Part 1: Screening of function

The main purpose of our project is to produce tyrian purple by biological method. The chemical nature of tyrian purple is 6,6'-dibromoindigo (6BrIG), so we plan to synthesize it from tryptophan in Escherichia coli. This process is mainly composed of halogenation, hydrolysis and oxidation of tryptophan. We found the corresponding functional enzymes, namely SttH, TnaA and MaFMO. They are tryptophan 6-halogenase SttH, tryptophanase TnaA and flavin-containing monooxygenase MaFMO.After studying the functional properties of the gene, in order to enhance the solubility of SttH, we designed linker to connect it with Fre.

Part2：Plasmid construction and strain transformation

We selected E. coli BL21 strain as the chassis organism for our work. In the application of computer in plasmid design, various aspects such as screening markers and expression intensity are comprehensively considered. Finally, we constructed three plasmids:pET28a-Fre-L-SttH, pACYC-Duet1-TnaA and pYES2-MaFMO. The construction of plasmid can be divided into two processes: the acquisition of target gene fragment and the connection of plasmid fragment. The restriction enzymes used are:Nhe1 and EcoR1, EcoN1 and Xba1,Kpn1 and EcoR1. The following figure is the verification diagram of our nucleic acid electrophoresis. After verifying that the constructed plasmid vector had no problem, we successfully transformed it into E. coli.

Fig. 1. Construction results of plasmid

Part3：Gene expression

The next step after the construction of functional strains is to induce gene expression. After induction, we tested it by SDS-PAGE. The results showed that our three functional enzyme genes were successfully expressed and had a considerable amount of expression, which was enough to support our dye production.

Part4：Production of dyes

After the functional enzyme was produced and verified, we fermented the product and analyzed the results by mass spectrometry.We applied MALDI-MASS assay to analysis the presence and purity of our product extracted from the cell. The analysis showed that the amount of the target product was close to the reference, and the purity was within the acceptable range of the preliminary experiment.

Fig. 3. Results of MS

Fig. 4. Results of MS(Enlarged)

Part5：Dyeing of cloth

After we successfully expressed three functional proteins in the constructed strain and verified the correct products by mass spectrometry, we cultivated the strain with high logarithmic phase and high activity. Then, after adding glucose, tryptophan and sodium bromide, the bacteria successfully produced Tyrian purple. We dyed the cloth with the tyrian purple powder which we bought from the enterprise, and the effect was good. After that, we also used heating and alcohol immersion to explore the sustainability of powder dye dyeing effect. The results show that the fabric still maintains good dyeing effect after heat treatment and solvent treatment.

Fig. 5. Cloth dyed with royal purple dye

Fig. 6. Heat treated and solvent treated cloth

Part6：Future work

Although we have succeeded in part of our work, the whole biological production of tyrian purple has not been completed perfectly. In the future, first of all, we will further transform and construct the strain to reduce the plasmid load and enhance its stability. Secondly, we will explore the optimal fermentation conditions, including temperature, oxygen, glucose and amino acid concentration. Finally, we will test the production under the actual industrial conditions in order to promote the industrialization of our work.