 ml centrifuge tube, add 15 μl of Ampicillin stock solution (1 ml + 1 μl, final concentration 50 μg/ml), mix well. Add 150 μl of DH5α bacterial culture.

Cap and remove from the biosafety cabinet, incubate in a shaker at 220 rpm, 37°C, overnight.

# 3 Fundamental Experiments in Genetic Engineering/Molecular Biology

## 3.1 Objective

The molecular biology/genetic engineering experiments on the 11th are conducted individually. Each student must perform the basic molecular biology/genetic engineering experiments. The experiments on the 12th will be conducted in groups.

## 3.2 Experimental Procedures

### 3.2.1 Plasmid Miniprep and Concentration Measurement

![](https://static.igem.wiki/teams/5374/experiments/fig3-2-1-1.png)
![](https://static.igem.wiki/teams/5374/experiments/fig3-2-1-3.png)
![](https://static.igem.wiki/teams/5374/experiments/fig3-2-1-4.png)
![](https://static.igem.wiki/teams/5374/experiments/fig3-2-1-5new.png)

### 3.2.2 PCR

#### 3.2.2.1 Fragment Preparation

PCR Conditions for Fragment

![](https://static.igem.wiki/teams/5374/experiments/fig3-2-2-1-1.png)

Estimated Time: 1 hour (Morning Session)

PCR Conditions for pet28a Fragment

![](https://static.igem.wiki/teams/5374/experiments/fig3-2-2-1-2.png)

Estimated Time: 1 hour (Noon Break)

![](https://static.igem.wiki/teams/5374/experiments/fig3-2-2-1-3.png)
![](https://static.igem.wiki/teams/5374/experiments/fig3-2-2-1-4.png)

#### 3.2.2.2 Purification of PCR Products

Due to the complexity of the PCR system, which contains templates, primers, enzymes, and other impurities that may hinder seamless cloning, purification is necessary to obtain the target gene fragment.

![](https://static.igem.wiki/teams/5374/experiments/fig3-2-2-2-1new.png)

After purification, measure DNA concentration again and calculate the yield of the purification process.

### 3.2.3 Agarose Gel Electrophoresis

Prepare the gel during the PCR amplification of the fragment. In the afternoon, add the loading buffer and then run the gel electrophoresis.

(1) Prepare the gel (do this during the morning PCR session; due to the limited number of gel trays, prepare two small gels per group).

**Agarose Gel Electrophoresis**

* Purpose: Used for the separation, identification, and extraction of nucleic acid fragments.
* Principle: Fragments of nucleic acids (DNA or RNA) are separated based on their size. After applying an electric field, the negatively charged DNA or RNA fragments migrate through the agarose gel.Smaller nucleic acid fragments migrate faster. Higher agarose concentration in the gel reduces pore size, affecting the separation. Higher agarose concentrations result in smaller pores, making it suitable for separating smaller fragments.

![](https://static.igem.wiki/teams/5374/experiments/fig3-2-2-2-3.png)

Our target gene and vector backbone are approximately 700 bp and 6000 bp, respectively, so prepare a 1% agarose gel.

Dissolve 1 g of agarose in 100 ml TAE buffer (not pure water!) in a 250 ml Erlenmeyer flask, cover with foil, and heat in a microwave until dissolved. Cool to approximately 60°C, add nucleic acid dye, mix, and pour into the gel tray. Cover with foil and allow to solidify in the dark.

（2）Preparation of Electrophoresis Samples

Mix a small amount of the purified PCR product with loading buffer (6X), in a 1:5 ratio.

Suggested: Take 10 µl each of the fragments in a 0.2 ml EP tube, add 2-3 µl loading buffer, and mix well.

（3）Loading Samples

（4）Electrophoresis and Analysis

Run at 90V. After verifying the molecular weight, store the samples at -20°C for use on the 12th.

# 4 Competent Cells, Seamless Cloning, and Transformation

## 4.1 Experimental Procedures

### 4.1.1 Preparation of Competent Cells

Upon arriving at the laboratory, first, pre-cool the calcium chloride solution prepared and sterilized on the 10th in an ice bath.

Transfer 1.25 ml of the bacterial culture into 1.5 ml centrifuge tubes (distributed into 8 tubes) and place them on ice for 10 minutes to cool to 0°C. Centrifuge the bacterial culture at 4°C, 4000 rpm for 10 minutes, and discard the supernatant (invert the tubes to drain the medium thoroughly).

Add 150 μL of ice-cold 0.1 M CaCl₂ to resuspend, combine two tubes into one, and place on ice for 30 minutes (resulting in 4 tubes total). Centrifuge again at 4°C, 4000 rpm for 10 minutes, discard the supernatant, and add 100 μL of ice-cold 0.1 M CaCl₂ to resuspend to obtain BL21 competent cells for immediate transformation. If not used immediately, add an equal volume of 50% glycerol, rapidly freeze in liquid nitrogen, and store at -80°C.

### 4.1.2 Seamless Cloning

Design the seamless cloning system according to the manual (classroom exercise; concentrations vary per individual, so each person's table will differ).

![](https://static.igem.wiki/teams/5374/experiments/fig4-1-2-1.png)

08/Experimental Procedure

08-1/Recombinant Reaction

#### 1. Concentration Determination of Linearized Vectors and Insert Fragments

If the linearized vector and insert fragments have been purified through a gel recovery kit, and the absorbance reading shows no apparent bands or smears remaining, you can use Nanodrop or similar spectrophotometers to measure concentration. However, the A260/A280 ratio should only be considered reliable between 1.8 and 2.0. Instruments like Nanodrop, Qubit, and PicoGreen are recommended for concentration measurement. When sample concentrations are below 10 ng/μl, different instrument models may yield significantly varying readings based on A260.

#### 2. Calculation of Vector and Insert Fragment Usage:

For single-fragment source recombination, the minimum recommended vector usage is 0.03 pmol, and the minimum recommended insert fragment usage is 0.06 pmol (with a vector-to-insert molar ratio of 1:2).

For multi-fragment source recombination, the minimum DNA usage for each fragment is 0.03 pmol (with a vector-to-insert molar ratio of 1:1).

The amount of DNA used in these calculations can be approximated using the following formulas:

* For Single-Fragment Source Recombination:
  + Optimal Cloning Vector Usage=[0.02\*Number of Base Pairs in the Cloning Vector]ng(0.03pmol）
  + The optimal amount of insert fragment=[0.04\* The number of base pairs in the insert fragment]ng(0.06pmol)
* For Multi-Fragment Source Recombination:
  + Optimal Cloning Vector Usage=[0.02\*Number of Base Pairs in the Cloning Vector]ng(0.03pmol）
  + The optimal usage amount for each fragment=[0.02\* The number of base pairs in each fragment]ng(0.03pmol)

* For single-fragment source recombination, the insert fragment usage should not exceed 20 ng. When the insert fragment is larger than the vector, it is advisable to calculate the appropriate mass conversion, treating the insert fragment as the vector and vice versa.
* For multi-fragment source recombination, each fragment should not exceed 10 ng. If the minimum recommended usage is below this value, directly use 10 ng/μl.
* In single-fragment source recombination, to avoid non-specific bands in PCR, do not use direct linearized DNA. Add no more than 1/5 of the total reaction volume (2 μl), or directly clone after gel extraction.

#### 3. Prepare the Following Reaction Systems on Ice:

![](https://static.igem.wiki/teams/5374/experiments/fig4-1-2-2.png)

1. X/Y is calculated based on the calculated vector and insert fragment amounts. To ensure amplification accuracy, vectors and insert fragments should be concentrated before reaction setup and used in no less than 1 μl volumes.
2. Positive Controls-1 and -2 employ plasmids as templates, with amplification lengths <3 kb. Primer Tm values should be >60°C.
3. Positive Control-3 uses genomic DNA as a template. Use high-fidelity polymerases for amplification.
4. Fragment length, GC content, and sequence complexity affect reaction time. High GC content in insert fragments can hinder the reaction; consider adding DMSO or betaine to enhance amplification.

#### 4. Gently mix using a pipette (avoid vortex mixing) and briefly centrifuge the reaction mixture to the bottom of the collection tube.

#### 5. Recombination Reaction Conditions:

Single-Fragment Recombination: Incubate at 50°C for 5 minutes; cool to 4°C or place on ice immediately.

2-3 Fragment Recombination: Incubate at 50°C for 15 minutes; cool to 4°C or place on ice immediately.

4-5 Fragment Recombination: Incubate at 50°C for 30 minutes; cool to 4°C or place on ice immediately.

* It is recommended to perform the reaction on instruments such as a PCR machine that offers precise temperature control.
* This protocol accommodates 0.01 - 0.25 pmol input of vectors and fragments. When the total vector and insert volume exceeds 5 μl, a lower input amount is acceptable, but avoid exceeding the recommended reaction time.
* Store the recombination product at -20°C for up to a week. Purify only when necessary for further transformation.

### 4.1.3 Transformation

#### Recombinant Product Transformation

1. Thaw chemically competent cells on ice (e.g., Fast-T1 Competent Cell, Vazyme #C505).
2. Add 5-10 μl of the recombinant product to 100 μl of competent cells. Gently flick the tube to mix (avoid vortexing) and incubate on ice for 30 minutes.
   * The volume of recombinant product should not exceed 1/10 of the volume of competent cells used.
3. Heat shock the cells at 42°C for 30 seconds, then immediately place them back on ice for 2-3 minutes.
4. Add 900 μl of SOC or LB medium (without antibiotics) and incubate at 37°C with shaking for 1 hour (rotation speed 200-250 rpm).
5. Pre-warm the required LB agar plates containing the appropriate antibiotic at 37°C in an incubator.
6. Centrifuge at 5,000 rpm (2,500 x g) for 5 minutes and discard 900 μl of the supernatant. Resuspend the bacterial pellet in the remaining medium and spread it gently on the pre-warmed agar plate with the antibiotic.
7. Invert the agar plates and incubate at 37°C for 12-16 hours.

Each experimental group will have four sets of samples (AC, AD, BC, BD), with each set plated on 1-2 LB plates and incubated overnight at 37°C.

# 5 Biotechnology Visit and Fermentation Inoculation

## 5.1 Fermentation (Inoculation)

Upon arrival in the morning, distribute the fermentation seed culture prepared on the 13th into the remaining three 250 ml sterilized culture bottles from step 2.2, adding at least 25 ml of seed culture to each (using sterile Pasteur pipettes). Incubate at 37°C, 220 rpm for 5-10 hours.

Afternoon: Visit to the biotechnology facility.

## 5.2 Fermentation (Induction)

After the visit in the afternoon, return to the laboratory and add 125 μl of IPTG stock solution to each bottle (the stock is 1 M, with a final concentration of 1 mM). Incubate overnight at 37°C, 220 rpm for induction.

# 6 Bacterial Collection, Ultrasonic Disruption, Separation of E. coli Lysate, Protein Electrophoresis

## 6.1 Collection and Resuspension

Fill the fermentation broth into 50 ml centrifuge tubes, not exceeding 40 ml per tube. Balance the tubes, and centrifuge multiple times, ensuring each group collects bacterial cells from at least 200 ml of fermentation broth. Centrifuge at 4000 x g for 10 minutes. Resuspend the collected bacterial pellet in solution A, at a ratio of 1 g of cells to 10 ml of solution A. Transfer 1 ml of resuspended cell culture to a new 1.5 ml EP tube for protein electrophoresis.

## 6.2 Ultrasonic Disruption

Disrupt the resuspended bacterial cells using ultrasonic waves. Ensure that the centrifuge tubes containing the cells are placed in an ice bath within a beaker. Use ultrasonic settings of 5 seconds on, 5 seconds off, for a total of 20 minutes.

## 6.3 Centrifugal Separation of E. coli Lysate

### 6.3.1 Experiment Objective

Our target protein is located in the E. coli lysate supernatant. However, cell disruption produces many insoluble impurities (e.g., cell wall debris), so the first purification step is centrifugation to separate the insoluble precipitate from the soluble protein solution.

### 6.3.2 Experimental Procedures

Centrifuge the E. coli lysate obtained above using 1.5 ml EP tubes, with 1 ml per tube, at 4°C, 17,000 x g for 40 minutes. After centrifugation, transfer the supernatant to a new centrifuge tube, and resuspend the precipitate with 1 ml of solution.

## 6.4 Protein Electrophoresis

### 6.4.1 Experiment Objective

To further confirm the expression status of our protein (the proportion of target protein in the lysate supernatant, solubility of the target protein), protein electrophoresis is required.

### 6.4.2 Experimental Procedures

#### Sample Preparation

For three 0.2 ml EP tubes, take 100 μl each of resuspended cell culture, lysate supernatant, and lysate precipitate. Add at least 20 μl of 6× protein loading buffer to each tube, and incubate in a PCR thermocycler for 20 minutes.

#### Loading

Use pre-cast gels. Each person should practice loading at least two wells. Each group should load samples that include the three prepared in 4.2.1 and a protein marker.

#### Electrophoresis

Run at 160V for 40 minutes.

#### Staining

Incubate the gel in staining solution on a shaker for 20 minutes to visualize bands.

# 7 Cell Culture and Passaging Protocol

## Materials Needed

* Tissue culture flasks or plates
* Cell culture media (appropriate for the cell line)
* Trypsin-EDTA solution
* Phosphate-buffered saline (PBS)
* Sterile pipettes and pipette tips
* Hemocytometer or cell counter (optional)
* Incubator set at 37°C with 5% CO₂
* Personal protective equipment (PPE)

## Steps

1. Preparation:

* Ensure that all materials and reagents are sterile.
* Warm the cell culture media, PBS, and trypsin-EDTA to 37°C in the incubator.

2. Observing Cells:

* Check the cells under an inverted microscope to assess their confluency.
  Ideally, cells should be 70-80% confluent for passaging.

3. Removing Old Media:

* Gently aspirate the old culture media from the flask or plate without disturbing the cell layer.

4. Washing Cells:

* Add 5-10 mL of PBS to wash the cells. Swirl gently and then aspirate the PBS.
  This step removes residual media and dead cells.

5. Adding Trypsin-EDTA:

* Add enough trypsin-EDTA solution to cover the cell layer (typically 1-2 mL for a T25 flask).
* Incubate at 37°C for 2-5 minutes. Monitor the cells under the microscope. Cells should start to round up and detach.

6. Stopping Trypsin Activity:

* Once most cells are detached, add an equal volume of fresh complete cell culture
  media (containing serum) to neutralize the trypsin.
* Gently pipette up and down to dislodge any remaining attached cells.

7. Collecting Cells:

* Transfer the cell suspension to a sterile centrifuge tube.
* If needed, count the cells using a hemocytometer or automated cell counter.

8. Dilution and Re-seeding:

* Dilute the cell suspension to the desired cell concentration with fresh media.
* Transfer the diluted cell suspension to new tissue culture flasks or plates (e.g., 1:5
  or 1:10 dilution for passaging).

9. Incubation:

* Place the newly seeded flasks or plates in the incubator set at 37°C with 5% CO₂.
* Allow cells to adhere and grow for 24-48 hours before the next observation.

10. Documentation:

* Record the date, cell passage number, and any observations regarding cell morphology and health.

## Notes

1. Always use aseptic techniques to prevent contamination.
2. Adjust the passaging frequency based on the specific growth characteristics of your cell line (e.g., every 2-4 days).
3. Dispose of waste properly in biohazard containers.
4. This protocol should guide you through the cell culture and passaging process efficiently. Let me know if you need any additional information!

# 8 Tube Formation Assay Protocol

## Materials Needed

* Matrigel or growth factor-reduced basement membrane matrix
* Complete endothelial cell culture media (e.g., EBM-2)
* Fetal Bovine Serum (FBS)
* Trypsin-EDTA solution
* Phosphate-buffered saline (PBS)
* 96-well plate or 24-well plate
* Incubator set at 37°C with 5% CO₂
* Personal protective equipment (PPE)
* Inverted microscope for observation

## Steps

1. Preparation:

* Thaw Matrigel overnight at 4°C. Keep it on ice until use.
* Warm complete endothelial cell media to 37°C.

2. Cell Culture:

* Culture HUVECs in complete media until they reach 70-80% confluency.
* Wash cells with PBS and detach using trypsin-EDTA. Neutralize trypsin with complete media.

3. Cell Counting:

* Count the cells using a hemocytometer or automated cell counter.
* Adjust the cell density to approximately 1 × 10⁶ cells/mL in complete media.

4. Matrigel Coating:

* Add 50-100 µL of Matrigel per well in a 96-well plate. Swirl gently to ensure even distribution.
* Allow the Matrigel to polymerize by incubating at 37°C for 30-60 minutes.

5. Seeding Cells:

* Add 100-200 µL of the HUVEC suspension (1 × 10⁶ cells/mL) onto the polymerized Matrigel.
* Gently swirl the plate to distribute the cells evenly.

6. Incubation:

* Place the plate in the incubator (37°C, 5% CO₂) and allow the cells to adhere and form tubes for 4-6 hours or overnight, depending on the experimental design.

7. Treatment (Optional):

* If testing the effects of treatments (e.g., CBD MMPs-VEGF), add the treatments to the
  cells during this incubation period.

8. Observation:

* After incubation, observe the plate under an inverted microscope.
* Capture images of the tube-like structures formed by the endothelial cells.

9. Analysis:

* Analyze the tube formation using quantitative parameters such
  as total tube length, number of junctions, and branching points. Use image analysis software if available.

10. Documentation:

* Record your observations, including any differences between control and treatment groups, and save images for further analysis.

## Notes

1. Ensure all materials and tools are sterile to prevent contamination.
2. Repeat the experiment in triplicates or more for statistical validity.
3. Adjust cell density or incubation time based on specific experimental requirements.
4. This protocol will guide you through conducting a Tube Formation Assay effectively. Let me know if you need any further details!

# 9 Alkaline Phosphatase (ALP) Staining Protocol

## Materials Needed

* Complete cell culture media
* PBS (Phosphate-buffered saline)
* 4% Paraformaldehyde (PFA)
* Alkaline Phosphatase Staining Kit (e.g., BCIP/NBT substrate)
* Incubator set at 37°C with 5% CO₂
* Sterile pipettes and pipette tips
* Personal protective equipment (PPE)

## Steps

1. Cell Culture and Differentiation:

* Culture MC3T3-E1 cells (or your cell line) in complete growth media.
* Induce osteogenic differentiation by switching to osteogenic media, if applicable, and continue culturing for 7-14 days (early differentiation stages).

2. Fixing Cells:

* After the desired differentiation period, aspirate the media from the cells.
* Wash the cells twice with PBS to remove any remaining media.
* Fix the cells by adding 4% paraformaldehyde (PFA) and incubating for 10-15 minutes at room temperature.
* Wash cells again with PBS to remove excess PFA.

3. Staining Procedure:

* Prepare the ALP staining solution according to the manufacturer’s
  instructions (e.g., BCIP/NBT substrate solution).
* Add the ALP staining solution to the fixed cells, covering the entire cell layer.
* Incubate at 37°C for 30-60 minutes or until the desired level of blue/purple color development is achieved.

4. Washing and Observation:

* Once staining is complete, aspirate the staining solution and wash the cells gently with PBS.
* Observe the stained cells under a microscope. ALP-positive cells will show blue/purple staining, indicating ALP activity.

5. Documentation:

* Take images of the stained cells for further analysis and quantification.

6. Optional:

* If desired, measure the staining intensity by solubilizing the dye and quantifying the absorbance using a spectrophotometer.

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{% block title %}Results{% endblock %}
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# 1 DBTL Cycle 1: Screening Collagen-Binding Domains (CBDs)

We constructed several **CBD-EGFP fusion proteins** and expressed them in bacterial systems to analyze their
activity.

We first commissioned a company to synthesize the gene sequences of each CBD, and then used seamless cloning to
connect the gene sequences of each CBD with the EGFP sequence already in the laboratory.

In this **stage**, the colony PCR results show successful amplification of the eight different **CBD-EGFP
fusion proteins**, confirming the correct construction of the engineered strains. The bands visible in the gel
correspond to the expected sizes of the fusion proteins:

* **SPARC/OD**, **DB**, and **DDR** are shorter sequences. After seamless cloning with **EGFP**, their
  expected PCR product size is approximately **800 bp**.
* The remaining five CBDs—**CBD MMPs, AGR, COMP, VWF-A, and FTD**—are longer tags. After seamless cloning with
  **EGFP**, their expected PCR product size is around **1200 bp**.

The PCR bands in the gel align well with these predicted sizes, demonstrating that each fusion construct was
correctly assembled, which is crucial for proceeding with protein expression and purification.

![](https://static.igem.wiki/teams/5374/results/fig1-1.png)

Fig1.1 Plasmid design

We then used these *E. coli* to express proteins and measured the fluorescence value of each protein host
bacteria at
the same bacterial concentration. Some of the fusion proteins were then purified by Ni-NTA for further analysis.

The fluorescence activity of each fusion protein was tested to evaluate whether the CBDs affected EGFP activity.
The relative fluorescence values of the eight **CBD-EGFP fusion proteins** were measured to assess the impact of
the collagen-binding domains (CBDs) on EGFP activity. As shown in the graph:

* **SPARC/OD-EGFP, CBD MMPs-EGFP, DB-EGFP, VWF-A Domain-EGFP**, and **FTD-EGFP** exhibited fluorescence
  values close to or higher than that of the control **EGFP**, indicating that these CBDs did not significantly
  affect the bioactivity of EGFP.
* **DDR-EGFP, AGR-EGFP,** and **COMP-EGFP** showed slightly lower fluorescence values, suggesting some
  impact on EGFP activity, but still maintaining measurable fluorescence.

Overall, the majority of CBDs retained sufficient EGFP activity, which confirms that they can be used in further
experiments for controlled release without significantly impairing the functionality of the fusion proteins. These
results support the selection of CBDs that balance collagen binding with maintaining protein bioactivity.

![](https://static.igem.wiki/teams/5374/results/fig1-2.png)

Fig1.2 The fluorescence activity of each fusion protein

Binding affinity tests (via microscale thermophoresis) were also conducted to determine which CBDs had high binding
affinity (for BMP-4) and low binding affinity (for VEGF). Based on the binding experiment shown in the graph, the
results illustrate the relative binding affinities of different **CBD-EGFP fusion proteins** to collagen, with
the binding affinity represented by the dissociation constant **(Kd)**. The **midpoint of each curve** on the
x-axis corresponds to the Kd value, indicating how tightly the CBDs bind to collagen.

* **FTD-EGFP** shows the strongest binding to collagen, with the lowest Kd value. From the graph, the FTD
  curve's midpoint is approximately **8.3\*10^-7 M**, indicating a high binding affinity.
* **CBD MMPs-EGFP** exhibits the weakest binding to collagen, with the highest Kd value. The midpoint of the
  CBD MMPs curve is approximately at **6.7\*10^-6 M**, indicating a lower binding affinity compared to the other
  CBDs.

These Kd values highlight that **FTD** is the most suitable for applications requiring strong binding, while **CBD
MMPs** may be used in situations where weaker, more transient binding is needed.

# 2 DBTL Cycle 2: Developing the Dual Cell Factor Release System

**FTD-BMP-4** proved more challenging. The presence of multiple disulfide bonds in BMP-4 led to the formation of
inclusion bodies when expressed in BL21, which made the protein insoluble and inactive. To overcome this, we
explored various optimized bacterial strains designed to facilitate proper folding of disulfide bond-containing
proteins.

Several strains were tested:

* ***E. coli* Origami (DE3)** improved disulfide bond formation but still resulted in considerable protein
  aggregation.
* ***E. coli* Shuffle T7-B** enhanced soluble expression, but its slow growth rate limited its practicality for
  large-scale production.
* Finally, **ArcticExpress (DE3) pRARE** was selected as the most suitable expression host, balancing both high
  protein yield and improved solubility of **FTD-BMP-4**.

The culmination of this stage was obtaining both fusion proteins in their active, soluble forms, laying the
foundation for further testing of their controlled release and biological activity in subsequent phases. In Fig3.2,
lanes 1 to 4, 5 to 8, and 9 to 12 are whole bacteria, supernatant after disruption, and precipitate after
disruption, respectively. Each group consists of four lanes, and the order within each group is BL21, ArcticExpress
(DE3) pRARE, Shuffle T7-B, Origami (DE3)

![](https://static.igem.wiki/teams/5374/results/fig2-1.png)

Fig2.1 Results of expression system optimization a) SDS-PAGE; b) semi-quantitative analysis
(Lanes 1 to 4, 5 to 8,
and 9 to 12 are whole bacteria, supernatant after disruption, and precipitate after disruption, respectively. Each
group consists of four lanes, and the order within each group is BL21, ArcticExpress (DE3) pRARE, Shuffle T7-B,
Origami (DE3))

In the gel (left) and the bar chart (right), ArcticExpress shows significantly improved soluble protein expression
compared to other strains like BL21, which primarily produces inclusion bodies. This makes ArcticExpress the optimal
choice for expressing soluble and active **FTD-BMP-4**, ensuring that a higher percentage of the total protein is
in the
desired soluble form.

In this experiment, the release profiles of **CBD MMPs-VEGF** and **FTD-BMP-4** from collagen hydrogels were
monitored over
time. The two fusion proteins were incorporated into collagen hydrogels, and their release was quantified using
ELISA assays. The hydrogels were incubated in PBS at 37°C, with supernatant samples collected at regular intervals
to measure the amount of protein released. After each sampling, fresh PBS was added to the hydrogels to maintain
consistent conditions throughout the experiment.

![](https://static.igem.wiki/teams/5374/results/fig2-2.png)

Fig2.2 Time-Dependent Release Profiles of CBD MMPs-VEGF and FTD-BMP-4 from Collagen
Hydrogel

The results reveal a significant difference in the release kinetics between the two cell factors. **CBD
MMPs-VEGF**
showed a rapid and early release pattern, starting with an accelerated increase in the release rate during the first
10 days. By day 10, more than 50% of the VEGF had already been released from the hydrogel. This sharp increase
continued, reaching approximately 75% release by day 20. Following this, the release rate gradually slowed, with the
curve plateauing as nearly 100% of the VEGF was released by day 30. The release curve of VEGF shows an initial rapid
release phase, followed by a stabilization period where the release rate gradually levels off.This fast release
behavior can be attributed to the lower binding affinity of the CBD MMPs domain, allowing VEGF to dissociate from
the collagen matrix more quickly.

In contrast, **FTD-BMP-4** exhibited a much slower and more controlled release. During the first 10 days, less
than 20%
of BMP-4 was released, indicating a more gradual release process. The release steadily increased over time, with the
curve following a linear trend. By day 20, around 30% of BMP-4 had been released, and by day 30, approximately 50%
was released. The release continued at a steady pace, reaching close to 100% around day 40. This extended release
profile reflects the higher binding affinity of the FTD domain to collagen, which ensures a slower and sustained
release of BMP-4 over the course of the experiment.

These contrasting release profiles demonstrate the effectiveness of using different collagen-binding domains to
control the timing and rate of cell factor release, with CBD MMPs facilitating a faster release for VEGF and FTD
ensuring a prolonged release for BMP-4.These results confirm that the controlled, sequential release of the two cell
factors can be achieved by leveraging the different binding affinities of the CBDs, with **CBD MMPs-VEGF**
releasing
faster to initiate angiogenesis, followed by the slower release of **FTD-BMP-4** to promote osteogenesis.

The **tube formation assay** is a widely used in vitro method for assessing the ability of endothelial cells,
such as
**HUVECs** (Human Umbilical Vein Endothelial Cells), to form capillary-like structures, mimicking the process of
angiogenesis. This assay is particularly useful for evaluating the angiogenic potential of **cell factors**,
small
molecules, or other treatments.

In the tube formation assay, human umbilical vein endothelial cells (HUVECs) were seeded onto a Matrigel-coated
plate to facilitate tube formation. The experimental group was treated with CBD MMPs-VEGF at a final concentration
of 100 ng/ml, while the control group received no VEGF. The cells were incubated at 37°C with 5% CO₂ for 4-6hours,
during which tube formation was observed. The formation of capillary-like structures was monitored under a
microscope to assess angiogenesis. The extent of tube formation between the experimental and control groups was
compared to determine the impact of CBD MMPs-VEGF on promoting angiogenesis.

![](https://static.igem.wiki/teams/5374/results/fig2-3.png)

Fig2.3 Comparison of Tube Formation in HUVECs a) Control; b)CBD MMPs-VEGF Treatment

Image a (Without CBD MMPs-VEGF): In the control group, where no CBD MMPs-VEGF was added, the HUVECs exhibited weak
angiogenic activity. Only a few scattered cell connections were observed, with minimal evidence of network
formation. Quantitatively, the tube formation rate (measured by parameters such as total tube length, number of
branch points, and closed loops or tube closure rate) was significantly reduced. The tube closure rate, which
measures the percentage of formed capillary-like loops, was close to zero, reflecting the lack of organized tube
structures. This result confirms that without the presence of VEGF, the cells are unable to initiate or sustain
effective angiogenesis.

Image b (CBD MMPs-VEGF at 100 ng/ml): In the experimental group treated with 100 ng/ml CBD MMPs-VEGF, the HUVECs
showed robust tube formation, with clearly defined and well-organized capillary-like structures. Quantitative
analysis revealed a dramatic increase in several angiogenesis indicators. The tube closure rate increased to
approximately 70-80%, indicating a high number of complete tube loops. Additionally, the total tube length and
number of branch points were significantly higher compared to the control, demonstrating enhanced network complexity
and cell connectivity. These findings suggest that the addition of VEGF significantly promoted angiogenesis, with
the cells forming a dense and interconnected tubular network, characteristic of active capillary formation.

This comparison highlights the essential role of CBD MMPs-VEGF in promoting angiogenesis, as quantified by standard
tube formation metrics. The increased tube closure rate, total tube length, and branch points in the experimental
group strongly indicate that the VEGF produced in the system retains its biological function and effectively
stimulates endothelial cell organization and network formation.The comparison between these two images confirms that
the CBD MMPs-VEGF produced in this study retains biological activity and effectively stimulates tube formation,
validating its functional ability to promote angiogenesis.

In our osteogenic differentiation experiments, **MC3T3-E1 cells**, a well-established **pre-osteoblast cell
line** derived from mouse calvaria, were used. These cells are commonly employed in bone research because they
have the inherent capacity to differentiate into mature osteoblasts when cultured under osteogenic conditions.
During the early stages of osteogenic differentiation, **MC3T3-E1 cells** express **alkaline phosphatase
(ALP)**, which is critical for mineralization. ALP staining can be used to measure the early osteogenic activity
in these cells. As differentiation progresses, **MC3T3-E1** cells begin to deposit calcium in the extracellular
matrix, which is a key indicator of bone formation. This can be detected using **Alizarin Red staining**, which
binds specifically to the calcium deposits and allows for the visualization of mineralization. Thus, by using both
**ALP staining** and **Alizarin Red staining**, the osteogenic differentiation process of **MC3T3-E1
cells** can be effectively tracked from early osteoblast activity (ALP expression) to late-stage bone formation
(calcium deposition).

Together, **ALP staining** and **Alizarin Red staining** offer a comprehensive view of osteogenic
differentiation. ALP staining identifies early differentiation events, while Alizarin Red staining confirms matrix
mineralization, showing that the cells are functionally mature osteoblasts capable of bone formation.

For the ALP staining, cells were cultured for 7 and 14 days, then washed with PBS and fixed with 4%
paraformaldehyde for 10-15 minutes at room temperature. After washing with PBS, the cells were incubated with ALP
staining solution (e.g., BCIP/NBT) for 30-60 minutes at 37°C. ALP activity, indicating early osteogenic
differentiation, was visualized as blue-purple staining under a microscope. For the Alizarin Red staining, cells
were cultured for 21 and 28 days, washed with PBS, and fixed with 4% paraformaldehyde for 10-15 minutes. After
washing with distilled water, Alizarin Red S solution (pH 4.1-4.3) was applied for 20-30 minutes to detect calcium
deposits. The cells were then rinsed with distilled water, and red staining was observed to indicate matrix
mineralization, a marker of mature osteoblast activity.

![](https://static.igem.wiki/teams/5374/results/fig2-4.png)

Fig2.4 ALP and Alizarin Red Staining of MC3T3-E1 Cells at Different Time Points During
Osteogenic
Differentiation

**ALP Staining Results (Day 7 and Day 14):** From the images provided, on **Day 7** (top-left images), the ALP
staining shows minimal blue-purple staining, suggesting that osteogenic differentiation had only just begun. By **Day
14** (bottom-left images), there is a noticeable increase in blue-purple staining, indicating elevated ALP
activity, which correlates with increased osteoblast differentiation. The stronger ALP activity seen on Day 14
demonstrates that the cells are undergoing early osteogenic differentiation, which is being effectively stimulated
by the BMP-4 cell factor, confirming its biological activity in promoting bone formation.

**Alizarin Red Staining Results (Day 21 and Day 28):** In the **Day 21** images (top-right), there are initial
signs of mineralization with scattered red deposits, indicating the onset of calcium deposition. By **Day 28**
(bottom-right), the staining intensity has significantly increased, with larger and more densely stained red areas,
showing substantial mineralization of the extracellular matrix. This confirms the later-stage osteogenic activity,
as BMP-4 continues to drive the maturation of osteoblasts and the deposition of calcium, which is a hallmark of bone
tissue formation.

These results confirm that the BMP-4 produced in our system retains its biological activity, as it successfully
stimulates both early (ALP activity) and late (mineralization) osteogenic differentiation.

# 3 DBTL Cycle 3: Development and Validation of the Collagen Hydrogel System

A collagen hydrogel system was designed to sequentially release two cell factors—VEGF and BMP-4—for bone tissue regeneration. Collagen was chosen as the base material due to its biocompatibility, biodegradability, and bioactivity, promoting cell adhesion and proliferation. The engineered Type I collagen was expressed using E. coli, and the hydrogel allowed for the rapid release of VEGF to stimulate angiogenesis, followed by the slower release of BMP-4 to support osteogenesis.

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