Difference between revisions of "Part:BBa K5236010"

Latest revision as of 13:12, 2 October 2024

BhrPETase N205G

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 N205G.

Fig.1 The BhrPETase-N205G protein structure predicted using Alphafold and ligand binding predicted using Autodock, mutation sites are marked in red in the images.

Fig.2 The the affinity of the top 15 positions of the BhrPETase enzyme to the microplastic molecules. More negative the affinity is, the better the mutant is.

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 BhrPETase N205G 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 BhrPETase N205G sequence, which is inserted between the promoter and terminator.

Fig.3 The illustration of BhrPETase N205G genetic pathway

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.4 The DNA gel electrophoresis result

Sequencing also demonstrated successful plasmid construction.

Fig.5 The result of N205G DNA sequencing

After confirming that the BhrPETase N205G is present in our chassis, we tested whether the bacteria can survive as usual with our genes. Thus, we coated the bacteria on petri dishes to observe their growth. After a night of incubation, E. coli grew over the plate, justifying that E. coli can survive with the gene encoding BhrPETase N205G.

Fig.6 BhrPETase-N205G was coated on spectinomycin plates and incubated overnight at 37 ℃

Next, we tested the synthesis of BhrPETase N205G in E.coli through SDS PAGE. The results show that the enzymes synthesized in E.coli an secreted are in fact BhrPETase N205G.

Fig.7 Protein electrophoresis result

To test the potential PET degradation efficiency of the BhrPETase N205G 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 BhrPETase WT and IsPETase WT compared to the mutations N191S, M57L, W229F, N205G. The mutation BhrPETase N205G has a slight higher enzyme activity than BhrPETase WT at 30 min; it has the potential to significantly surpass the efficiency of BhrPETase if given more time.

Fig.8 Mutated BhrPETase Dynamic Curve

The relative enzyme efficiency (A415/protein concentration) that we are looking at takes into consideration both the efficiency of the enzyme itself and the PETase synthesis rate of the chassis, since our end goal is to implement the engineered organism in a self-sufficient PET degrading system as a whole.

To further measure the BhrPETase N205G efficiency we used a scanning electron microscope to directly observe changes in the plastic after degradation. Different plastic samples were placed in the culture medium of the engineered E. coli. After two weeks, the samples were observed under an SEM for any alterations in the surface of the plastic by technicians at Shenzhen University. The results demonstrated that plastic with low level of crystallinity were degraded under the exposure to PETase synthesized by our engineered E. coli. Further, the fact that plastic with high crystallinity did not show any significant changes addresses our hypothesis in Cycle 1: PET degradation is affected by the crystallinity of the plastic, which varies depending on its manufacturing process.The SEM allows us to see the changes of plastic pieces with our bare eyes.

SEM procedure:

1 Cultivate the bacteria with PET for 14 days.

2 Remove and clean with water

3 Soak in 72% ethanol for 10 minutes (sterilization)

4 Soak in 100% ethanol for 10 minutes

5 Replace the ethanol and soak again in 100% ethanol for half an hour.

6 Dry well in an ultra-clean bench

7 Hand over to engineer

Fig.9 N205G Scanning Elctron Microscope result

After determining that our enzyme BhrPETase N205G expressed in E.coli is actually more efficient than wild type BhrPETase N205G, we moved on to expressed the gene in Synechococcus elongates PCC 7942. To achieve this we inserted the signal peptide and BhrPETase N205G sequence between the PpsbA2 and terminator Bba_B0015 of a transfer vector.

Fig.10 The illustration of BhrPETase N205G genetic pathway

The reconstructed plasmids were transformed into cyanobacteria and coated on BG11 plates containing the antibiotic spectacularionomycin. Single colonies containing the BhrPETase N205G gene appeared after two weeks of incubation.

Fig.11 N205G in cyanobacteria coating result

For more direct indication of PET degradation by BhrPETase N205G, we conducted another test using high-performance liquid chromatography. HPLC is a method to directly detect the presence of specific compound in a chemical mixture. During the degradation of PET by PETase, TPA is created as a byproduct; hence, the presence of TPA in the final product indicates degradation occurred. The results show that TPA is present after a 2 week incubation of PET with the engineered bacteria.

Fig.12 High-performance liquid chromatography analysis.

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]

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.

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K5236010 short</partinfo>
-Since plastic pollution poses a serious global environmental problem, one of the potential solution, enzyme degradation, would be a suitable approach of dealing with plastic wastes. And among all of the plastic pollutions, more than 10% of them are Polyethylene terephthalate (PET). Thus, our team has been looking for possible PET hydrolase to deal with 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--in a synthetic biology way--manually mutate wild-type BhrPETase, which 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, to enlarge the acceptable range of temperature and pH level for PET hydrolases to function more efficiently and degrade more PET to solve global plastic pollution as soon as possible. As one of our most-confident mutants, this basic part encodes mutated BhrPETase N205G and was constructed in Escherichia coli in our lab.
+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 N205G.
 <center><html><img src ="https://static.igem.wiki/teams/5236/model-pics/bhrpet-n205g.gif" width = "50%"><br></html></center>
-<center>Fig.1 The the affinity of the top 19 positions of the BhrPETase enzyme to the microplastic molecules. More negative the affinity is, the better the mutant is. </center>
+<center>Fig.1 The BhrPETase-N205G protein structure predicted using Alphafold and ligand binding predicted using Autodock, mutation sites are marked in red in the images.</center>
 <center><html><img src ="https://static.igem.wiki/teams/5236/model-pics/wechatimg132.jpg" width = "50%"><br></html></center>
-<center>Fig.2 The BhrPETase-N205G protein structure predicted using Alphafold and ligand binding predicted using Autodock, mutation sites are marked in red in the images.</center>
+<center>Fig.2 The the affinity of the top 15 positions of the BhrPETase enzyme to the microplastic molecules. More negative the affinity is, the better the mutant is. </center>
 ===Usage and Biology===
-Initially, we've 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 there are only changes in nucleotides but not in amino acids, functions, or structures, to ensure that our mutants have some postive impacts. For further imformation, please check the  model page on our wiki. https://2024.igem.wiki/basis-china/model
+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
-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 N205G 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.
+The BhrPETase N205G 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 BhrPETase N205G sequence, which is inserted between the promoter and terminator.
+<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/n205g-plasmid.png" width = "50%"><br></html></center>
+<center>Fig.3 The illustration of BhrPETase N205G genetic pathway </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.
+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.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/marker-colony-pcr.png" width = "50%"><br></html></center>
-<center>Fig.3 The DNA gel electrophoresis result </center>
+<center>Fig.4 The DNA gel electrophoresis result </center>
+Sequencing also demonstrated successful plasmid construction.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/n205g.png" width = "50%"><br></html></center>
-<center>Fig.4 The illustration of BhrPETase N205G genetic pathway  </center>
+<center>Fig.5 The result of N205G 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 confirming that the BhrPETase N205G is present in our chassis, we tested whether the bacteria can survive as usual with our genes. Thus, we coated the bacteria on petri dishes to observe their growth. After a night of incubation, E. coli grew over the plate, justifying that E. coli can survive with the gene encoding BhrPETase N205G.
-The result show that chassis carrying our PETase could survive.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/n205g-plate-coating.png" width = "50%"><br></html></center>
-<center>Fig.5 BhrPETase-N205G was coated on spectinomycin plates and incubated overnight at 37 ℃ </center>
+<center>Fig.6 BhrPETase-N205G was coated on spectinomycin plates and incubated overnight at 37 ℃ </center>
-We tested whether the bacteria could translate for our protein, and we examined whether our mutated enzyme (N205G, W229F, M57L, N191S) is more efficient. For this section, we analyzed two results as well. First, the electrophoresis result of our protein proves that our enzyme can be successfully coded by the parts we designed. Second, the dynamic curve of our enzyme shows its high efficiency in degrading rate (x-axis stops at 30min because that's what the professional research teams did).
+Next, we tested the synthesis of BhrPETase N205G in E.coli through SDS PAGE. The results show that the enzymes synthesized in E.coli an secreted are in fact BhrPETase N205G.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/n205g-sds-page-new.png" width = "50%"><br></html></center>
 <center>Fig.7 Protein electrophoresis result </center>
-This dynamic curve shows that only N205G's efficiency is able to exceed wild-type after 30 minute, proving that mutated PET hydrolase does have an increase in efficiency.
+To test the potential PET degradation efficiency of the BhrPETase N205G 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 BhrPETase WT and IsPETase WT compared to the mutations N191S, M57L, W229F, N205G. The mutation BhrPETase N205G has a slight higher enzyme activity than BhrPETase WT at 30 min; it has the potential to significantly surpass the efficiency of BhrPETase if given more time.
-<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/bhrpetase-dynamic-curve-no-units.jpg" width = "50%"><br></html></center>
+<center><html><img src ="https://static.igem.wiki/teams/5236/yh/bhrpet-relative-activity.jpg" width = "50%"><br></html></center>
-<center>Fig.6 Mutated BhrPETase Dynamic Curve </center>
+<center>Fig.8 Mutated BhrPETase Dynamic Curve </center>
+The relative enzyme efficiency (A415/protein concentration) that we are looking at takes into consideration both the efficiency of the enzyme itself and the PETase synthesis rate of the chassis, since our end goal is to implement the engineered organism in a self-sufficient PET degrading system as a whole.
-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——scanning electron microscope and high performance liquid chromatography
-Different plastic samples were placed in the culture medium of the engineered E. Coli. After two weeks, the samples were observed under an SEM for any alterations in the surface of the plastic by technicians at Shenzhen University. The results demonstrated that plastic with low level of crystallinity were degraded under the exposure to PETase synthesized by our engineered E. coli. Further, the fact that plastic with high crystallinity did not show any significant changes addresses our hypothesis in Cycle 1:  PET degradation is affected by the crystallinity of the plastic, which varies depending on its manufacturing process.The SEM allows us to see the changes of plastic pieces with our bare eyes.
+To further measure the BhrPETase N205G efficiency we used a scanning electron microscope to directly observe changes in the plastic after degradation. Different plastic samples were placed in the culture medium of the engineered E. coli. After two weeks, the samples were observed under an SEM for any alterations in the surface of the plastic by technicians at Shenzhen University. The results demonstrated that plastic with low level of crystallinity were degraded under the exposure to PETase synthesized by our engineered E. coli. Further, the fact that plastic with high crystallinity did not show any significant changes addresses our hypothesis in Cycle 1:  PET degradation is affected by the crystallinity of the plastic, which varies depending on its manufacturing process.The SEM allows us to see the changes of plastic pieces with our bare eyes.
 SEM procedure:
 Cultivate the bacteria with PET for 14 days.
 Remove and clean with water
-Soak in 72% ethanol for 10 minutes (sterilization)
-Soak in 100% ethanol for 10 minutes
+Soak in 72% ethanol for 10 minutes (sterilization)
-Replace the ethanol and soak again in 100% ethanol for half an hour.
-Dry well in an ultra-clean bench
+Soak in 100% ethanol for 10 minutes
-Hand over to engineer
+Replace the ethanol and soak again in 100% ethanol for half an hour.
+Dry well in an ultra-clean bench
+Hand over to engineer
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/n205g-esm.png" width = "50%"><br></html></center>
-<center>Fig.8 N205G Scanning Elctron Microscope result </center>
+<center>Fig.9 N205G Scanning Elctron Microscope result </center>
+After determining that our enzyme BhrPETase N205G expressed in E.coli is actually more efficient than wild type BhrPETase N205G, we moved on to expressed the gene in Synechococcus elongates PCC 7942. To achieve this we inserted the signal peptide and BhrPETase N205G sequence between the PpsbA2 and terminator Bba_B0015 of a transfer vector.
+<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/n205g-plasmid.png" width = "50%"><br></html></center>
+<center>Fig.10 The illustration of BhrPETase N205G genetic pathway </center>
+The reconstructed plasmids were transformed into cyanobacteria and coated on BG11 plates containing the antibiotic spectacularionomycin. Single colonies containing the BhrPETase N205G gene appeared after two weeks of incubation.
+<center><html><img src ="https://static.igem.wiki/teams/5236/part-images/n205g-cyano-plate.png" width = "50%"><br></html></center>
+<center>Fig.11 N205G in cyanobacteria coating result </center>
-However, pure observations are not enough to prove the effectiveness of our enzymes. Thus, we conducted another experiment. Therefore, we tried to use HPLC for compositional identification. HPLC is a method to directly detect the presence of specific compound in a chemical mixture. During the degradation of PET by PETase, TPA is created as a byproduct; hence, the presence of TPA in the final product indicates degradation occurred.
+For more direct indication of PET degradation by BhrPETase N205G, we conducted another test using high-performance liquid chromatography. HPLC is a method to directly detect the presence of specific compound in a chemical mixture. During the degradation of PET by PETase, TPA is created as a byproduct; hence, the presence of TPA in the final product indicates degradation occurred. The results show that TPA is present after a 2 week incubation of PET with the engineered bacteria.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/n205g-hplc.png" width = "50%"><br></html></center>
-<center>Fig.9 High-performance liquid chromatography analysis. The results show that TPA is present after a 2 week incubation of PET with the engineered bacteria. </center>
+<center>Fig.12 High-performance liquid chromatography analysis. </center>
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@@ Line 76: / Line 96: @@
 ===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.