Each year, seven lakh tons of dyes are produced in factories, and a large amount of them were discharged in waste stream without proper treatment. These emissions could lead diseases of human beings or even due irreversible harm the ecosystem. Our team sets our goal to come out with a system that could effectively decompose azo dyes.

Azo dyes’ pollution

First, we researched about previous ways to decompose azo dyes. There are three existing ways to decompose azo dyes: Chemical method, photocatalytic method, and microbiological method. First, chemical method uses carbon-based sorbents to adsorb synthetic dyes. However, it is energy consuming to make such substance. Consequently, this method is too expensive for smaller business owners, so they would never use such method. Second, photocatalytic method involves several catalysis and require special lighting equipment. The reaction tends to be very slow due to most dyes were designed to tolerate strong lights. Thus, this method is time consuming. Third, microbiological decomposition process is relatively cheap and this method does generate any toxic by-products. Nevertheless, bacteria cell that could flow every in the treatment apparatus is still an issue. We decided to improve the on-going microbiological method. We need to create a system that holds all the cells in one piece and could decompose azo dyes.

Focusing on that, we soon located our first key component of our system: The Biofilm. Biofilm is a network of individual cells which act not only as a net that holds everything in one piece, but also as a shield for individual cells to the harsh environment condition. As we all know, bacteria cannot grow as well in medias as in harsh environments like flowing sewage water.

The typical biofilm we researched on is formed by gene csgA on the genome of E. coli MG1655 wild type. Cells were stuck together by a kind of fiber protein known as curli fiber that is assembled by CsgA protein on the cell’s surface. By the way, csgA was a previous iGEM part and we mead improvements to it. We also read about adding the sequence of SpyTag after csgA gene could produce curli fiber with SpyTags on them.

Figure of csgA - SpyTag

We are now confidence that we can fix anything we want to the surface of a biofilm. But we now need a substance that could decompose the azo dyes. After some more researching, we found laccase, a kind of blue multi-copper oxidases, has strong ability to oxidize and decompose azo dyes. Gene cotA, initially found in Bacillus subtilis, can produce protein with high laccase activity. Gene cotA has a very low translation rate in natural hosts. However, when cotA is produced in specialist strains like BL21 – DE3 (after T7 promoter), it shown up a relatively high rate. As mentioned, laccase belong to multi-copper oxidases, so that CotA protein still need to combine with Cu2+ to become active laccase. The solution is quiet simple, we can just ultrasonic cells and mix whatever is left with 0.1 mM/L of CuSO4.

Up till now, we have designed a biofilm with SpyTag and have located a kind of powerful enzyme. In order to fix our powerful enzyme to the biofilm, we added a SpyCatcher sequence after the cotA gene. So that these proteins could fix onto the curli fibers of the biofilm csgA - SpyTag using isopeptide bonds. These bonds are covalent bonds which is relatively hard to broke.

cotA - SpyCatcher

Signal peptides - CotA

We then came out with another solution of how to get CotA protein out of the cells. By adding signal peptide sequence ahead of cotA – SpyCatcher sequence, we could let the cells secrete CotA – SpyCatcher protein in to the media. Then we can let CotA – SpyCatcher combine with Cu2+.

Project Overview