Difference between revisions of "Part:BBa K5236025"

Revision as of 13:24, 1 October 2024

BhrPETase

This basic part encoding the BhrPETase, which has been sequence predicted and optimized by Wu et al. And was constructed and modified as WT BhrPETase in our project.

Usage and Biology

To insert the parts into plasmids, we’ve designed primers and performed PCRs. Then, our genes were recombined into plasmids and transformed into chassis. By conducting colony PCR, we are able to test if our parts have been transformed into E.coli 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 798 base pairs and the results are in the right location.

Fig.1 The DNA gel electrophoresis result
Fig.2 The result of DNA sequencing

When we had completed the plasmid construction and transformation. We need to construct and test the enzymatic activity. .

Fig.3 Mutated BhrPETase Dynamic Curve

Fig.4 Protein electrophoresis result

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]

@@ Line 7: / Line 7: @@
 ===Usage and Biology===
-To insert the 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 BhrPETase. We want and whether the enzyme works, so we need to construct and test the enzymatic activity.
+To insert the parts into plasmids, we’ve designed primers and performed PCRs. Then, our genes were recombined into plasmids and transformed into chassis. By conducting colony PCR, we are able to test if our parts have been transformed into E.coli 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 798 base pairs and the results are in the right location.
-By conducting colony PCR, we are able to test if our parts have been transformed into E.coli 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 798 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>
@@ Line 17: / Line 15: @@
 <center>Fig.2 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.
+When we had completed the plasmid construction and transformation. We need to construct and test the enzymatic activity.
 .
-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.
 <center><html><img src ="https://static.igem.wiki/teams/5236/part-images/bhrpetase-mutation-efficiency-line-graph-1.jpg" width = "50%"><br></html></center>
@@ Line 29: / Line 26: @@
 <center>Fig.4 Protein electrophoresis result </center>
-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, 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.
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