GLF protein purification
Design
To verify the successful expression of GLF in the plasmid transferred to Escherichia coli, we purified GLF with reference to the standard protein purification protocol.
Test
Figure 1. GLF expression validation via SDS-PAGE with Coomassie blue staining. There was no lanes in E1-E6, which indicates that no protein was successfully purified.CL (Cell lysate), FT (Flow through), W1-3 (wash 1-3), Protein Marker, E1-6 (elution 1-6).
Failed to extract GLF protein.
Learn
After completing plasmid transformation, Escherichia coli could grow on kanamycin-resistant plates, indicating that the plasmid was successfully introduced into Escherichia coli, but the protein could not be successfully purified.
Two possible reasons:
1. Plasmid was not expressed after entering Escherichia coli.
2. Expressed protein could not be extracted using methods above.
Therefore, we further consulted the relevant information about GLF protein. We found that GLF is a glucose dislodgment carrier, located on the cell membrane that belongs to the membrane protein. Membrane proteins can be discarded following cell debris after sonication. This may be one of the important reasons why we failed to achieve GLF purification.
Re-design
We therefore looked for ways to purify membrane proteins and finally decided to replace ultrasonic fragmentation method with triton X-100, which can lyse the cell membrane and thus more fully solubilize membrane proteins into buffer without discarding them with cell debris (standard protein purification protocol).
Re-test
Figure 2. GLF expression validation via SDS-PAGE with Coomassie blue staining. The results suggest that the expressions of glf were successfully detected, which was indicated by the corresponding bands around 50kDa, 53kDa, 28kDa and 25kDa. CL (Cell lysate), FT (Flow through), W1-3 (wash 1-3), Protein Marker, E1-6 (elution 1-6).
Red border marked in the figure: successfully purified 50kDa target protein GLF.
Learn
Our previous learning about GLF is correct. Since GLF is a membrane protein, the ultrasonic fragmentation method cannot effectively separate target protein from cells. The triton X-100 method can successfully extract GLF. But we also extracted lots of other proteins, which are probably membrane proteins with his-tag removed from cell membrane by triton X-100.
Aqueous-organic two-phase system
Design
Since α-pinene oxide is toxic and insoluble in inorganic solutions, but the degradation of α-pinene by bacteria introduced into the prα-pol plasmid needs to be carried out in a way that fully maintains the activity of the cells, the bacteria need to live in LB medium to complete the degradation. How to dissolve organic α-pinene oxide in inorganic LB medium is a problem that requires special consideration. Therefore, we designed a biphasic device in which the organic and inorganic phases were placed in a conical flask and shaken by a shaker to mix them well. It allows the engineering bacteria in the inorganic phase to have full access to the α-pinene oxide in the organic phase for efficient degradation, while avoiding bacterial death due to continuous contact between the engineering bacteria and α-pinene oxide.
Build
Please see Lab Notebook
Test
Our Erlenmeyer flask containing aqueous-organic two-phase system was placed in a communal shaker. However, α-pinene oxide continued to leak through the pore of the sealing film. Experiment was aborted.
Learn
α-pinene oxide is volatile, which is accelerated by shaking and heating, but normal life of E. coli requires a constant supply of oxygen, and we could not completely seal the container., We chose to use the decolorization shaker after communicating with PIs to solve this problem. The decolorization shaker was placed in the fume cupboard, so that the escaping α-pinene oxide would be absorbed without affecting experimental safety.
Re-design
We adjusted the aqueous-organic two-phase system as follows:
Adding 4ml of bacterial solution containing Escherichia coli with the introduced prα-pol plasmid and 1ml of pure α-pinene oxide to the culture tubes.
Figure 3. The structure of simplified aqueous-organic two-phase system. The components of the device are indicated in the figure.
Re-test
In this device, the organic and inorganic phases can be fully mixed and play the same mixing role as that of the thermostatic shaker, but it is impossible to maintain the temperature at 37℃. Therefore, we chose to incubate the bacteria in the thermostatic shaker until the OD value reaches about 0.5, and then take it out and add it into the culture tube to incubate with shaking for 3.5h.
Learn
This incubation was successful and we removed the organic phase after 3.5h and sent it to the Chemical Analysis Centre for GC-MS, where α-pinene oxide was finally detected.
Figure 4. The mass spectrum was generated for analysis. This revealed that both substrates, α-pinene oxides (A), and product,isonovaval (B) were detected in the GC-MS analysis.
Figure 5. 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.
37℃ is the optimal temperature for the degradation of α-pinene oxide by E. coli introduced into the prα-pol plasmid, there is no way to keep the temperature at 37℃ using this device, so it will have a certain impact on the survival of the engineered bacteria and the degradation process, and subsequently, we consider the use of a constant temperature shaker to optimise the degradation environment.
Co-transformation
Design
In order to express both GLF plasmid and P450-GlcDH plasmid in the same E. coli, we referred to the protocol for chemical transformation (add a hyperlink to the protocol for chemical transformation) and added half of the total plasmid amount of GLF plasmid and P450-GlcDH plasmid, respectively, and carried out chemical transformation.
Test
Figure 6. Colony growth on the plate after chemical cotransformation. We transferred GLF plasmid and P450-GlcDH plasmid into BL21 and took 200μL respectively on LB, kanamycin resistant, streptomycin resistant and dual-resistant plates one hour after resuscitation. a. Non-resistant (LB) plates have colony growth. b. Kanamycin resistant plates had colony growth. c. Streptomycin resistant plates have colony growth. d. Double resistance (kanamycin resistance and streptomycin resistance) on the plate without colony growth.
Learn
Considering the inefficiency of chemical transformation, which may be responsible for the fact that there is no way for two plasmids to enter a bacterium at the same time, we plan to use the more efficient electroshock transformation.
Re-design
We referred to the protocol for electrotransformation and added half of the total plasmid amount of GLF plasmid and P450-GlcDH plasmid, respectively, for electrotransformation.
Re-test
Figure 7. Growth of bacterial colonies on the plate after electroporation of E. coli. A total of four groups of electroporation (1) P450-GlcDH plasmid (2) GLF plasmid (3)(4) P450-GlcDH plasmid and GLF plasmid were transformed into E.coli BL21, respectively. After electroporation, the bacteria were inoculated into liquid LB medium and shaken in a constant temperature incubator for one hour. (1) (2) (3) After centrifugation, 800μL supernatant was discarded, leaving 200μL suspensible bacteria. (1) was inoculated on a streptomycin resistant plate (a), (2) was inoculated on a kanamycin resistant plate, and (3) was inoculated on a streptomycin, kanamycin double resistant plate (b). (4) After centrifugation, the 200μL supernatant was discarded, leaving 800μL heavy suspension bacteria, and divided into four parts, respectively, on (d) non-resistant LB plate, (e) streptomycin resistant plate, (f) kanamycin resistant plate, and (g) double resistant plate. All plates were cultured in a constant temperature incubator at 37°C for 12h.
Colonies grew only when BL21 E. coli introduced with the glf single plasmid was applied to kanamycin-resistant plates, and when BL21 E. coli introduced with the GLF plasmid and the P450-GlcDH plasmid was applied to non-resistant plates, suggesting that electrotransformation also did not enable the two plasmids to be introduced into a single bacterium at the same time.
Learn
According to the results, the P450 plasmid did not manage to enter the bacteria even during the electrotransformation of a single plasmid, so we considered using the chemical transformation method first by introducing the P450 plasmid into BL21 E. coli, then restoring the sensory state of these E. coli, and then transfecting the glf plasmid into the same tube of BL21 E. coli by electrotransformation.
