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Team:Berkeley/Project/Introduction
From 2013.igem.org
The world needs to dye 3 billion pairs of jeans with indigo annually. The current industrial dyeing method involves many steps ,first producing indigo from the petroleum product, benzene, and then solubilizing indigo to allow it to adhere to cloth. The chemicals used in the process include strong acids, strong bases, and the reducing agent sodium dithionite. The table shown below shows the NFPA diamonds for each chemical highlighting their reactivity and hazards. Given the potential for a greener, biosynthetic alternative to denim dyeing, we started our project – Project Blue Genes.
Upon beginning our search for biosynthetic routes for dyeing jeans, we learned that certain species of plants produce indigo under stressed conditions. Below is polygonum tinctorium, an indigo producing plant. In the healthy, green part of the leaf, this plant naturally sequesters a toxic compound produced in plants called indole by using a glucosyltransferase (GT) enzyme to glucosylate into indican. Indican, a soluble and non-reactive chemical, is harmlessly stored in the vacuoles of plant cells.
However when we sprayed the leaf with ethanol (simulating stress conditions) cell compartmentalization fails and the vacuole degrades. De-compartmentalization exposes the previously isolated indican to another enzyme in the cell called a glucosidase (GLU), which cleaves the glucose moiety from indican. This produces an unstable intermediate called indoxyl which quickly oxidizes and dimerizes to indigo, turning the leaf blue.
Drawing on our knowledge of the indigo chemistry in plants, we believed we could take advantage of the indican production pathway for jeans dyeing. Because indican is soluble, it adheres to jeans and can be mass produced as a dyeing agent. After jeans are coated in the soluble indican, they can then be exposed to a glucosidase, similar to the mechanism in stressed plants, producing dyed jeans. This process would use renewable resources as inputs and circumvent many of the hazardous chemicals used industrially to produce blue jeans.
We aim to establish a pathway in E. coli to use soluble indican to dye jeans. Our pathway begins with indole, a toxic intermediate generated in E. coli as well as plants, and uses a heterologously expressed FMO enzyme to produce indoxyl which would then be taken to indican using a glucosyltransferase (GT). After coating fabric with indican as desired, a B-glucosidase enzyme (GLU) would cleave the sugar from indican to generate indoxyl which oxidizes to blue indigo.
