Difference between revisions of "Team:HK GTC/Contribution"
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| − | <p> | + | <p>In the existing part BBa_K2982005, we added Scanning Electron Microscope (SEM) photos of PET films after |
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being digested by S245I PETase mutant. The usage of SEM can accurately visualize how serious the PET film is | being digested by S245I PETase mutant. The usage of SEM can accurately visualize how serious the PET film is | ||
| − | digested by the enzyme. | + | digested by the enzyme. Besides, we added HPLC profile of products released from PET film digested with |
| + | S245I PETase to show the extent of PET hydrolysis. | ||
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</p> | </p> | ||
| − | <h2>SEM images of digested PET films</h2> | + | <h2>(A) SEM images of digested PET films</h2> |
| − | <p>We performed PET film digestion to study the PET hydrolytic activity of S245I PETase. After incubating 9μg of purified proteins with PET film at 30°C for 96 hours, we viewed the digested PET film under a SEM. | + | <p>We performed PET film digestion to study the PET hydrolytic activity of S245I PETase. After incubating 9μg |
| + | of purified proteins with PET film at 30°C for 96 hours, we viewed the digested PET film under a SEM. | ||
</p> | </p> | ||
<div class="single-image-with-desc"> | <div class="single-image-with-desc"> | ||
<center><img src="https://static.igem.org/mediawiki/2021/c/ce/T--HK_GTC--contribution_1.png" alt=""></center> | <center><img src="https://static.igem.org/mediawiki/2021/c/ce/T--HK_GTC--contribution_1.png" alt=""></center> | ||
| − | + | </div> | |
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| + | <p>The pitting resulting from the digestion of S245I PETase mutant can be visualized, and the buffer-only SEM | ||
| + | photo acts as a control. | ||
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| + | </p> | ||
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| + | <p>(B) HPLC profile of the products released from the PET films</p> | ||
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| + | <div class="single-image-with-desc"> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2021/c/ce/T--HK_GTC--Contribution--a.png" alt=""></center> | ||
| + | <p>Figure 2a. HPLC spectrum of TPA standard. </p> | ||
| + | </div> | ||
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| + | <div class="single-image-with-desc"> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2021/5/58/T--HK_GTC--2b.png" alt=""></center> | ||
| + | <p> Figure 2b. HPLC spectrum of MHET standard.</p> | ||
| + | </div> | ||
| + | |||
| + | <div class="single-image-with-desc"> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2021/9/99/T--HK_GTC--2d.png" alt=""></center> | ||
| + | <p> Figure 2c. HPLC spectrum of products released from the PET film digested with S245I PETase.</p> | ||
| + | </div> | ||
| + | |||
| + | <p>HPLC profiles demonstrated that the detection peaks representing TPA monomer and MHET intermediate product | ||
| + | formed during PET film digestion have retention times at 4.64 minutes (Figure 2a) and 5.17 minutes (Figure | ||
| + | 2b) respectively. <br> | ||
| + | HPLC profiles of products released from PET film digestions using S245I PETase revealed incomplete PET | ||
| + | hydrolysis as considerable amounts of intermediate product, MHET which showed a peak at 5.07 minutes were | ||
| + | detected (Figure 2c). <br> | ||
| + | Therefore, we can conclude that S245I PETase exhibits hydrolytic activity for PET depolymerization. | ||
| + | </p> | ||
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</div> | </div> | ||
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Latest revision as of 14:22, 20 October 2021