Difference between revisions of "Part:BBa K5236003"
 
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<partinfo>BBa_K5236003 short</partinfo>
 
<partinfo>BBa_K5236003 short</partinfo>
  
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This basic part encodes mutated IsPETase K95S 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. As one of the most-confident mutants created in our lab, this basic part encodes mutated IsPETase K95S.
  
 
===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 K95S 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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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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The IsPETase K95S 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 K95S 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/k95s-plasmid.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.1 M10L complete pathway in plasmid </center>
  
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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/k95f-sequnce-cycle-3.png" width = "50%"><br></html></center>
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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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<center>Fig.2 The result of IsPETase K95S 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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<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.2 The DNA gel electrophoresis result </center>
  
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Sequencing also demonstrated successful plasmid construction.
  
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<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/k95f-sequnce-cycle-3.png" width = "50%"><br></html></center>
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<center>Fig.3 The result of IsPETase K95S DNA sequencing  </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.
 
  
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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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To test the potential PET degradation efficiency of the IsPETase K95S synthesized in E.coli we applied the p-nitrophenyl butryte degradation assay from the iGEM19_Toronto team (for more details, please see protocols). The following graph shows the enzyme activities of IsPETase WT compared to the mutations. The mutation IsPETase K95S has a higher enzyme activity than BhrPETase WT at 30 min, and has certain potential to surpass the efficiency of IsPETase if given more time.
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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><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.4 Protein electrophoresis result </center>
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<center>Fig.4 Mutated IsPETase Dynamic Curve </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. Through HPLC, we are able to see the enzyme and waste product curves after plastic degradation via our enzyme.
 
  
 
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<partinfo>BBa_K5236003 parameters</partinfo>
 
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===Reference===
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Lu, Hongyuan, et al. “Machine Learning-Aided Engineering of Hydrolases for Pet Depolymerization.” Nature News, Nature Publishing Group, 27 Apr. 2022, www.nature.com/articles/s41586-022-04599-z. Kato, Shingo, et al. “Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle.” Microbes and Environments, vol. 33, no. 1, Jan. 2018, pp. 107–10. https://doi.org/10.1264/jsme2.me17165. 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/

Latest revision as of 12:52, 2 October 2024

IsPETase K95S

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. As one of the most-confident mutants created in our lab, this basic part encodes mutated IsPETase K95S.

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 K95S 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 K95S sequence, which is inserted between the promoter and terminator.


Fig.1 M10L complete pathway in plasmid

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.2 The DNA gel electrophoresis result

Sequencing also demonstrated successful plasmid construction.


Fig.3 The result of IsPETase K95S DNA sequencing


To test the potential PET degradation efficiency of the IsPETase K95S synthesized in E.coli we applied the p-nitrophenyl butryte degradation assay from the iGEM19_Toronto team (for more details, please see protocols). The following graph shows the enzyme activities of IsPETase WT compared to the mutations. The mutation IsPETase K95S has a higher enzyme activity than BhrPETase WT at 30 min, and has certain potential to surpass the efficiency of IsPETase if given more time.


Fig.4 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]



Reference

Lu, Hongyuan, et al. “Machine Learning-Aided Engineering of Hydrolases for Pet Depolymerization.” Nature News, Nature Publishing Group, 27 Apr. 2022, www.nature.com/articles/s41586-022-04599-z. Kato, Shingo, et al. “Long-Term Cultivation and Metagenomics Reveal Ecophysiology of Previously Uncultivated Thermophiles Involved in Biogeochemical Nitrogen Cycle.” Microbes and Environments, vol. 33, no. 1, Jan. 2018, pp. 107–10. https://doi.org/10.1264/jsme2.me17165. 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/