Team:HKU-HKBU/Polar Expression Design

=Design=

When we were formulating the design of our BactoMotor, we were well aware of the existance of previous models of similar bacterial driven motors. However, the previous designs, though receiving a lot of attention, lack some key components an ideal motor should have:


 * 1) Low efficiency while transforming vectorial differences in Brownian Motion into rotation [1]
 * 2) Lack unidirectional movement of bacteria [2]
 * 3) A small percentage of motors rotated in undesired direction [2]
 * 4) Involve chemical rather than genetic modification of surface proteins [2]
 * 5) No speed control mechanism (discussed in Speed Controller module) [1] [2]

To overcome such problems, we designed a 'Direction Controller' module which we have implemented into our bacteria. The use of this module is to overcome the problems listed above as well as introducing several more benifits:


 * 1) Achieve strong selective binding of Bacteria onto the BactoMotor
 * 2) The genetic modification (biobricks) is flexible and stable compared to chemical treatment
 * 3) Future application for medical purposes by expression of desired ligands or receptors

The mechanism by which the Direction Controller operate will be discussed below.

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 desired interaction between streptavidin and biotin. The two systems used in our design go as follows:

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 protein to the membrane region of the bacteria; eGFP is integrated within the plasmid for detection; Streptavidin specifically binds to biotin, which mediates the orientated binding of the bacteria and to biotin coated motor; AIDA is a transmembrane protein, which leads to the polar expression of the protein. However, AIDA protein can only be expressed in a LPS complete strain, which is a constraint when choosing the bacteria strains.
 * AIDA System


 * 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 crucial task.

Strain Selection

 * For AIDA System

To achieve polar expression, it is essential to screen and select bacteria species and strains equipped with outstanding propelling 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(AIDA) The candidates included ''Escherichia. coli'' strains: BL21, NCM3722, MG1655, YBE03, and YBE01, and YBS01.


 * For Lpp-OmpA System

Lpp-OmpA system can only achieve outer membrane surface expression. To select the strain which can polarly express the desired proteins, Lpp-OmpA-GFP-Streptavidin was transformed into the strains above. GFP conferred this system the ability to be detected easily under fluorescent microscope.

Reference

 * 1) Luca Angelani, Roberto Di Leonardo, and Giancarlo Ruocco, Self-Starting Micromotors in a Bacterial Bath, Phys. Rev. Lett., 102:048104 (2009), doi:10.1103, PhysRevLett.102.048104
 * 2) Yuichi Hiratsuka, Makoto Miyata, Tetsuya Tada, and Taro Q. P. Uyeda, A microrotary motor powered by bacteria, PNAS, 103:13618-13623 (2006), doi:10.1073, pnas.0604122103
 * 3) Maurien M. A. Olsthoorn, Bent O. Petersen, Siegfried Schlecht, Johan Haverkamp, Klaus Bock, Jane E. Thomas-Oates and Otto Holst, Identification of a Novel Core Type in Salmonella Lipopolysaccharide, The Journal of Biological Chemistry, 1998, 273(7):3817-3829