Difference between revisions of "Part:BBa K5236009"

Revision as of 11:57, 2 October 2024

BhrPETase W229F

Plastic pollution poses a serious threat to the global environment. One of the potential solutions, enzyme degradation, would be a suitable approach of dealing with plastic wastes. Among all plastic pollutions, more than 10% of them are Polyethylene terephthalate (PET). Thus, our team has been searching for possible PET hydrolases to break down PET. However, according to Nature's publishment on April 27, 2022, traditional PET hydrolases' enzymatic ability of degrading PET are easily affected by the fluctuation of temperature and pH value. Therefore, we decided to artificially mutate wild-type BhrPETase to increase the enzyme’s range of tolerance so that it can efficiently degrade PET under a wider range of environmental conditions, thereby enhance its potential application. BhrPETase was identified by the Shingo group in a metagenomic study on uncultured thermophiles and was deposited into the NCBI database by the group in 2018 and annotated as a PET hydrolase. As one of the most-confident mutants created in our lab, this basic part encodes mutated BhrPETase W229F.

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 BhrPETase W229F 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 Contructed 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 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 BhrPETase Dynamic Curve

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.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 226
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 136
1000
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K5236009 short</partinfo>
-This basic part encodes mutated BhrPETase W229F and constructed in Escherichia coli.
+Plastic pollution poses a serious threat to the global environment. One of the potential solutions, enzyme degradation, would be a suitable approach of dealing with plastic wastes. Among all plastic pollutions, more than 10% of them are Polyethylene terephthalate (PET). Thus, our team has been searching for possible PET hydrolases to break down PET. However, according to Nature's publishment on April 27, 2022, traditional PET hydrolases' enzymatic ability of degrading PET are easily affected by the fluctuation of temperature and pH value. Therefore, we decided to artificially mutate wild-type BhrPETase to increase the enzyme’s range of tolerance so that it can efficiently degrade PET under a wider range of environmental conditions, thereby enhance its potential application. BhrPETase was identified by the Shingo group in a metagenomic study on uncultured thermophiles and was deposited into the NCBI database by the group in 2018 and annotated as a PET hydrolase. As one of the most-confident mutants created in our lab, this basic part encodes mutated BhrPETase W229F.
 ===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 BhrPETase W229F 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.
+<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/w229f.png" width = "50%"><br></html></center>
+<center>Fig.1 Contructed 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.
 <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.2 The DNA gel electrophoresis result </center>
 <center><html><img src ="" width = "50%"><br></html></center>
-<center>Fig.2 The result of DNA sequencing  </center>
+<center>Fig.3 The result of 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.
@@ Line 22: / Line 26: @@
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/ispetase-mutation-efficiency-line-graph.jpg" width = "50%"><br></html></center>
-<center>Fig.3 Mutated BhrPETase Dynamic Curve </center>
+<center>Fig.4 Mutated BhrPETase Dynamic Curve </center>
+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.
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