Difference between revisions of "Part:BBa K5236004"
 
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<partinfo>BBa_K5236004 short</partinfo>
 
<partinfo>BBa_K5236004 short</partinfo>
  
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This basic part encodes mutated IsPETase H104W and constructed in Escherichia coli.
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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 H104W and constructed in Escherichia coli.
  
 
===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 mutated IsPETase H104W 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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To generate mutated variants, we have trained a Transformer AI model. This model predicts the top 10 potential mutation sites, which are likely to have significant impacts on the enzyme's structure and function. Next, we analyzed the top 10 potential sites via Meta's ESM-1b model to eliminate the silent mutations, which involve changes in nucleotides that do not altering the corresponding amino acids. This ensures that the mutations result in changes in the enzyme's structure and thereby its function. For further information, please check the model page on our wiki. https://2024.igem.wiki/basis-china/model
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The IsPETase H104W sequence is expressed in E.coli BL21(DE3) using the pET28a vector. The pET-28a is a classical plasmid vector used for protein expression in E.coli. This vector contains the T7 promoter, the lac operator, a ribosome binding site, the 6xHis sequence, and the T7 terminator. The T7 promoter is a strong promoter recognizable by T7 RNA polymerase, used to regulate gene expression of recombinant proteins. The lac operator can be activated by IPTG and used to control gene expression. The 6xHis sequence encodes for a tag that facilitates protein purification. Asides from the features included in the plasmid backbone, we added a signal peptide sequence — pELB — before the IsPETase H104W sequence, which is inserted between the promoter and terminator.
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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/ispetase-h104w.png" width = "50%"><br></html></center>
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<center>Fig.1 The Constructed Plasmid </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/h104w-sequence-cycle3.png" width = "50%"><br></html></center>
 
<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/h104w-sequence-cycle3.png" width = "50%"><br></html></center>
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<center>Fig.2 The result of IsPETase H104W DNA sequencing  </center>
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<center>Fig.3 The result of IsPETase H104W 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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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.
 
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><html><img src ="https://static.igem.wiki/teams/5236/yh/ispet-relative-activity.jpg" width = "50%"><br></html></center>
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<center>Fig.3 Mutated IsPETase Dynamic Curve </center>
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<center>Fig.4 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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Reference
  
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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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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/
  
 
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Latest revision as of 12:51, 2 October 2024

IsPETase H104W

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 H104W and constructed in Escherichia coli.

Usage and Biology

To generate mutated variants, we have trained a Transformer AI model. This model predicts the top 10 potential mutation sites, which are likely to have significant impacts on the enzyme's structure and function. Next, we analyzed the top 10 potential sites via Meta's ESM-1b model to eliminate the silent mutations, which involve changes in nucleotides that do not altering the corresponding amino acids. This ensures that the mutations result in changes in the enzyme's structure and thereby its function. For further information, please check the model page on our wiki. https://2024.igem.wiki/basis-china/model

The IsPETase H104W sequence is expressed in E.coli BL21(DE3) using the pET28a vector. The pET-28a is a classical plasmid vector used for protein expression in E.coli. This vector contains the T7 promoter, the lac operator, a ribosome binding site, the 6xHis sequence, and the T7 terminator. The T7 promoter is a strong promoter recognizable by T7 RNA polymerase, used to regulate gene expression of recombinant proteins. The lac operator can be activated by IPTG and used to control gene expression. The 6xHis sequence encodes for a tag that facilitates protein purification. Asides from the features included in the plasmid backbone, we added a signal peptide sequence — pELB — before the IsPETase H104W sequence, which is inserted between the promoter and terminator.


Fig.1 The Constructed Plasmid

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 H104W 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.


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.


Fig.4 Mutated IsPETase Dynamic Curve

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/

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]