Part:BBa_K3059618

Staphylococcus Aureus SuhB Protein (SaSuhB)

Description and Characterization

This is a SaSuhB fiber isolated from E. coli culture grown in LB for 18 hours. The macroscopic nature of the fiber is quite apparent.

Sequence Confirmation

The sequence for SaSuhB was confirmed in all of the circuits in which it was used in.

Congo Red Assay

SaSuhB is a protein native to Staphylococcus aureus. While it is characterized as an inositol monophosphatase enzyme, it is also necessary for unimpaired pia-independent biofilm formation in S. aureus [1]. Moreover, further study of this protein has revealed that it is capable of forming macroscopic amyloid fibers when expressed in E. coli [2]. The fibers were characterized as being incredibly sticky, causing the researchers great trouble in trying to wash the fibers off of glass and plastic ware [2]. Additionally, SEM imagery revealed that bacteria were readily adhered to these fibers [2]. This combination of traits makes SaSuhB perfect for our application of forming strong, robust biofilms.

The tube on the left is uninduced, while the tube on the right is induced.

As can be seen in the graph and picture above, when SaSuhB is expressed in E. coli after performing a congo red assay there is a significant decrease in the A490 value of the solution, indicating that there is an amyloid protein being expressed that is absorbing the congo red dye.

Microscopy

Microscopy Images of a SaSuhB fiber at 200x magnification with varying polarization of light. The top left image is without polarization, the top right is with polarization setting 1, the bottom left is with polarization setting 2, and the bottom right image is with polarization setting 3.

The left image is a SaSuhB fiber under fluorescence microscopy with blue excitation and green emission at 200x magnification. The right image is the same fiber under transmission microscopy, also at 200x magnification. A length bar in the right image is used to measure a fiber width around 138um at the selected location.

As can be seen in these images, SaSuhB exhibits a very robust, fibrous structure.

References

[1] Boles, B. R., Thoendel, M., Roth, A. J., & Horswill, A. R. (2010). Identification of genes involved in polysaccharide-independent Staphylococcus aureus biofilm formation. PloS one, 5(4), e10146.

[2] Dutta, A., Bhattacharyya, S., Kundu, A., Dutta, D., & Das, A. K. (2016). Macroscopic amyloid fiber formation by staphylococcal biofilm associated SuhB protein. Biophysical chemistry, 217, 32-41.