Part:BBa_K5291034
pAB1-cypY96F-vgb
VHb, the first discovered bacterial hemoglobin, is a soluble heme-binding protein with a faster rate of oxygen dissociation. It can enhance cell growth, product synthesis and stress tolerance. And CYPY96F, the P450cam mutant with higher alkane oxidation activity. This composite part is the fusion protein of both.
Usage and Biology
In degradation module we constructed pAB1-CYPY96F-VHB to ensure the expression of emzymes related to degradation.
Fig.1 The map of plasmid pAB1-CYPY96F-VHb.
We transferred the constructed plasmid into Escherichia coli DH5α strain, conducted colony PCR and obtained the correct result.
Fig.2 P.aeryginosa PAO1 colony PCR results of CYPY96F-VHB.
Moreover,CYPY96F proteins was successfully expressed in the strains. SDS-PAGE was performed and the following results were obtained.
Fig.2 The SDS-PAGE result of CYPY96F.
In order to verify that the engineering bacteria did degrade the microplastics faster, a 7-day co-incubation experiment was conducted. When the co-culture was done, microplastics were carefully separated from the culture medium and weighed. Fourier infrared (FTIR) detection was performed 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.3 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.4 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.
It can be seen 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. In the enlarged images the microplastics co-incubated with the engineering bacteria displayed a rough surface, while the microplastic in the control group is rather smooth. All of these features suggest that engineering bacteria degrade plastics more efficiently.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 604
Illegal BamHI site found at 684
Illegal BamHI site found at 1513 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 401
Illegal NgoMIV site found at 569 - 1000COMPATIBLE WITH RFC[1000]
| None |