iGEM 2023 — Team shsbnu-china

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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Engineering

Current Page： **Home**

**Engineering**

# Engineering

### Engineering success for SHSBNU\_China

The development of synthetic biology has solved many problems for mankind, one of which is to provide high-performance polymers. Not only do the biosynthesized polymers exhibit higher strength, toughness and better damping energy, they also avoid from producing toxic byproducts in the whole process. This year, SHSBNU\_China concentrated on improving the performance of artificial heart valves with megadalton titin polymers produced by E.coli. We believe our project will expand the connection between synthetic biology and biomedical field.

###  4XT: BBa\_K4672000

To do this, we read the published paper and notice that titin is a muscle protein, in which the most essential part is hundreds of folded immunoglobulin (Ig) domains in repetitive sequence. Following the paper, we chose to polymerize a relatively rigid subunit consisting of four Ig domains from the I-band of the rabbit soleus muscle titin, which has been uploaded as part BBa\_K4672000 and named as 4XT.

##### Protocol we use:

We asked the biological company to synthesize 4XT sequence at first, the sequence was then constructed onto pET28a(+) plasmids, which is proper for protein purification and expression.
We further transformed the plasmids into E. coli BL21(DE3) strains.
The colony was grown up on the plate, and we picked a single colony using a sterile tip.
The single colony and tip were added it into 4 ml LB medium with the kanamycin and then cultured overnight.
When the bacteria solution reached OD600=0.6, we added 0.5 mM IPTG for induction and shook at 16℃ for 20 h or 37℃ for 4 h. We also set a control group and didn’t add IPTG into it.
After the expression was completed, we centrifuged the bacterial solution at 12000 rpm, discarded the supernatant, and then used RIPA as a lysis buffer to resuspend the bacteria.
For the cell lysate, we added loading buffer to heat at 96℃ for 10 min.
Finally we underwent SDS-PAGE assay and Coomassie brilliant blue staining for expression test.
As shown in the figure, both the two environment showed the expression bands of pET28a(+)-4XT, which only appeared in the group with IPTG. The molecular weight of this band was in line with the expectation at around 43 kDa. Besides, the expression for 20 h under 16℃ had better performance. The above results confirmed our success in titin monomer expression, which preferred a lower temperature and longer induction time.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/en1.jpg)

###  BBa\_K4672001

After the monomer expression of [4XT](http://parts.igem.org/Part%3ABBa_K3264000) , we designed a new plasmid to realize polymerize, in which we fused the C- and N-terminal of a fast-reacting SI pair to 4XT, named IntC-4XT-IntN (Part number BBa\_K4672001). According to the published papers, SIs catalyze splicing reactions spontaneously, which lead to the covalent link between fusion partners through peptide bonds.

##### Protocol we use:

We asked the biological company to synthesize IntC-4XT-IntN sequence at first, the sequence was then constructed onto pET28a(+) plasmids, which is proper for protein purification and expression.
We further transformed the plasmids into E. coli BL21(DE3) strains.
The colony was grown up on the plate, and we picked a single colony using a sterile tip.
The single colony and tip were added it into 4 ml LB medium with the kanamycin and then cultured overnight.
When the bacteria solution reached OD600=0.6, we added 0.5 mM IPTG for induction and shook at 16℃ for 20 h or 37℃ for 4 h. We also set a control group and didn’t add IPTG into it.
After the expression was completed, we centrifuged the bacterial solution at 12000 rpm, discarded the supernatant, and then used RIPA as a lysis buffer to resuspend the bacteria.
For the cell lysate, we added loading buffer to heat at 96℃ for 10 min.
Finally we underwent SDS-PAGE assay and Coomassie brilliant blue staining for expression test.
The pET28a(+)-IntC-4XT-IntN worked well, as we observed cells produced a cluster of ultra-high molecular weight (UHMW) products up to and above 400 kDa in the top. Consistently with the former result, expression for 20 h under 16℃ had better performance. Taken together, we successfully produced titin polymer and found a better expression condition for UHMW production.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/bba-k4672001-silver-2.png)

###  BBa\_K4672002

Besides of the monomer part [4XT](parts.igem.org/Part%3ABBa_K4672000) and polymer part [IntC-4XT-IntN](parts.igem.org/Part%3ABBa_K4672001), we also constructed pET28a(+)-IntC-8XT-IntN plasmid in which the subunit consisting of eight Ig domains to see whether more subunit could help with better polymerization

##### Protocol we use:

We asked the biological company to synthesize IntC-8XT-IntN sequence at first, the sequence was then constructed onto pET28a(+) plasmids, which is proper for protein purification and expression.
We further transformed the plasmids into E. coli BL21(DE3) strains.
The colony was grown up on the plate, and we picked a single colony using a sterile tip.
The single colony and tip were added it into 4 ml LB medium with the kanamycin and then cultured overnight.
When the bacteria solution reached OD600=0.6, we added 0.5 mM IPTG for induction and shook at 16℃ for 20 h or 37℃ for 4 h. We also set a control group and didn’t add IPTG into it.
After the expression was completed, we centrifuged the bacterial solution at 12000 rpm, discarded the supernatant, and then used RIPA as a lysis buffer to resuspend the bacteria.
For the cell lysate, we added loading buffer to heat at 96℃ for 10 min.
Finally we underwent SDS-PAGE assay and Coomassie brilliant blue staining for expression test.
However, this result is not ideal. We further asked for help and underwent a Western blot assay to see whether this is because of the low expression level, but western blot does not present bands either. We feel confused about the result and will fix it later.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/bba-k4672002-silver-wb.png)

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# Experiments

Current Page： **Home**

**Experiments**

* [Experiments](#p0-5)

### Cultivation of Bacteria

(a Extract the plasmid from the original bacteria

(b Transform the plasmid into the competent cell BL21 and Dh5α

(c Place in solid LB which contain kanamycin to culture the bacteria in 37°C constant temperature incubator for 12h

(d Pick the best monoclonal and put into liquid LB which contain kanamycin to amplify bacteria in 37°C constant temperature incubator for 12h

### PCR

(a Add necessary buffers and mixs and primers to the template
(b Put the 20 μl tubes into the PCR machine
(c Set the temperature at 95°C for 30s, and then cycle the process of 95°C for 15s, 58.5°C for 15s, 72°C for 1min 35 cycles, and at last set 72°C for 5mins.

### DNA electrophoresis

(a Make a 10% agarose gel by using 0.4g agarose and 40ml TAE with 100ml water
(b Add the products of PCR to the agarose gel
(c Move the agarose gel into the DNA electrophoresis device and run for 30mins
(d Move the agarose to under the UV light and photograph the result

### SDS-Page

(a Add 0.5mM IPTG and 15 ml liquid LB and kanamycin to 15ml transformed BL21
(b Separate the 30ml solution into 10 test tubes equally, and cultivate separately in 16°C incubator for 6h, 12h, 18h, 24h, 30h and 36h
(c Extract the protein form the solution
(d Use ddH\_2O, 30%Acr-Bis, separate the gel buffer, 10%APS, TEMED to make a piece of SDS-Page gel
(e Add the protein to the SDS-Page gel and add the Loading Buffer to the device
(f Open the power supply for 1h
(g Take out the SDS-Page gel and add the dye for 30mins
(h Wash the SDS-Page gel until can see the result

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# Results

Current Page:**Home**

**Results<**

# Results<

Synthetic biology has made significant strides in recent years, offering immense benefits to humanity. One of its most notable achievements addresses the issue of high-performance polymers. Traditionally, these polymers have been non-biodegradable, sourced from petroleum feedstocks, and manufactured through environmentally harmful processes that generate toxic byproducts. This year, the SHSBNU\_China team set its sights on the microbial synthesis of these high-performance polymeric materials.
Employing E. coli, we managed to produce megadalton titin polymers. These polymers have proven to yield high-performance fibers characterized by an array of desirable properties, including superior strength and toughness, as well as enhanced energy damping capabilities. Building on this success, we integrated our megadalton muscle titin polymers into the domain of healthcare, specifically to augment the performance of artificial heart valves. This innovation not only boosts the bio-compatibility of these valves but also significantly improves their mechanical strength.
It is our aspiration that our project will underscore the revolutionary potential of synthetic biology, especially in addressing complex challenges in biomedical engineering—challenges that remain elusive to conventional materials and methodologies.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/re1.png)

Project plan from synthetic biology component such as genetic engineering to hardware implementation.

### Expression of titin monomer

Many high-performance natural materials are protein-based and their advantages originate from hierarchically assembling ultra-high molecular weight (UHMW) proteins consisting highly repetitive amino acid sequences. Titin, the muscle protein, endows muscles with damping capacity, passive strength, and rapid mechanical recovery derived from its UHMW (>3 MDa) and hundreds of folded immunoglobulin (Ig) domains in repetitive sequence. As suggested in the paper, we chose to polymerize a relatively rigid subunit consisting of four Ig domains from the I-band of the rabbit soleus muscle titin, which has been uploaded as part BBA\_K4672000（http:// parts.igem.org/Part:BBa\_K4672000）and named as 4XT.
In order to express 4XT, we used the pET28a(+) plasmids as backbone and transfected it into E.coli BL21(DE3).

![Figure 1](https://static.igem.wiki/teams/4672/wiki/re2.png)

After IPTG induction, bacteria were lysed for SDS-PAGE and Coomassie brilliant blue staining. The result showed our plasmids pET28a(+)-4XT worked pretty well. We repeated the experiment in different condition:
1) expression for 20 h under 16℃
2) expression for 4h under 37℃.
As shown in the figure below, both the two environment showed the expression bands of pET28a(+)-4XT, which is around 43 kDa as expected, but expression for 20 h under 16℃ had better performance. The above results confirmed our success in monomer expression, which preferred a lower temperature and longer induction time.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/en1.jpg)

**We communicated our ideas and experimental results with Mr. BoXiang Wang, the instructor of the 2019 GreatBay\_SZ iGEM team. Mr. Wang was very surprised and emphasized that many spinning proteins had problems in solubleness. We successfully expressed soluble proteins under such experimental conditions, and he encouraged us to continue the experiment. It is also suggested by him that we could refer to the 2019 project to further improve our experiment.**

### Expression of titin polymer

To realize titin polymerization, we designed a new plasmid, in which we fused the C- and N-terminal of a fast-reacting SI pair to 4XT, named IntC-4Ig-IntN (Part number BBA\_K4672001, http:// parts.igem.org/Part:BBa\_K4672001). According to some published papers, SIs could catalyze splicing reactions spontaneously, leading to the covalent link of their fusion partners via peptide bonds, which showing minimal effect on the properties of the resulting products.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/re3.png)

We then transformed the new plasmid into E.coli BL21(DE3). Consistent with the previous procedure, after IPTG induction, bacteria were lysed for SDS-PAGE and Coomassie brilliant blue staining. The pET28a(+)-IntC-4XT-IntN worked well too, as we observed cells produced a cluster of UHMW products up to and above 400 kDa in the top. We also tested different condition for protein polymerization:
1) expression for 20 h under 16℃
2) expression for 4h under 37℃.

Consistently with the former result, expression for 20 h under 16℃ had better performance. Taken together, we successfully produced titin polymer and found a better expression condition for UHMW production.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/bba-k4672001-silver-2.png)

Besides, we also constructed pET28a(+)-IntC-8XT-IntN plasmid in which the subunit consisting of eight Ig domains to see whether it could help with the polymerization. However, the expression result is not ideal.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/re4.png)

**We feel confused about the result and proposed two hypotheses: first, there was protein expression and aggregation in the cell, but the protein expression was too low to be detected by SDS-PAGE; second, there is no protein expression and aggregation in the cell. In order to find out the reason, we interviewed with Mr. Hu from Minzu University of China, who suggested us to use the more sensitive Western Blot method and assisted us in carrying out the experiment. However, the results showed that there was no protein expression in our system, which maybe indicated that four Ig domains have the best effect.**

![Figure 1](https://static.igem.wiki/teams/4672/wiki/bba-k4672002-silver-wb.png)

### Wet Spinning for Production of fibrous threads

Wet spinning is a technique employed to convert proteins or polymers into fibers. The process involves extruding a solution of the protein or polymer into a coagulation bath, where the dissolved substance precipitates or solidifies to form continuous fibers. For the synthesis of Titin protein fibers, it's crucial that the protein solution is injected into the coagulating medium at a consistent rate to ensure the resultant fibers have uniform diameter and properties. This is where the need for mechanical assistance, like the use of a stepper motor-driven injection system, comes into play.
To establish a reliable wet spinning setup, we conceptualized a system where the protein solution could be driven by a stepper motor attached to a syringe. The motor ensures precision in the extrusion rate, allowing us to regulate the diameter and consistency of the resulting fibers.
However, this motorized system requires a sturdy framework. Hence, we designed and built a frame using customized metal bars and metal brackets. The metal bars provided the structural integrity, ensuring stability and resistance to vibrations which could disrupt the delicate extrusion process. Brackets were incorporated to secure the bars together, forming a solid yet adjustable structure. The placement of these bars and brackets was meticulously planned so as to hold the motor and syringe in optimal position for the wet spinning process.
We took advantage to use 3D-printing available in our school’s engineering lab, for the creation of connector parts. These connectors fix the stepper motor to the frame, and they are designed to hold the syringe in alignment, allowing for precise downward movement. This guarantees that the protein injection carry on with uniformity.
Following the spinning process, we can employ traditional plain weaving techniques to convert these fibers into fabric. This fabric is then ideally suited to act as the intermediary layer in a sandwich structure, boasting the combined benefits of flexibility, strength, and bio-compatibility.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/re5.png)

Our hand-drawn design of a combination of the stepper motor and frame. We constructed the frame with customized metal bars and brackets, and 3D-printed the connector parts, and eventually assemble the injection drive.

### Wet Spinning: Injection Condition Testing

Aiming to produce finer fibers, extensive testing and adjusting the injection speeds is necessary. Recognizing that our protein production is still experimental and not yet scalable enough to generate vast quantities needed for repeated hardware testing, we followed expert suggestion and used generic protein fibers as a stand-in to evaluate the spinning equipment's performance.
This approach would enable us to refine the equipment settings and ensure the resulting fibers meet the desired specifications. By using a readily available protein as a proxy, we can gain insights into the behavior of our specialized protein under similar conditions without depleting our limited resources. Furthermore, the use of a common protein can also provide a baseline for performance, making it easier to identify any unique challenges or advantages presented by our proprietary protein when it's eventually used.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/re6.png)

Video: Protein Solution Injecting into Cold Coagulation Bath

![Figure 1](https://static.igem.wiki/teams/4672/wiki/re7.png)

Testing wet spinning and injection conditions, such as temperature, solution viscosity, and injection diameter/speed.

### Designing the Shape of the Heart Valve

The heart facilitates a more laminar flow, which reduces the risk of clot formation. In contrast, traditional mechanical valves often induce turbulent flow, which can lead to clot formation.
After consultations with cardiac specialists, we decided to adopt a tricuspid valve shape for our artificial heart valve design. The tricuspid design incorporates three leaflets inspired by the natural anatomy of the heart's right atrioventricular valve. This shape is versatile to adapt and fit into various blood vessel configurations in the heart, ensuring a broad applicability.
To translate our conceptual design into reality, we utilized 3D printing technology in our school’s physics and engineering lab. To demonstrate its functionality and visualize the flow patterns, we mounted the 3D-printed valve in a transparent plastic tube simulating a blood vessel. By testing water flow through this system, we could closely observe the flow dynamics and ensure our design met the desired criteria.

![Figure 1](https://static.igem.wiki/teams/4672/wiki/re8.png)

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