Difference between revisions of "Part:BBa K5236006"
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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 mutated IsPETase M154L 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.   
 
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 mutated IsPETase M154L 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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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/ispetase-m154l.png" width = "50%"><br></html></center>
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<center>Fig.1 The DNA gel electrophoresis result </center>
  
 
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.   
 
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.   
  
 
<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>
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<center>Fig.1 The DNA gel electrophoresis result </center>
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<center>Fig.2 The DNA gel electrophoresis result </center>
  
 
<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/m154l-sequence-cycle-3.png" width = "50%"><br></html></center>
 
<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/m154l-sequence-cycle-3.png" width = "50%"><br></html></center>
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<center>Fig.2 The result of IsPETase M154L DNA sequencing  </center>
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<center>Fig.3 The result of IsPETase M154L DNA sequencing  </center>
  
 
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.   
 
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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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/ispetase-mutation-efficiency-line-graph.jpg" width = "50%"><br></html></center>
 
<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 The Dynamic Curve of the Enzyme </center>
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<center>Fig.4 The Dynamic Curve of the Enzyme </center>
  
  

Revision as of 11:31, 2 October 2024

IsPETase M154L

IsPETase is friendly to enviorment and energy-saving to chemical recycling of PET. However, the temperature for it to react is even lower than glass transition temperature of PET. This basic part encodes mutated IsPETase M154L and constructed in Escherichia coli.


Usage and Biology

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 mutated IsPETase M154L 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.


Fig.1 The DNA gel electrophoresis result

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.


Fig.2 The DNA gel electrophoresis result

Fig.3 The result of IsPETase M154L DNA sequencing

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.

The result show that chasis carrying our PETase could survive.


Fig.4 The Dynamic Curve of the Enzyme


Reference

Brott, S., Pfaff, L., Schuricht, J., Schwarz, J.-N., Böttcher, D., Badenhorst, C. P. S., Wei, R., & Bornscheuer, U. T. (2021, November 29). Engineering and evaluation of thermostable isPETASE variants for PET degradation. Engineering in life sciences. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8961046/


Fig.3 Mutated IsPETase Dynamic Curve


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]