Difference between revisions of "Part:BBa K5236007"
 
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The IsPETase W159HF229Y 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 W159HF229Y sequence, which is inserted between the promoter and terminator.
 
The IsPETase W159HF229Y 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 W159HF229Y 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/colony-pcr.png" width = "50%"><br></html></center>
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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/w159-plasmid.png" width = "50%"><br></html></center>
 
<center>Fig.1 Constructed Plasmid </center>
 
<center>Fig.1 Constructed Plasmid </center>
  
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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>
 
<center>Fig.4 Mutated IsPETase Dynamic Curve </center>
 
<center>Fig.4 Mutated IsPETase Dynamic Curve </center>
  

Latest revision as of 12:46, 2 October 2024

IsPETase W159HF229Y

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 W159HF229Y, which comes from academic essays but is constructed by us 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 W159HF229Y 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 W159HF229Y sequence, which is inserted between the promoter and terminator.


Fig.1 Constructed Plasmid

Fig.2 The DNA gel electrophoresis result

Fig.3 The result of IsPETase W159HF229Y 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 chassis carrying our PETase could survive.


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

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 864
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 864
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 864
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 864
    Illegal AgeI site found at 627
  • 1000
    COMPATIBLE WITH RFC[1000]



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/