Introduction
We have made a lot of innovations in multiple aspects, including software, hardware and experience for wet lab. Numerous basic and composite parts were also constructed, contributing to the proliferation of synthetic biology. We hope our project can give some help and guidance to the future iGEM teams.
Parts
Biology is different from other engineering fields due to a lack of modular nature of the structure and function, which means the expression of gene will change when transferred from one species to another. What synthetic biology is dedicated to is the modularization and standardization of functional genes. Our project has registered 19 basic parts and 56 composite parts, all qualified for standardized requirements of RFC[10]. We have also tested these parts. And we hope that everyone can utilize our parts based on our research through registration and further improve the information of our parts to make more people can use them. For more details please refer to parts.
Experience of wet lab
During our experiment, we summarized the experience in genetic engineering, heterologous expression of yeast, hair dyeing and perm and so on. In addition we have compiled a protocol by our team and a yeast handbook by collaborate with the Yeast alliance.
genetic engineering:
In the process of constructing the whole pathway, We first replaced the promoter of this component by restriction endonuclease digestion and T4 ligase ligation. However, when we carried out enzyme digestion, we found that the efficiency of double digestion of the two adjacent sites was very low, so we improved the double enzyme digestion system, increased the amount of endonuclease, prolonged the enzyme digestion time (did overnight enzyme digestion).
When we were doing enzyme digestion and enzyme linking, we used the method of plasmid enzyme digestion. But we met the situation in which the concentration of the plasmid vector was very low. Therefore, we suggest that the products of multiple enzyme digestion should be recovered into the same system after gel electrophoresis to increase the concentration of the vector. The fragments amplified by PCR need to be further purified otherwise salt ions will affect the efficiency of enzyme digestion. In addition, the fragments also need to be further purified after enzymatic digestion to avoid affecting the efficiency of ligation.
Secondly, when we were going to carry out seamless cloning and splicing, we had never been able to succeed. The fragments we connected have long repetitions. We speculate that the principle of seamless cloning enzymes of some companies may be different from that of Gibson assembly. The operation and result according to the instructions provided by these companies failed to meet expectations. Therefore, it is recommended that if seamless cloning is needed in the design of the project, try to ensure that there are no repetitions in fragments or use the seamless cloning kit of Gibson assembly.
genetic engineering of yeast: In the process of expression, we used α-factor as a signal peptide for extracellular expression, but some proteins could not be expressed successfully. Extracellular expression of eukaryotic system is a very complex process, the adaptations between signal peptide and protein, signal peptide and chassis, protein and chassis are very important, but there is no accurate method for the mutual selection of the three. After removing the signal peptide for intracellular expression, we successfully expressed most of the proteins that could not be expressed with the signal peptide.
The Best Condition of hair dye:
| Dye/Condition | Time | temperature | Dyeing aid ingredients | concentration(g/L) | comment |
|---|---|---|---|---|---|
| indigo | 2min | Room temperature | none | 2 | The color deepens significantly while dyeing for multiple times |
| curcumin | 30min | 50℃ | Ethyl alcohol | 0.5 | |
| lycopene | 30min | Room temperature | alum | 2 |
And we found that as for the three colors selected for the experiment, bleach the hair to 8 degrees could achieve a bright coloring effect.
The best condition of perming:
1)immerse 10 hairs in SDS 3 times to remove charges on hair surface.
2)temperature:50℃
3)time: 1 hour
For more information please refer to engineering and result.
Software
1)MCN Counter:
Figure 1: MCN Counter
We customized a pair of data bases and data tags about the micronucleus counting of broad bean root tips. This data base includes images of hundreds of typical chromosomes and micronuclei, and a model was trained based on this to conduct the counting of micronuclei in root tips of broad beans, which was used to determine the toxicity of multiple dyes.
This tool has greatly liberated manpower and gives the heavy work of processing images to the computer. We have opened the access of this model on GITHUB and detailed the customization process in the MODEL section (if you are only using micronuclei identification) to ensure that each user can train a counting model that is appropriate for their individual experimental conditions.
2)Staining predictor
Figure 2: Staining predictor
We set up several BP neural networks in the environment of python3+tensorflow+keras, which can be used to predict hair dyeing results according to the type, concentration and time of pigments. This network applies to virtually all molecular attachment systems. Therefore, it is possible for later groups to customize their own predictive models according to this network structure, thus enriching the predictive models including fiber staining, macromolecular surface adsorption, etc. The contribution of this model lies more in the mathematical part, which is determining the data labels and the layer activation functions. You can learn more details in the MODEL section. For more information please refer to Modeling.
Hardware:
Figure 3: Fermentation tank
Hardware includes the fermentation bottle and the dyeing comb. Together, they make up a product of Mr.Tony, providing the customers with a powerful device that allows them to dye their hair entirely on their own. What's more fascinating is that our fermentation bottle is not limited to producing our own product. Theoretically, any fermentation work that can be carried out in our fermentation flask at room temperature and oxygen. The bottle owns the basic needed for a fermentation device: the air pump, the agitator, and the dam-boards. Additionally, what makes the bottle amazing is that as long as you turn the bottle upside down, you can quickly get a pure (yeast-free) product. The specially designed filter could filter out the yeasts and let the product into the storage chamber. This bottle provides a possible solution to conveniently purify fermentation products. Also, the bottle is portable, making it possible for scientists and customers to bring it to anywhere they like. For more information please refer to hardware.
Wow! The team contributes to synthetic biology in multiple ways! I was told that their achievement is not only related to the dedication of the team members, but also can be attributed to human practice from which they drew expertise and inspiration.