iGEM 2023 — Team swuer-china


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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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**Engineering**

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(1)

## New gene selection

### **Get the protein added to the spider silk can enhance the performance**

(2)

## Wire drawing

### **Obtain the experimental material:The use of rotating instruments to drive wire drawing, tweezers from the tail of the spider to take out the spider silk fixed to the instrument, the instrument rotates, constantly pull out the spider silk.**

![#](https://static.igem.wiki/teams/4915/wiki/eng2.png)

Fig. 1 Schematic diagram of spider drawing steps. (Arndt T et al, Adv Funct Mater, 2022)

Additionally, as an innovation point, we designed a wire-drawing machine. After extensive literature review and experimental design, according to the rich experimental experience in the field of spider silk material research, we designed such an experimental device which is convenient for drawing wire (Figure 2) . The visible silk thread is pulled out from the fixed spider tail with tweezers and placed on the white roller. After power is applied, the roller will turn clockwise at a suitable speed so that the spider silk can be continuously pulled out, freeing the hands of the experimenter and saving the experiment time.

![#](https://static.igem.wiki/teams/4915/wiki/eng3.png)

Fig. 2 Spider drawing device real thing.

(3)

## Dissolve silk fibroin

### **Degumming after preparing solution with chemical reagents, and then dissolved with lithium bromide at 4 nights.**

![#](https://static.igem.wiki/teams/4915/wiki/eng4.png)

Fig. 3 Dissolution of silk fibroids.

(4)

## Dialysis

### **Leave it to the company,doing something about fibroin protein purification by dialysis.**

(5)

## Performance measurement

### **(1)Slide detection**

**Observing changes under an electron microscope:** Wash the slide, cover slide and wipe them clean. After the egg whites have melted, mix them with the silk fibroin solution and drop at least on the slide. Cover the cover slide and place it under the microscope for observation and photo recording. The average size and diameter of the aggregated protein crystals were measured by scanning electron microscopy.

![#](https://static.igem.wiki/teams/4915/wiki/eng8.png)

Fig. 4 Microscopic examination apparatus for slide experiments.
Fig. 5 Schematic diagram of the slide experimental testing process.

### **(2) Raman spectra**

**Raman spectrum statistics of secondary structure changes:** Raman spectroscopy is very sensitive to molecular bonding and the structure of the sample, so each molecule or sample has its own spectral signature. These markers can be used for further study and analysis of the secondary structure of different samples in the experimental group.

![#](https://static.igem.wiki/teams/4915/wiki/eng7.png)

Fig. 6 Raman spectrum statistics of secondary structure changes.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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**Experiments**

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## **Preparation**

## I. Adopt a mild degumming method II. Dissolution and desalination of silk fiber

• 2.5 g degummed silk fiber is stirred for 1 hour in a 45◦C water bath in 25 mL 56% (w/w) of ZnCl2 aqueous solution.
• The two proportional configuration of zinc chloride aqueous solutions cannot completely dissolve silk.

![#](https://static.igem.wiki/teams/4915/wiki/exp1.png)

## Experimental procedures

* ![#](https://static.igem.wiki/teams/4915/wiki/steps-v1-shape1.png)

  ## 01

  ## First

  Preparation of materials: we collected the special equipments and a large number of spiders with particular characters, then we extracted a lot of gossamer of these spiders.
* ![#](https://static.igem.wiki/teams/4915/wiki/steps-v1-shape2.png)

  ## 02

  ## Second

  Designing different condition and operation: we concocted buffer solution with PH gradient and dissolve quantitative gossamer in the different solutions respectively.
* ## 03

  ## Third

  Microscopic examination: we observed structural changes of spider silk protein before and after the reaction between the protein and solutions.

### **Last, adding other substances: we added particular molecules with particular performances which were found through our established model into the compound, and test whether there are some new structures and naval functions of spider silk protein or not.**

![#](https://static.igem.wiki/teams/4915/wiki/exp2.png)

![#](https://static.igem.wiki/teams/4915/wiki/jt1.png)

![#](https://static.igem.wiki/teams/4915/wiki/exp4.png)

## Slide experiment

![#](https://static.igem.wiki/teams/4915/wiki/exp5.png)

![#](https://static.igem.wiki/teams/4915/wiki/exp3.png)

Mix preparingA, B and C together, and then place the mixture under the microscope to observe. Then we can get the measurements.

## Microscopic examination process

### Clean the slide and cover glass and wipe it clean. After the protein melts, mix it with the silk solution, drop it on the slide at least, cover the cover glass and place it under the microscope to observe the photo record.

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## Preparation

Previous studies have shown that the binding of the new gene expression protein to MaSp2 can double the performance of the silk, and the results of the Ma three-segment proteome show that two new genes exist in the anterior middle of the silk gland and the traction filament.

![](https://static.igem.wiki/teams/4915/wiki/results5.png)

From:Wenbo Hu et al, Nat commun, 2023

## Result one

Blastp showed that these two proteins were present in several Araneus species, but not in non-Araneus species

![](https://static.igem.wiki/teams/4915/wiki/results3.png)

Fig.1 Plot of sequence alignment results

At last,we choose these two proteins as the added proteins in the experiment,named as protein 1 and protein 2 respectively in the forward experiment.

## Result two

1. Irregular aggregation was formed in both control group and blank control group
2. Protein 1, formed dense filament-like fibers relative to the blank control group and the control group
3. Protein 2, formed some foamy clumps of fibers compared with the blank control group and the control group
4. Protein 1 and protein 2 were added to form dense filament-like fibers and some foamy clumps of fibers compared with blank control group and control group

![](https://static.igem.wiki/teams/4915/wiki/results4.png)

Fig.2 Schematic diagram of slide test results

The addition of protein 1 can form dense filamentous fibers compared to the blank control group and the control group, the addition of protein 2 can form some foam-like clusters of fibers compared to the blank control group and the control group, and the addition of protein 1 and protein 2 can form both dense filamentous fibers and some foam-like clusters of fibers compared to the blank control group and the control group. These results show that protein 1 and protein 2 can indeed enhance the performance of spider silk, at least improve its fiber forming ability, which is conducive to improving toughness, and can be better applied to the manufacture of automobile tires, aviation clothing, etc.