Difference between revisions of "Part:BBa K5236008"

Revision as of 06:00, 2 October 2024

IsPETase-S121E/R224Q/N233K

Because of the substantial amount of microplastic pollution contaminating our oceans, our project aims to use biosynthetic methods to achieve effective degradation of these compounds in ambient temperatures. Whilst there are existent chemical and biological measures for microplastic degradation, however, chemical means often release chemical byproducts and contaminate the environment, whilst enzymatic degradation can only be done in an environment with specific pH, temperature, and humidity levels.[1] IsPETase-S121E/R224Q/N233K comes from Lu. et al. 2022 but is constructed by us in Escherichia coli. It breaks down polyethylene terephthalate into terephthalic acid through hydrolysis. IsPETase is an enzyme found in Iodeonella sakaiensis that possesses the ability to degrade PET[2].

Fig.1 The IsPETase-S121E/R224Q/N233K protein structure predicted using Alphafold and ligand binding predicted using Autodock, mutation sites are marked in red in the images.

Usage and Biology

IsPETase is an esterase, specifically designed to break down ester bonds between polyethylene terephthalate, a polymer, into terephthalic acid, a monomer. IsPETase-S121E/R224Q/N233K is a mutated sequence of PETase which enhances its degradation efficiency. It contains mutations at sites S121E, R224Q, and N233K. The mutation at position 121 involves a negative side chain, which enhances its catalytic efficiency. The mutation at position 224 replaces arginine with glutamine, facilitating better substrate access. The mutation at position 233 replaces asparagine by lysin which increases the thermal stability. The mutations were derived from a Transformer model on 1007 homologous PETase protein sequences derived from the UniProt Database using the masked language model (MLM) training method.

Our experimental design was aimed at proving the functionality of our mutated sequences, the viability of E.coli while harboring our sequences, the translational capacity of E.coli and the successful degradation of our mutated enzyme. To validate the functionality of our mutated sequences, we first inserted our genes into plasmid vectors. This process was initiated with the design of specific primers, followed by PCRs. Then, our sequences were recombined into plasmids and transformed into the chassis. In order to assess the part sequences for the mutated IsPETase variants S121E/R224Q/N233K and the functionality of the enzyme, we designed our experiment into two sub-parts. The first is plasmid construction. The second is to test the enzymatic activity. Through conducting colony PCR, we tested whether our parts had been transformed into chassis successfully. The following result of electrophoresis proves that we’ve inserted genes into the chassis since the sequence containing our mutated genes has a total of 891 base pairs with the sequence at the corresponding locations of the marker.

Fig.2 The DNA gel electrophoresis result.

Fig.3 The result of IsPETase-S121ER224QN233K DNA sequencing, the results showed that N205G and signaling peptide pelB sequences have been correctly assembled.

After confirming the integration of IsPETase into our chassis, we proceeded to test whether E.coli could maintain its viability while harboring our genes. Thus, we cultured the bacteria on a petri dish. After a 24-hour incubation period, E. coli grew over the plate, indicating that it can harbor our genes.

Fig.4 The result of plate coating. The results show that the chassis carrying our IsPETase mutation could survive.

We assessed the translational capacity of E.coli and the efficiency of our mutated enzyme. This section presents an analysis of two key results. 1. Enzyme Kinetic: the dynamic curve of our enzyme shows a high efficiency in degrading rate, in which S121E/R224Q/N233K, M10L, H104W, W159H/F229Y outperformed WT IsPETase. We stopped our testing phase at 30 minutes because 2. SDS-PAGE Electrophoresis: the electrophoresis result of our protein validates that our enzyme can successfully synthesize the proteins.

Fig.5 The enzymatic activity of mutated IsPETase was measured using p-nitrophenyl butyrate. The results showed that the IsPETase-S121E/R224Q/N233K have approximately 2.5-fold higher activity than WT group

Fig.6 Protein electrophoresis result.The Cell Culture group demonstrated the expression of the proteins, while we also tested the medium where cells were centrifuged and discarded. Proving the presence of IsPETase extracellular.

After confirming the enhanced efficiency of our enzymes, we have reached the most essential part of our part examination, which is to test if our mutated enzyme is capable of degrading microplastics. We designed two main approaches to validate it. First, we employed the scanning electron microscope to observe the morphological changes of the plastic after exposure to E.coli with IsPETase. The results confirm the degradation capacity of the enzyme, as there is a notable change on the surface of the plastic. However, observations are insufficient to prove the effectiveness of our enzymes. Consequently, we conducted an additional experiment utilizing High-Performance Liquid Chromatography (HPLC). We measured the amount of TPA in the cell culture medium, the peak of IsPETase S121E/R224Q/N233K in E.coli and cyanobacteria reflects the peak of the TPA standard sample, which proves the effectiveness of the microplastic degradation capabilities of our enzymes.

Fig.7 S121ER224QN233K Electron Scanning Microscope result

Fig.8 The HPLC results after 14 days of incubation of IsPETase-S121E/R224Q/N233K and PET. The results showed that the concentration of TPA, a degradation product of PET, was elevated in the cultures compared to the control group, implying that the degradation of PET

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

Lukaszewicz, Paulina, et al. "Plastic Waste Degradation in Landfill Conditions: The Problem with Microplastics, and Their Direct and Indirect Environmental Effects." International Journal of Environmental Research and Public Health, vol. 19, no. 12, 2022, pp. 12345-12367. National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9602440/.

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.

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K5236008 short</partinfo>
-This basic part encodes mutated IsPETase-S121E/R224Q/N233K, which comes from <i>Lu.et al. 2022</i> but is constructed by us in Escherichia coli. IsPETase is an enzyme found in Iodeonella sakaiensis that possesses the ability to degrade PET[1].
+Because of the substantial amount of microplastic pollution contaminating our oceans, our project aims to use biosynthetic methods to achieve effective degradation of these compounds in ambient temperatures. Whilst there are existent chemical and biological measures for microplastic degradation, however, chemical means often release chemical byproducts and contaminate the environment, whilst enzymatic degradation can only be done in an environment with specific pH, temperature, and humidity levels.[1] IsPETase-S121E/R224Q/N233K comes from Lu. et al. 2022 but is constructed by us in Escherichia coli. It breaks down polyethylene terephthalate into terephthalic acid through hydrolysis. IsPETase is an enzyme found in Iodeonella sakaiensis that possesses the ability to degrade PET[2].
 <center><html><img src ="https://static.igem.wiki/teams/5236/model-pics/ispet.gif" width = "50%"><br></html></center>
 <center><html><img src ="https://static.igem.wiki/teams/5236/model-pics/20241001191302.png" width = "50%"><br></html></center>
@@ Line 8: / Line 8: @@
 ===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 IsPETase S121E/R224Q/N233K 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.
+IsPETase is an esterase, specifically designed to break down ester bonds between polyethylene terephthalate, a polymer, into terephthalic acid, a monomer. IsPETase-S121E/R224Q/N233K is a mutated sequence of PETase which enhances its degradation efficiency. It contains mutations at sites S121E, R224Q, and N233K. The mutation at position 121 involves a negative side chain, which enhances its catalytic efficiency. The mutation at position 224 replaces arginine with glutamine, facilitating better substrate access. The mutation at position 233 replaces asparagine by lysin which increases the thermal stability. The mutations were derived from a Transformer model on 1007 homologous PETase protein sequences derived from the UniProt Database using the masked language model (MLM) training method.
-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.
+Our experimental design was aimed at proving the functionality of our mutated sequences, the viability of E.coli while harboring our sequences, the translational capacity of E.coli and the successful degradation of our mutated enzyme.
+To validate the functionality of our mutated sequences, we first inserted our genes into plasmid vectors. This process was initiated with the design of specific primers, followed by PCRs. Then, our sequences were recombined into plasmids and transformed into the chassis. In order to assess the part sequences for the mutated IsPETase variants S121E/R224Q/N233K and the functionality of the enzyme, we designed our experiment into two sub-parts. The first is plasmid construction. The second is to test the enzymatic activity.
+Through conducting colony PCR, we tested whether our parts had been transformed into chassis successfully. The following result of electrophoresis proves that we’ve inserted genes into the chassis since the sequence containing our mutated genes has a total of 891 base pairs with the sequence at the corresponding locations of the marker.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/colony-pcr.png" width = "50%"><br></html></center>
 <center>Fig.2 The DNA gel electrophoresis result. </center>
@@ Line 18: / Line 19: @@
 <center>Fig.3 The result of IsPETase-S121ER224QN233K DNA sequencing, the results showed that N205G and signaling peptide pelB sequences have been correctly assembled.</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 cultured the bacteria on a petri dish. After a 24 hour incubation period, it was found that E. coli grew over the plate, indicating that E. coli can survive with the presence of our genes.
+After confirming the integration of IsPETase into our chassis, we proceeded to test whether E.coli could maintain its viability while harboring our genes. Thus, we cultured the bacteria on a petri dish. After a 24-hour incubation period, E. coli grew over the plate, indicating that it can harbor our genes.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/s121e-r224q-n233k-plate-coating.jpg" width = "50%"><br></html></center>
 <center>Fig.4 The result of plate coating. The results show that the chassis carrying our IsPETase mutation could survive.</center>
+We assessed the translational capacity of E.coli and the efficiency of our mutated enzyme. This section presents an analysis of two key results.
-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.
+. Enzyme Kinetic: the dynamic curve of our enzyme shows a high efficiency in degrading rate, in which S121E/R224Q/N233K, M10L, H104W, W159H/F229Y outperformed WT IsPETase. We stopped our testing phase at 30 minutes because
+. SDS-PAGE Electrophoresis: the electrophoresis result of our protein validates that our enzyme can successfully synthesize the proteins.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/ispetase-mutation-efficiency-line-graph.jpg" width = "50%"><br></html></center>
@@ Line 32: / Line 34: @@
 <center>Fig.6 Protein electrophoresis result.The Cell Culture group demonstrated the expression of the proteins, while we also tested the medium where cells were centrifuged and discarded. Proving the presence of IsPETase extracellular. </center>
+After confirming the enhanced efficiency of our enzymes, we have reached the most essential part of our part examination, which is to test if our mutated enzyme is capable of degrading microplastics. We designed two main approaches to validate it.
-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.
+First, we employed the scanning electron microscope to observe the morphological changes of the plastic after exposure to E.coli with IsPETase. The results confirm the degradation capacity of the enzyme, as there is a notable change on the surface of the plastic. However,  observations are insufficient to prove the effectiveness of our enzymes. Consequently, we conducted an additional experiment utilizing High-Performance Liquid Chromatography (HPLC). We measured the amount of TPA in the cell culture medium, the peak of IsPETase S121E/R224Q/N233K in E.coli and cyanobacteria reflects the peak of the TPA standard sample, which proves the effectiveness of the microplastic degradation capabilities of our enzymes.
-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.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/s121e-esm.png" width = "50%"><br></html></center>
 <center>Fig.7 S121ER224QN233K Electron Scanning Microscope result </center>
@@ Line 55: / Line 54: @@
 ===Reference===
+Lukaszewicz, Paulina, et al. "Plastic Waste Degradation in Landfill Conditions: The Problem with Microplastics, and Their Direct and Indirect Environmental Effects." International Journal of Environmental Research and Public Health, vol. 19, no. 12, 2022, pp. 12345-12367. National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9602440/.
 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.