Difference between revisions of "Team:Tianjin/Notes"
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| − | + | <p style="font-size:20px">As is known to all, the activity of enzyme depends on the interaction of enzyme molecules and substrate molecules. We made the active sites more exposed so that the reaction will be easier to take place. Second, we should increase the hydrophobicity near the active sites because of the high hydrophobicity of PET surface. We totally designed 22 site-directed mutants and expressed them in Saccharomyces cerevisiae and then assay their activity to determine which design is valid.<a href="https://2016.igem.org/Team:Tianjin/Note/Protein_Engineering" target="_blank"> Clicking this link to see details.</a> </p> | |
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Latest revision as of 13:53, 19 October 2016
Overview About Notes
Our experiment started from July after designing and discussing for several months. Every day after finishing the experiment, we wrote down what we did this day briefly but clearly. After all the experiments came to a end, we summaried all of our experiment notes and divided them into five groups. The detailed notes can be accessed by clicking the link below.
R-R (Reporting and regulation) system consists of the inclusion body reporting system, the inclusion body induced cell lysis system and the TPA positive feedback regulation system. The experiment of this part started at end of August and lasted for 6 weeks. Clicking this link to see details.
After Yoshida and his co-workers found and isolated Ideonella sakaiensis 201-F6, which produced two enzymes to degrades PET, we kept very high interests at their works and also came up with many ordinary ideas to increase the efficiency of degradation reaction. Bacterial consortium is one of the most creative ideas. The inspiration of this idea comes from nature and also learns from nature. Actually, bacteria never exist alone in our nature, they co-work and cooperate together to achieve an aim or live better in a special condition. Thinking from this point, we established a special bacteria consortium for this enzyme catalysis reaction. Clicking this link to see details.
As is known to all, the activity of enzyme depends on the interaction of enzyme molecules and substrate molecules. We made the active sites more exposed so that the reaction will be easier to take place. Second, we should increase the hydrophobicity near the active sites because of the high hydrophobicity of PET surface. We totally designed 22 site-directed mutants and expressed them in Saccharomyces cerevisiae and then assay their activity to determine which design is valid. Clicking this link to see details.
In order to achieve high-throughput screening for the mutants of PETase. We used the E.coli CFPS (Cell-Free Protein Synthesis) to express modifided PETase and compared them to the wild type as a assay method. The PETase gene is fused with a CFP (Cyan Fluorescence Protein) gene so that the cyan fluorescence signal can act as a reporter of PETase expression level. And than we used the enzymes we had got to degrade PET and detected the degradation products. . Clicking this link to see details.
The Nickel-inducible lysis system is composed of two parts: lysis genes and a nickel responding signal operon. After the previous material preparation, we began to construct the system wishing to make Synechocystis sp.PCC 6803 lysis to our bidding. Clicking this link to see details.
