Team:SDSZ China/Descr
Project description
Fecal water pollution is always associated with pathogen transmission and serious human diseases among the world. As one of the most common pollutants of fecal-polluted water, somatic coliphage is widely used in various protocols as the indicator of fecal and viral pollution. Our team plan to design a biosynthetic system that greatly simplifies the existing Bluephage system and improves detection efficiency.
By substituting Bluephage system’s downstream sequence of glucuronidase with the sequence of North American firefly luciferase and polyphenol oxidase, we plan to cultivate clones with plasmid pET 28a and select the successful clones for detection. After corresponding substrates are added to the culture medium, enzymatic reactions occur when cell lysis happens due to phage attack, and according enzymes expressed by E.coli. leak from the lysed cells and react with their substrates in the medium. Fluorescence and observable colour change of the enzymatic reactions will then be tested and recorded for the identification of the sample’s phage density, and thus the degree of fecal pollution of the tested sample will be quantified through data analysis.
Aiming to simplify Bluephage system by eliminating gene-knockout and recombination, we carefully considered the properties of the possible candidates of the downstream proteins, and chose those whose substrates, including themselves, do not pass cell membranes and do not involve in sites of inter-membrane proteins.
Apart from building clones, we are also planning to design a computer program that allows automatic identification and counting of phage plaque that increases detection efficiency in a different measure.
Project inspiration
Team SDSZ_China has taken part in iGEM competition for three years until now and has always aimed for bringing meaningful impacts to the world with synthetic Biology technology. In our last year’s project, members of the team worked together to synthesize effective enzymes that can successfully relieve water pollution caused by chemically producing chitosan with chitin. After communicating with related stakeholders such as factory staffs and villagers who catch lobsters for living, we had a deeper understanding of the severeness of water pollution’s impacts on citizens’ health in China.
Thus, in this year’s project, we still wish to contribute to the improvement of water treatment method. In team leader Rebecca’s hometown, there was a stream by her grandma’s house by which she used to play with her childhood friends. When she revisited her hometown in 2019, she surprisingly found out that the stream was no longer clear as before. Stinky and murky, it was severely polluted during the six years. After asking villagers nearby, she realized that the pollution was caused by villagers pouring livestock excretion into the stream. Some even mentioned that there were cases of villagers getting diseases because of drinking the water from the stream.
Greatly shocked by the phenomenon, the team leader and members held discussions and finally decided to focus the aim of the project on the detection of fecal pollution. If we can make it easier and more efficient to detect the pollution level of water and clearly show the detection result, people will no longer suffer from mistakenly drinking polluted water. Through further research, we found out that as one of the most common pollutants of fecal-polluted water, somatic coliphage is widely used in various protocols as the indicator of fecal and viral pollution. According to a current promising detection method Bluephage, our team designed a biosynthetic system that greatly simplified Bluephage system and improved detection efficiency.
By substituting Bluephage system’s downstream sequence of glucuronidase with sequence of firefly luciferase and polyphenol oxidase, we planned to cultivate clones with plasmid pET 28a and pick the successful clones for detection. By adding appropriate substrates to LB medium, enzymatic reactions occur when cell lysis happens due to phage attack, and according enzymes expressed by E.coli. leak from the broken cells, then meet their substrates in the medium. Fluorescence and observable colour change of the enzymatic reactions will then be tested and recorded for the identification of the sample’s phage density, and thus the degree of fecal pollution of the tested sample can be indicated.
Aiming to simplify Bluephage system by eliminating gene-knockout and recombination, we carefully considered the properties of the possible candidates of the downstream proteins, and chose those whose substrates and themselves cannot pass cell membranes and do not involve in sites of inter-membrane proteins.