Team:ZJU-China/BasicPart

<!DOCTYPE html>

BEST BASIC PART  
  

Usage and Biology

CsgA is the monomer of curli, which is an important component of excellular matrix of Enterobacteriaceae[1]. Curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation, they are also used for studies of amyloid fiber formation in human diseases, such as Alzheimer’s, Huntington’s, and prion diseases.

In consideration of curli is an important component of excellular matrix and biofilms, confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof.to the biofilm matrix.[2]In our project, we modified csgA to construct a brand new enzyme fixation matrix or surface display platform, which works as expected and proved the great biological potential.Furthermore, the cell-surface display has many potential applications, including biodegradation,live vaccine development, peptide library screening, bioconversion using whole cell biocatalyst and bioadsorption.

In short, csgA provides huge potentials for future igem communicity and we are so glad to see the future application!

Design

This year we made use of the design of cell-surface display and modified the csgA protein by fusing spytag to the N-terminal, making it a dorable platform to form enzyme complex and increasd the electrical conductivity because of the histag in the C-terminal. Therefore, we finally constructed our available platform for protein scaffolding, which has been proved more stronger to fix enzymes on electrodes.

Approach and Results

Cango Red(CR) dye plates and deletion assay shows success induction of over-expressing and the amploid protein characteristic.To see the results, please click here to change to part page.

Fig. 1
Quantitative Congo Red (CR) Binding Assays to verify the successful expression of Curli. (A) a is a wild-type E.coli strain that normally expresses csgA protein. b is the PHL628-△csgA cells (csgA knock out) strain. It can be seen that there is almost no expression of csgA after knocking out. (B) a is the wild type strain, b is the csgA overexpressing strain (a plasmid carrying a csga protein is expressed in wild type E.coli), and c is the csga KO cell. By adding Congo red dye to the bacteria mixture, centrifugation and shaking, we can compare the expression level of curli by observing the color depth of the precipitate. (C) Figure C is an inverted view of the EP tube in Figure B, showing the color of the sediment at the bottom of the centrifuge tube. a – WT. b – csgA KO, c – csgA overexpressing.

References

[1]Barnhart, M.M. and M.R. Chapman, Curli Biogenesis and Function - Annual Review of Microbiology, 60(1):131. Amyloid Cellulose Enteric Biofilm Extracellular Matrix.

[2]Nguyen, P.Q., et al., Programmable biofilm-based materials from engineered curli nanofibres. Nature Communications, 2014. 5: p. 4945.