Team:QHFZ/Design
Design
Abstract
In this page, we will introduce our design of the gene circuit for the production of 6’,6’-dibromoindigo (6BrlG), known as Tyrian Purple. More detailed information can be found in proof of concept page. We also give a overall demonstration of our project background.
In 2020, a study published on Nature Chemical Biology reported a new method using synthetic biology to produce the expensive Tyrian purple dye in a harmless way. However, the study also mentioned that problems of this type of production still exist, for example, lack of expensive material, intricate making process, etc.
During the Human Practice we found out that in industrial production, toxic chemical process takes the lead. In our dedication to protect the environment, we are determined to promote the biological approach to the Tyrian Purple production.
(1)Lack of expensive material: The original approach uses tryptophan as the raw material, however it is expensive and not produced by the bacteria itself.
(2)The time-consuming 2-cell system: In order to separate 2 making processes, former researchers conducted the experiments in 2 different cells.
(3)Low production efficiency.
Based on the information above, we believe we are capable of making the production more environmentally friendly, easier and cheaper. Therefore, we planned to use inducible promoters and only one cell to achieve the process.
Design Of the Tyrian Purple Production Circuit
We engineered E.coli cells (BL21) to express SttH-Linker-Fre, MaFMO, and TnaA with the idea of synthetic biology. The 3 genes are the core components of the circuit. The production depends on 4 steps of reaction. Firstly, Trp 6-Hal (SttH-Linker-Fre) turns tryptophan into bromotryptophan: Trp 6-Hal requires oxygen, bromide salt and FADH2. Flavin reductase (Fre) regenerates FAD to FADH2 using NAD(P)H. Then TnaA cleaves 6-Br-Trp into 6-Br-indole ,pyruvate and ammonia. Thirdly, Monooxygenase (MaFMO) hydroxylates the C3 position of the indole ring and forms 6-Br-indoxyl. 6BrIG is a dimer of 6-Br-indoxyl formed via an auto-oxidation reaction.
Optimization
Before our synthesis begin, we tried dying different kinds of fabric using Tyrian Purple and Indigo blue, we found out that when using magnetic rotors wrapped around the fabric and the solution heated to around 80 Celsius, the results are most satisfying. After we conducted the experiments, we considered transferring 2 plasmids (TnaA, MaFMO) into one.