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. With this process in mind, what if there was a better way to dye jeans? One that doesn't use oil, that doesn't use harsh chemicals, that doesn't require additional solubilization, and that doesn't result in environmental damage? We figured that there might be such a process... and so we began our project - Blue Genes.

Using biology to address some of our energy and chemical needs has become an increasingly popular route. Because biosynthetic processes begin with renewable resources like sugar and amino acids, engineering biology can potentially resolve our continual reliance on oil. As we searched for a biosynthetic route to dye jeans, we learned that certain species of plants produce indigo under stressed conditions. One of these plants is polygonum tinctorium. In the healthy, green part of the leaf, the plant sequesters a naturally produced toxin called indole by using a glucosyltransferase (GT) enzyme to glucosylate indole and produce indican - a soluble and non-reactive chemical which is harmlessly stored in the vacuoles of plant cells.

However when we sprayed the leaf with ethanol, the leaf begins to die, cell compartmentalization fails, and the vacuoles degrade. De-compartmentalization exposes the previously isolated indican to another enzyme in the cell called B-glucosidase (GLU), which cleaves the glucose moiety from indican. This cleavage produces an unstable intermediate called indoxyl which quickly oxidizes and dimerizes to indigo, turning the leaf blue as shown below. Indican is a suitable solubilized indigo that we can make to dye jeans. It can be created and converted to indigo enzymatically.

Because indican is soluble, it adheres to jeans and can be mass produced as a dyeing agent. This would avoid the current problem of making an insoluble indigo dye and then solubilizing it to facilitate dyeing. 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.

Putting all of our research together, we came up with a novel enzymatic dyeing process to dye jeans completely biologically, using the E. coli chassis. Our pathway begins with indole, an 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. In the subsequent pages, we identify and characterize each of the three highlighted enzymes in this pathway. To the best of our knowledge, our Berkeley iGEM 2013 team is the first to generate indican from a recombinant enzyme in literature. Beyond this, we show that we can dye cloth permanently using indican. Keep reading to find out more about the functionality of each part of our process!