We hope to develop a more environmentally friendly 、safe and interesting biological dye——Neovio dye. To this end, we have made efforts in five aspects:

1.Yield improvement module：Production enhancement module: We adhere to the idea from micro to macro, using codon optimization, protein semi-rational design, RNA skeleton assisted phase separation, metabolic modification of E. coli and other multi-dimensional to improve the production of violacein.

2. Communication module：We design an orthogonal quorum sensing system, using salicylic acid and C4-HSL as signal molecules to regulate pigment production of producer, in order to achieve a richer dyeing pattern.

3.Painting module：We intend to design a light-induced pattern rendering model, including the use of blue-light-Inducible recombinases to burn pattern information on the controller.

4.blocking module：We try to modify HYER(Hydrolytic Endonucleolytic Ribozymes) to destroy its hydrolytic activity while retaining the recognition function and enhancing its specificity, allowing us to employ it as a blocker of expression leakage .

5.biosafety module：We use KillerRed to make engineered bacteria that accidentally enter the environment commit suicide to achieve biosafety.

We choose E. coli k12 as the chassis organism. Because we have a more thorough understanding of its metabolic model, it is convenient for us to use mathematical modeling and software design calculations to make our project more environmentally friendly and efficient.

See our model, hardware and software

Obviously, a key to successful dyeing and reducing post-production costs is ensuring an adequate supply of the "dye" violacein. In this module, we propose a multi-level biological design optimization scheme from molecules to networks, and from micro to macro.

(1) Construction of producing strain At the gene expression level, we found in the literature the sequences of a set of violacein synthases derived from violaceum. Considering that Bacillus purpurea and E. coli have different codon bias, we performed codon optimization on these sequences and obtained gene sequences adapted to E. coli K12.

(2) Semi-rational design of enzymes
At the biological macromolecule level, we found vioE, a key enzyme in the pigment synthesis pathway, as a target for protein design. Our team identified the relatively conserved homologous domain of the vioE sequence through Tbtool gene family analysis, and also attempted to predict the composition of the enzyme catalytic pocket through molecular docking. We expect to obtain a batch of amino acid sites with reasonable design potential through the above two strategies.
In current research on computationally based rational design of proteins, integrating various molecular properties and shaping energy landscapes and extracting precise features is a major challenge[1], and traditional multi-objective optimization methods are susceptible to interference from confounding variables. So in order to explore the causal relationship between the contribution of various protein structural properties to enzyme activity, we used an optimized sampling method that can eliminate confounding factors with the help of software (see the software page for details) to help us predict the possible maximum enzyme activity. The range of living molecular structures. Finally, we were able to screen the samples with the largest increase in enzyme activity from a larger virtual mutant library through this method. We also performed molecular dynamics simulation analysis on the final optimized mutants to analyze the mechanism behind the increase in enzyme activity from a more precise perspective.

(3) RNA skeleton assists phase separation
At the spatial distribution level, although enzymes for pigment production are well expressed inside our chassis, they tend to distribute dispersively, hurting efficiency, for they have to catalyze substrates in order. To solve this problem, we design the RNA scaffold to pull enzymes together so that they can function consecutively.
Based on specific interaction between the RNA hairpin, boxB, and the adaptor, λ peptide, we try to trap enzymes for pigment production to co-localize them[1]. Meanwhile, we form condensed beads resulting from spontaneously aggregated 47-CAG-repeat RNA sequence and thus gather more enzymes.

(4) Metabolic modification of E. coli
At the individual metabolism level[2], we hope to look at the influence of allogenic pathway of ionin synthesis on the overall metabolism of E.coli. Therefore, we try to construct a metabolic model based on the whole genome of E.coli. After introducing the heterologous synthesis pathway, a batch of genes with transformation potential can be screened out through the FBA model based on the metabolic flux balance theory, and the metabolic flow optimization of the chassis microorganisms could be realized through gene editing.

Quorum sensing system is an important signaling mode in bacteria. Quorum sensing systems include signaling molecules and receptors that bind to signaling molecules. When the receptor binds to the signaling molecule, its ability to induce expression of the corresponding inducible promoter will be changed.

According to the synthesis pathway of violacein, if vioA, vioB and vioD are normally expressed, vioE is expressed while vioC is not, green pigment will be produced. If vioE and vioC are expressed together, purple pigments will be produced. In order to make the strains with vioA, vioB, vioC, vioD and vioE be controlled to take different synthetic pathways and thus produce different colors of pigments[3], we will divide the engineered strains into two types, one is the production strain carrying two kinds of quorum sensing receptors,the other one is a control strain capable of producing two quorum sensing signaling molecules. By producing different signaling molecules, the production strains can be induced to produce different pigments.

However, AHL molecules produced by the quorum sensing signaling molecule synthase in Gram-negative bacteria tend to have multiple types. Due to the differences in the metabolic networks of different species of bacteria, heterogeneously expressing AHL synthetase of other bacteria in E.coli may produce different AHL from the original host, and may produce multiple different AHL, so it may be difficult to obtain an orthogonal quorum sensing system. Therefore, on the basis of the traditional LuxI/LuxR type quorum sensing system, we chose pchBA/nahR with salicylic acid as a signaling molecule as another quorum sensing system.

DNA information storage has unique advantages and development prospects in information encryption, information transmission and storage by virtue of its high storage density, longevity and energy efficiency[4]. However, the traditional information decoding method is realized through DNA sequencing, which has drawbacks of poor timeliness and complex operation.

Inspired by the concept design of "cell disk"[5], ZJU-China tried to combine Neovio Dye to develop a "WYSIWYG"(What You See Is What You Get) DNA memory module. We attempt to "write in" information using repolymerization of light-induced sorting DNA recombinases by cutting recognition sites. What’s more, different toehold switches before the reporters mark different “storage disk unit”, which means that only certain times can be turned on by specific trigger RNA to achieve specific "read out".

Protein VVD is a biological clock regulation protein, and homologous dimerization occurs under blue light induction. Based on this characteristic, scientists have developed blue light induced fission enzyme technology[6], especially the modification of DNA recombination enzyme cre. Through the selection of specific cleavage sites, the proteases were separated into N- and -C segments at non-critical functional domains, so that they did not have biological enzyme activity when they existed alone, but had full enzyme activity when they were close to each other in space. In this year's project, ZJU-China 2024 is trying to build and test split-cre-loxP systems at different split sites to achieve a stable and efficient blue-induced DNA editing system.

On the other hand, in order to look for keys to open different archives, we catch our eyes on toehold switch, which is a kind of highly programmable engineered RNA-based gene switches. Inspired by the work of iGEM18_Vilnius-Lithuania-OG

we applied different toehold switches in different "disk unit" configurations, and tried to further verify the orthogonality and switching effect of these switches.

Since we introduce salicylic acid and AHL specific promoter as control elements and we’d better find a way to avoid leakage, we want to endow specific sites in vectors a “cover”. dCas9 can serve as a solution, but the protein may leave too heavy metabolic load on bacteria, and it’s unstable without constant target gRNA concentration level in the environment.

HYER(HYdrolytic Endonucleolytic Ribozymes)[7] is a lately identified ribozyme of the size of ~630nt, with DNA sequence recognition specificity and DNA cleavage activity. Researchers also successfully extend recognition site with another 14-nt. With target recognition site(TRS) and 14-nt recruiting sequence(RS), we may localize HYER to target site specifically.