Difference between revisions of "Part:BBa K5236013"
 
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===Usage and Biology===
 
===Usage and Biology===
  
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To insert our parts into plasmids, we’ve designed primers and performed PCRs. Then, our genes were recombined into plasmids and transformed into chassis. To test if our part codes for the PETase LCCICCG we want and whether the enzyme works, we've completed two large experimental processes. The first step is plasmid construction. And the second is to test the enzymatic activity. 
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We tested for successful plasmid construction and transformation into E.coli through colony PCR and gel electrophoresis. The following gel result demonstrates that the plasmid transformed into E.coli are correct. The plasmid should have a total of 891 base pairs and the results match.
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By conducting colony PCR, we are able to test if our parts have been transformed into chassis successfully. The following result of electrophoresis proves that we’ve inserted genes into chassis since the sequence containing our mutated genes has a total of 891 base pairs and the results are in the right location.
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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/colony-pcr.png" width = "50%"><br></html></center>
 
<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/colony-pcr.png" width = "50%"><br></html></center>
 
<center>Fig.1 The DNA gel electrophoresis result </center>
 
<center>Fig.1 The DNA gel electrophoresis result </center>
  
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<center><html><img src ="" width = "50%"><br></html></center>
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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/lcciccg-sequence.png" width = "50%"><br></html></center>
 
<center>Fig.2 The result of DNA sequencing  </center>
 
<center>Fig.2 The result of DNA sequencing  </center>
  
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After proving that our genes existed in chassis, we need to test if the bacteria can survive as usual with our genes. Thus, we’ve coated the bacteria on nutritional petri dish. And after a night, E. coli grew over the plate our plate, justifying that E. coli can survive with the gene of our part. 
 
  
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The result show that chasis carrying our PETase could survive.
 
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We tested whether the bacteria could translate for our protein, and we examined whether our mutated enzyme is more efficient. For this section, we analyzed two results as well. First, the dynamic curve of our enzyme shows its high efficiency in degrading rate. Second, the electrophoresis result of our protein proves that our enzyme can be successfully coded by the parts we designed.
 
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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/ispetase-mutation-efficiency-line-graph.jpg" width = "50%"><br></html></center>
 
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<center>Fig.3 Mutated IsPETase Dynamic Curve </center>
 
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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/sds-page.png" width = "50%"><br></html></center>
 
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<center>Fig.4 Protein electrophoresis result </center>
 
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After proving that our enzyme are more effiicient, we moved on to test the ultimate and the most essential part of our part examination, which is to test if our mutated enzyme can actually degrade plastics. For this large step of process, we also designed two approaches. 
 
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First, the scanning electron microscope allows us to see the changes of plastic pieces with our bare eyes. However, pure observations are not enough to prove the effectiveness of our enzymes. Thus, we conducted another experiment. Though HPLC, we are able to see the enzyme and waste product curves after plastic degradation via our enzyme.
 
  
 
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<partinfo>BBa_K5236013 parameters</partinfo>
 
<partinfo>BBa_K5236013 parameters</partinfo>
 
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===Reference===
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Ding, Zundan, et al. “Rational Redesign of Thermophilic Pet Hydrolase LCCICCG to Enhance Hydrolysis of High Crystallinity Polyethylene Terephthalates.” Journal of Hazardous Materials, Elsevier, 7 Apr. 2023, www.sciencedirect.com/science/article/pii/S0304389423006696.

Latest revision as of 12:31, 2 October 2024

LCCICCG

Cutinase (EC 3.1.1.74) is a lipolytic/esterolytic enzyme that hydrolyzes not only cutin, which is a major component of plant cuticl, but also water-soluble esters and insoluble triglycerides. Leaf-branch compost cutinase (LCC) was first reported in 2012 and its activity and thermostability improved via four mutations yielding LCC-ICCG.

Usage and Biology

We tested for successful plasmid construction and transformation into E.coli through colony PCR and gel electrophoresis. The following gel result demonstrates that the plasmid transformed into E.coli are correct. The plasmid should have a total of 891 base pairs and the results match.


Fig.1 The DNA gel electrophoresis result

Fig.2 The result of DNA sequencing


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 258
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 258
    Illegal NheI site found at 190
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 258
    Illegal XhoI site found at 775
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 258
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 258
  • 1000
    COMPATIBLE WITH RFC[1000]



Reference

Ding, Zundan, et al. “Rational Redesign of Thermophilic Pet Hydrolase LCCICCG to Enhance Hydrolysis of High Crystallinity Polyethylene Terephthalates.” Journal of Hazardous Materials, Elsevier, 7 Apr. 2023, www.sciencedirect.com/science/article/pii/S0304389423006696.