Team:Worldshaper-XSHS/Design
Design
According to our previous study, interview and social investigation results, we planned to develop an efficient and affordable method that decolorizes azo dyes. It would be used by publics, especially for factories. After collaborating a great number of related researches and reading a plenty of documents, we eventually discovered the laccases and azo reductases . The first process is performed by azo reductases under anaerobic or low oxygen conditions. The second step is formed by oxidative enzymes that involves laccases. Based on this discovery, our minds came up with a practical idea ——whether it could be possible for us to use the synthetic biological methods to construct an E. coli strain into a sewage purifier. With putting a huge amount effort on researching and instructed by professors, we got an answer: Yes! This will be possible!
During the whole research process, we made several changes which is shown in the following 3 versions.
In the original design, refer to a large number of literature, the T7-lac operater regulatory unit is popular for protein inducing. it is regulated by IPTG and making protein expression in control. The his tag is also taken into consideration for future protein purification. Fortunately , we found a ideal prokaryotic expression vector -- pET24a that can be purchased from biological company, it contains all necessary funtional unit we need for our experiment like T7 promoter, lac operator , RBS , T7 terminator, his tag, and general restriction enzyme sites. We insert our protein gene into pET24a and transformed recombined plasmids into BL21(DE3), a excellent E.coli cell for protein expression.
We aim to express two laccases and two azo reductases by using this kind of method, and pick one of this two kinds of enzymes respectively for future use.
After picking out one laccase and one azo reductase, we want to investigate the synergistic action of azoreductase and laccase. The laccase will be added to the downsteam of azo reductase. The fusion protein will express in BL21(DE3) and work together.
To increase the rate of dye decolorization, we plan to secrete the expressed protein out of cell, which means the enzymes will be able to move directly out of cell to work and they might work better than work inside of cell. After searching in the igem website, we eventually decided to use OsmY( BBa_K1519123) in our part, one will be able to secret a protein of interest. One of our laccase will added after OsmY, and we will compare it’s intracellular and extracellular functions.
