Part:BBa_K5291033
pAB1-alkB2-Rd45-adhA
The system AlkB2-Rd45-AdhA is designed to accelerate the degradation rate by improving the absorbing and hydroxylation of PE monomer.
Usage and Biology
AlkB2-Rd45-AdhA is designed to accelerate the degradation rate by improving the absorbing and hydroxylation of PE monomer. In order to express above the enzymes in our chassis，pAB1-alkB2-Rd45-adhA was constructed.
Fig.1 The overview of the alkB2-Rd45-adhA system.
Fig.2 The map of the plasmid pAB1-alkB2-Rd45-adhA.
We transferred the constructed plasmid into Escherichia coli DH5αstrain and conducted colony PCR. After obtained the correct result we amplified and extracted the constructed plasmids. Then we transferred these plasmids into Pseudomonas aeruginosa PAO1 strain and E. coli BL21 strain and obtained correct colony PCR results, indicating that we successfully constructed strains containing the plasmid pAB1-pAB1-alkB2-Rd45-adhA.
Fig.3 The P.aeryginosa PAO1 colony PCR results of AlkB2-Rd45-AdhA.
In order to verify whether AlkB2-Rd45-AdhA proteins was successfully expressed in the strains, SDS-PAGE was performed and the following results were obtained.
Fig.4 SDS-PAGE result of AlkB2-Rd45-AdhA.
Plasmid pAB1-AlkB2-Rd45-AdhA is expected to express a large fusion protein AlkB2-Rd45-AdhA with a molecular weight of 94.59kDa. In the figure, it can be seen that there were an extra bands in the experimental group than in the control group at a position slightly less than 100kDa, suggesting that AlkB2-Rd45-AdhA protein was successfully expressed.
After that, we also conducted experiments of effect verification. We cultivated the engineered bacteria PAO1::alkB2-Rd45-adhA with microplastic and ourier infrared (FTIR) detection was performed after 7 days on the incubated microplastics to check the property changes of the microplastics, and scanning electron microscopy was also used to detect the morphologic change on the surface of the microplastics.
Fig.5 FTIR results of co-cultured PE microplastics. (*The peak around 2400 cm-1 is caused by the instrument itself, that it lacks of argon.)
Compared to its line, the bacteria input group’s peaks have decreased in the intensity level, indicating the decrease in chemical bonds. Especially, the engineered PAO1 have shown lower intensity than the wild type of PAO1. The decrease of chemical bonds indicates that the PAO1 is able to degradant PE, and our plasmids have improved its ability.
Fig.6 From left to right, PAO1::alkB2-Rd45-adhA, PAO1::cypY96F-vgb, wild type PAO1, blank control, respectively. In the first row, the experimental group is magnified 900 times, the control group is magnified 300 times. In the second row, the experimental group was magnified 5,000 times and the control group 2,500 times.
We can know from these figures that the overall particle size of microplastics degraded by engineering bacteria is generally smaller than that of the control group and microplastics co-incubated with Engineering bacteria were broken into small fragments while control group maintains complete microplastic particles. All of these features suggest that engineering bacteria degrade plastics more efficiently.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 902
Illegal NgoMIV site found at 2433 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1476
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