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Team:HKU-HKBU/polar expression results
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Polar Expression - Results
E. coli polar expression system
Fluorescent Microscope
Plasmid in the BL21 strain showed the polar expression of GFP protein under the fluorescent microscope. In the merge picture, the fluorescent proteins were showed at one end of the bacteria.
However, in the E.coli 2443 with the plasmid sample, the polar expression of GFP couldn’t be observerd.
SDS PAGE and Western Blotting
Via SDS PAGE and Western Blotting, the relatively higher GFP protein concentration was observed on the outer membrane, which corresponded with the expected result of the specific expression of proteins on the outer membrane.
When observing the moving strains in the microscope, we could find that all the GFP were expressed at the forehead end of the rod shaped bacteria. Therefore, this system was a perfect choice for binding with biotin-coated motor. It can express streptavidin at pole(s), which allows cells to adhere in the same orientation to a microrotatory motor through biotin-streptavidin interaction.
Salmonella polar expression system
Fluorescent Microscope
plasmids showed the expression of GFP in the bacteria and the polar expression pattern of the GFP protein. The left panel is a scan of salmonella 7207 with Lpp-OmpA-HA, which acts as a control. The right panel can show the successful expression of GFP in the bacteria.
Western Blotting
To verify this hypothesis in a more convincing way, Western Blotting was conducted, which could reflect the surface expression by studying the relative concentrations of expressed proteins. The relative higher concentrations of specific proteins on the outer membrane show that the GFP proteins are particularly expressed on the outer membrane.
Unfortunately, the expression of GFP at the two ends is of equal opportunity, which couldn’t be an effective propelling system to push motor’s directional rotation. It’s better for proteins to specifically express proteins at the head of the bacteria, which ensures the pushing force to the motion of the motor.
