Overview

Our project contributions include enriching libraries, improving organic pigment extraction techniques, modeling microbial metabolism, building high-precision light-controlled devices covering the full spectral range, and facilitating knowledge exchange within the iGEM community.

Through rational design of vioE enzyme, we enrich the iGEM library and enhance its catalytic ability. In addition, our projects often have to extract and measure the yield of organic pigments, and after many experiments, we provide useful solutions for other teams. We also contribute to the field of antibody engineering by generating antibody sequence models and measuring nanoantibody binding effects in the following ways. In addition, we promote access through our educational programs, reaching out to diverse communities and addressing inequalities. Our low-cost fluorescence microscope designs and stamping and punching modules further facilitate accessibility to essential laboratory equipment.

We sincerely hope that our work this year will inspire and help more iGEMer and synthetic biologists." At the same time, we hope that our project can expand the impact to a wider audience, so that people have a deeper understanding of the meaning of "fashion".

Enrich iGEM Parts Library

A total of 5 enzymes are required for the synthesis of violacein, and vioE plays the most critical role in the entire synthesis process. Combined with our early experimental data and software model, we screened out the sample group with the greatest potential for enzyme activity enhancement from a larger virtual mutant library. In the predicted region (pocket size 600±100 or 1200±100, channel length 6±2), the most likely mutant with a high increase in enzyme activity was obtained, so we conducted further experimental verification, and obtained a mutant with a maximum increase in enzyme activity efficiency of 30%.

Fig 1 vioE mutant and WT enzyme activity diagram

For more details, please see our Part page.

Measurement of Organic Pigment Yield

In this year's iGEM project, we often needed to measure pigment production to determine if we had a mutant with higher enzymatic activity, but because violacein is an organic pigment, it is almost insoluble in water. Therefore, on the basis of literature review, we obtained a relatively stable measurement method for measuring the production of organic pigments by E. coli, which can be used as a reference for other teams in the iGEM community.

For more details, please see our protocol page.

Salicylic acid and C4-HSL Receptor Sensitivity Model Construction

We constructed the promoters corresponding to RhlR and nahR in front of sfGFP, which are induced by C4-HSL and salicylic acid respectively. Meanwhile, we constructed a plasmid containing corresponding receptors.

To measure the ability of salicylic acid and C4-HSL to induce the promoters we used, we used fluorescent microplates to detect fluorescence intensity at different signaling molecule concentrations. The results showed that when the concentration of salicylic acid was between 2e-6M~2.6e-4M, the fluorescence intensity increased gradually. However, when the concentration of salicylic acid was above 2.6e-4M, the fluorescence intensity gradually decreased, which may be due to the inhibition of E. coli growth caused by high concentration of salicylic acid. Meanwhile, the higher the concentration of C4-HSL, the higher the fluorescence intensity.

Fig2a:Plasmid containing RhlR and nahR
Fig2b:RhlR sensitivity detection
Fig2c:nahR sensitivity detection

High Precision Light Control System with Full Spectrum Coverage

Light-sensitive promoters are crucial in gene expression regulation and bioengineering due to their ability to non-invasively control gene expression, reducing side effects and external interference caused by chemical inducers. This advantage is significant in in vivo experiments and therapies. However, existing light-controlled devices are limited by their narrow wavelength range, restricting the utility of light- sensitive promoters. To address this and make light-control devices accessible for all laboratories, we have designed, constructed, tested, and open-sourced Suncraft, a high-precision projector capable of full-spectrum coverage(See Fig2a、2b). Suncraft reliably controls illumination in designated areas and creates an environment conducive to sample reactions.

Fig 3a Suncraft final product

Fig 3b Optical Path

For more details, please see our hardware page.

Causality Analysis: a new solution to system identification

Inspired by the Software EXPMEASURE from ZJU-China IGEM 2022, we plan to introduce a new mathematical method to analyze experimental data and address the "explanatory crisis" in complex biological systems.

Using causal analysis methods for statistical analysis is a solution to the crisis. We introduced causal graph model testing and discovery, breakpoint analysis, and multi-objective optimization methods, establishing a statistical analysis framework to quantify causal effects and assist in experimental design.

The framework is provided in two forms: a Python library and a highly integrated web application, to ensure that, it can be easily accessed and widely distributed in our biosynthesis community.

This software is designed to assist iGEMers and other researchers:

1. Identify patterns from complex intervention data (e.g. identification of metabolic pathways);
2. Testing of engineering assumptions;
3. Predict what the outcome of an intervention will be

Fig 4

For more details, please see our software page.

Modeling the future: models for bacterial growth and pigment diffusion

The model simulates the entire process of pigment production and precipitation, from the microscopic to the macroscopic scale, making a significant contribution to synthetic biology. By modeling the metabolic network, bacterial growth and reproduction, as well as the process of pigment production, diffusion, and precipitation, this project has achieved a complete visualization of the impact of gene knockouts on pigment production.

The modeling component utilizes the CobraToolBox to construct a metabolic network model of *E. coli* and employs a genetic algorithm to compute the flux distribution after gene knockout. A cellular automaton (CA) model with cubic nonlinear factors and time-dependent terms is used to simulate bacterial growth, death, and diffusion. This is coupled with a coefficient that links to the pigment diffusion PDE model. The pigment diffusion equation extends the basic convection-diffusion equation by incorporating Darcy's law and pressure field effects, which are solved using the Poisson equation. The model provides a modifiable pigment production coefficient, and the flux correction coefficient obtained from the metabolic network model allows for the visualization of pigment production changes after gene knockout.

Fig 5

For more details, please see our model page.

Promoting understanding of synthetic biology

One of the main contributions of our project to synthetic biology is to broaden people's understanding of fashion. By highlighting the complex systems of pigment production processes to enhance the richness of dyeing processes, our projects shed light on the basic biology of these processes and contribute to greater awareness and appreciation of fashion.

In addition, our project aims to break down stereotypes about biology as boring and boring, allowing people to break down the barriers to cutting-edge knowledge in synthetic biology and other fields. By consulting with education experts and taking their advice, as well as providing an engaging and memorable education program.

Through multi-faceted communication with the society, our project has opened up a broad platform for the exchange of innovative and entrepreneurial ideas and biological knowledge, and collected feedback from the interviewees for continuous improvement of project design and communication methods, promoting program iteration and responding to social needs.

In short, our project makes synthetic biology more accessible by raising awareness of global issues, and further, we aim to improve people's understanding of fashion itself. Together, we hope that our project has contributed to synthetic biology and ultimately promoted a greater understanding and appreciation of the field of fashion and synthetic biology.

Fig 6

For more details, please see our Human Practice page.