Result & Discussion
Our group takes a long time to accomplish the whole process. What we have performed is shown below. Detailed information including experimental design and protocols are described on the Experiment page.
We want to:
Oxidize α-pinene efficiently by P450BM-3 QM combined with NADH regenerative system, which is constructed by a membrane-bound protein GLF and intracellular enzyme GlcDH-II. Degrade α-pinene oxide through an enzyme, Prα-POL from Pseudomonas rhodesiensis CIP107491.
Figure 1. The process of the whole project, starting from engineered bacteria construction to the determination of the degradation efficiency.
How did we do it?
Gibson assembly
Figure 2. The general principles of Gibson assembly. This depends on the recombination of the overlap between the target gene and the vector. The result of transformation is checked by the LB agar plate with corresponding antibiotics.
Competent cell transformation
Three recombinant plasmids were transformed E. coli BL21 star (DE3) for gene expression respectively. The colonies shown on the agar plates containing antibiotics indicated that the three plasmids were all successfully transformed into E. coli BL21 star (DE3) (Figure 3).
Figure 3. Transformation results of three plasmids in the LB agar plate. Three plasmids were all successfully transformed into bacteria, indicated by the colonies on the agar plate. (A) E. coli BL21 star (DE3) with pQE-80L-Kan glf; (B) E. coli BL21 star (DE3) with pCDFDUET-1 p450bm-3qm glcdh-II; (C) E. coli BL21 star (DE3) pET-28a prα-pol.
Prokaryotic protein expression
To induce protein expression, IPTG was added into the bacteria culture when OD600 equaled 0.5-0.6, approximately 4 h after inoculation and then cultured for another 4 h (Figure 4).
Figure 4. The general pattern of E. coli growth. The change of OD600 of the bacterial culture along with time was shown. OD600 was recorded at 0, 1, 2, 3 and 4 h, and IPTG was added at 4 h.
Protein purification and SDS-PAGE
The proteins are all conjugated to a 6xhis tag, which can be purified by His-tag Protein Purification Kit. We can observe the target bands through SDS-PAGE with Coomassie brilliant blue staining.
Figure 5. Glf, P450BM-3 QM, GLcDH-II, Prα-POL expression
validation via SDS-PAGE with Coomassie blue staining. The results suggest that the expressions of glf,
P450BM-3 QM, GLcDH-II, Prα-POL were successfully detected, which was indicated by the corresponding bands around
50kDa, 53kDa, 28kDa and 25kDa. However, the bands of protein GLF were relatively blurry in all lanes.
CL (Cell lysate), FT (Flow through), W1-3 (wash 1-3), Protein Marker, E1-6 (elution 1-6).
Detection of engineered bacterial susceptibility to α-pinene oxides
To better determine the concentration of α-pinene oxides used in aqueous-organic two-phase system, corn oil was used as solvent to dilute α-pinene oxides. Kirby-Bauer test was used to detect the engineered bacterial sensitivity to α-pinene oxides. The results revealed that E. coli BL21 star (DE3) pET-28a prα-pol showed low-sensitivity to α-pinene oxides.
Figure 6. Results of preliminary experiment. (A) .The two
centrifugal tubes originally contained 1 ml corn oil, and 0.8 ml α-pinene oxides was added twice each time into
the left tubes. No stratification was observed. (B)Kirby-Bauer test. A total of three groups of repeats
were performed. 75% and 100% concentrations (v/v) of α-pinene oxides dissolved in corn oil were used.
Antibacterial rings were less than 3 mm in all groups, and the 75% and 100% experimental groups had similar
antibacterial rings in each repeat group.
Aqueous-organic two-phase system with GC-MS analysis
In order to test whether the engineered bacteria can degrade the target substances efficiently, an aqueous-organic two-phase system was constructed. The mass spectrum showed that both substrates, α-pinene oxides, and product, isonovaval were detected. In addition, an obvious difference can be seen between 6-8 min and 13-16 min in the Total ion chromatogram (TIC) of the IPTG group. This suggests that the certain reaction that converts α-pinene oxides to isonovaval may appear in the engineered bacteria.
Figure 7. The mass spectrum was generated for analysis.This
revealed that both substrate, α-pinene oxides(A), and product, isonovaval (B) were detected in the
GC-MS analysis.
Figure 8. TIC spectrum of four groups. During The ion
abundance was the highest in the IPTG group (A)compared to that in the group without IPTG induction
(D) during 6-8 min and 13-16 min. However, the ion abundance was similar in the PBS group (C) and
pure α-pinene oxides group (B) both of which were lower than the other groups.
Discussion
We have successfully cloned and expressed three plasmids into E. coli BL21 star (DE3) and validated the expression of each protein. However, the bands of GLF were not as clear as other bands, which can be due to the properties of GLF protein. GLF is an insoluble membrane-bound protein and may also be expressed at a low level, thus hard to be purified by our protocols. In addition, further analysis is required to determine the degradation efficiency of both engineered bacteria.
