"
Team:HKU-HKBU/Polar Expression Design
From 2009.igem.org
Design
The biggest advantage of our Bactomotor is that the expression of streptavidin is specifically at the pole of the bacteria, which allows cells to adhere in the same orientation to the motor. Once bacterial cells adhere to the biotin-coated micromotor, the propulsion force would thereafter be generated by bacterial swimming. Therefore, this synthetic device is capable to convert biological energy into mechanical work.
The polar expression of the streptavidin at one pole of the bacteria is crucial in our project. It's well known that there're some systems could express proteins evenly on the outer membrane of the bacteria, or concentratedly at single poles under some certain conditions. This surface expression characteristics could ensure the precise structures and functions of the expressed protein streptavidin, which could lead to strong interaction between streptavidin and biotin. The two systems used in our design go as follows:
- AIDA System
In the biobrick pT7-rbs-(sp_eGFP_streptavidin_AIDAc)-Terminators(BBa_K283001), promoter(T7) needs T7 polymerase to promote its function; Signal peptide(SP) could bring the plasmid to the membrane region of the bacteria; eGFP is integrated within the plasmid for detection; Streptavidin is specifically binding with biotin, which can achieve the combination of the bacteria and biotin coated motor; AIDA is a transmembrane protein, which leads to the polar expression of the whole plasmid. However, AIDA protein can only be expressed in a LPS complete strain, which is a constraint when choosing the bacteria strains.
- Lpp-OmpA System
The other biobrick we used is pTet-rbs-lpp_ompA_eGFP_streptavidin-double Terminators(BBa_K283000), which can be polarly expressed on one side of rod-shaped bacteria. Lpp functions as a signal peptide; OmpA is the player which can achieve the surface expression of specific proteins; GFP-Strp acts the same role as above.
After we successfully constructed two polar expression systems, which bacteria strain and which system to be used became our crutial task.
Strain Selection
- For AIDA System
To achieve polar expression, it is essential to screen and select bacteria species and strains equipped with outstanding swimming abilities as well as a complete LPS (lipopolysaccharide) layer. This layer, present in the bacterial cell wall, is required for polar expression of certain proteins(AIDI) The candidates included Escherichia. Coli strains: BL21, NCM3722, MG1655, MG3, and YBE01 , and YBS01.
- For Lpp-OmpA System
Lpp-OmpA system can only achieve outer membrane surface expression. To select the strain which can polar express this plasmid, Lpp-OmpA-GFP-Streptavidin was transformed into the strains above. GFP equipped this plasmid the ability to be detected easily under fluorescent microscope.
