Part:BBa_K5291035
pAB1-pS-PEBP-PEase
PEBP is able to bind to PE microplastics non-specific, non-covalent hydrogen bonding and non-polar interactions. The PEase fused to PEBP can degrade PE when the engineered bacteria adhere to PE.
Usage and Biology
In degradation module we constructed pAB1-pS-PEBP-PEase to depolymerize PE microplastics.
Fig.1 The map of plasmid pAB1-pS-PEBP-PEase.
The bands of PEBP-PEase from PCR were identical to the theoretical lengths estimated by the designed primer locations, which could demonstrate that we successfully amplified our target fragments.The band was identical to the expected length of 3231bp.
Fig.2 The PCR result of PEBP-PEase fragment.
We transferred the constructed plasmid into Escherichia coli DH5αstrain and conducted colony PCR. Then we transferred these plasmids into Pseudomonas aeruginosa PAO1 strain and obtained correct colony PCR results, indicating that we successfully constructed strain containing the plasmid pAB1-pS-PEBP-PEase.
Fig.3 P.aeryginosa PAO1 colony PCR results of PEBP-PEase.
The plasmid PAB1-pS-PEBP-PEase was expected to express a protein with a size of 188.18kDa. Unfortunately, we did not see the corresponding bands, so the protein may have failed to be expressed in bacteria. We hypothesized that the protein could not be expressed correctly due to its large molecular weight. Therefore, we will verify the related effects of PEBP mainly by engineering bacteria with plasmid pAB1-pS-PEBP-GFP.(You can refer to Part:BBa K5291051).For the parts of the PEBP-PEase protein that are not verified, we will use modeling methods to predict.
For PEase, we tested its effect through moduling. We conducted structure predicting of PEBP-PEase. The predicting module clearly displayed a membrane spanning domain and an enzyme domain, which we exactly expected our bacteria to produce. After that molecular docking was conducted to study the interactions between the PEase enzyme and both large and small alkane molecules. It can be observed that the docking sites and the binding pockets are generally located within the same binding pocket, with multiple amino acid residues (such as LEU246, ILE250, VAL589, LEU622, etc.) surrounding the long-chain alkane. These amino acids are mostly hydrophobic residues, which likely stabilize the alkane molecules through hydrophobic interactions. Their affinity suggests that PEase may have the ability to bind to and degrade PE microplastics.
Fig.4 Predicted structure of PEBP-PEase protein.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 110
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 110
Illegal NotI site found at 247 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 2381
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 110
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 110
Illegal NgoMIV site found at 2695
Illegal NgoMIV site found at 3064
Illegal AgeI site found at 1363
Illegal AgeI site found at 2347
Illegal AgeI site found at 2410 - 1000COMPATIBLE WITH RFC[1000]
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