What’s more, biofilm can automatically grow by static adherence, which facilitates regeneration and recycling in mass production in industry. Startlingly, biofilms can also serve as a synthetic nonconductive biological platform for self-assembling function materials. The amyloid protein CsgA, which is the dominant component in E.Coli, can be programmed to append small peptide domain and successfully secreted with biological functions. Also, it has been tested that CsgA subunits fused with not too large peptide can be tolerated by curli export machinery and maintain the self-assembly function as always. (Neel S. Joshi, 2014)<p></p>

Congo Red dye is a classic method to detect amyloid protein (Alan Marcus, 2012). Amyloid can be visualized and quantified through the staining of Congo Red because Congo Red molecules obtain an oriented arrangement on amyloid fibrils. This property can be ascribed to the hydroxyl groups on the amyloid and hydrogen bonding on the Congo Red (Puchtler, 1962). It only takes approximately 20 minutes to dye so it is indeed a good practice in lab to crudely test the expression of biofilms.<p></p>

Crystal violet is a triarylmethane dye used as a histological stain to classify biomass. This is a simple assay practical and useful for obtaining quantitative data about the relative quantity of cells which adhere to multi-wells cluster dishes. After solubilization, the amount of dye taken up by the monolayer can be quantitated in a plate reader. (Soares, n.d.)<p></p>

The series of Congo Red assay are aim to visualize the expression of biofilm. To produce curli, we spread the CsgA-Histag mutant E.coli onto a low-nutrition culture medium, YESCA- CR plates (Neel S. Joshi, 2014), containing 10 g l-1 of casmino acids, 1 gl-1 of yeast extract and 20 gl-1 of agar, supplemented with 34 μg ml-1 of chloromycetin,  5 μg ml-1 of Congo Red and 5 μg ml-1 of Brilliant Blue. (Details in protocol 链接) Red staining indicates amyloid production.<p></p>

The figures shown above point out that the CsgA-Histag mutant induced by 0.25 μg ml-1 of aTc will produce amyloid structures which are dyed to red by CR in comparison to the negative control. This assay indicates the success in expression of the self-assembly to curli fibers. <p></p>

After CR dye, the figure indicates that the His-CsgA-SpyCatcher-Histag mutant induced by 0.25 μg ml-1 of aTc successfully secreted a thin-layer biofilm on the plate which are stained to brown-red color by CR, compared to the negative control with no inducer.  (Because the ratio between Congo Red dye and Brilliant Blue dye is not in the best state which leads to the unapparent phenomenon through the lens, the brown red biofilm is easy to be identified visually.) This assay also proved that the new and challenging construction of appending a large protein onto CsgA subunits will work accurately and effectively.<p></p>

After 72h culture, we scratch the biofilm down from the well and apply 25 μg ml-1 of Congo Red into solution. Then centrifuged and washed by PBS for several times, we get the result: newly His-CsgA-SpyCatcher mutant induced by 0.25 μg ml-1 of aTc was stained to bright-red color by CR, compared to the negative control with no inducer and the color can’t be washed away. This assay also manifested the success in construction of His-CsgA-SpyCatcher mutant and add versatility to our biofilm platform.<p></p>

               <h3 >SpyTag and SpyCatcher  (Zsofia Botyanszki, 2015)</h3>

We want to attach enzyme to biofilm, so we turn to a widely applied linkage system, SpyTag and SpyCatcher, originally discovered from Streptococcus pyogenes. By splitting its fibronectin-binding protein FbaB domain, we obtain a relatively small peptide SpyTag with 13 amino acids and a bigger protein partner, SpyCatcher, with 138 amino acids (Bijan Zakeria, 2012). The advantage of this system lies in the following three aspects. Firstly, they can spontaneously form a covalently stable bond with each other which guarantee the viability of the permanent linkage. The second point is quick reaction within 10 min, which will stand out by its efficiency in industrial application. Besides, the whole process proceeds in mild condition (room temperature), thus set lower requirement for reaction both in lab and future practice. Therefore, we design to leverage this advantageous system to achieve the binding of biofilm with specific enzyme. <p></p>

We cultured all E.coli mutants in multi-wells with increasing inducer gradient. The result demonstrated in accordance that 0.25 μg ml-1 of aTc will induce the best expression performance of biofilm. The possible reason for higher concentration of inducer strangely leading into low production of biofilm might lie in that aTc, a kind of antibiotic, can be harmful to protein synthesis in bacteria. We speculate there’s an antagonism between the effect of promoting expression and impeding growth brought by aTc and 0.25 μg ml-1 of aTc just reach the optimal point.<p></p>

<ul>

<li> Alan MarcusEvita Sadimin, Maurice Richardson, Lauri Goodell,and Billie Fyfe. (2012). Fluorescence Microscopy Is Superior to Polarized Microscopy for Detecting Amyloid Deposits in Congo Red–Stained Trephine Bone Marrow Biopsy Specimens. Am J Clin Pathol. </li>

<li> Bijan Zakeria, J. O.-L. (2012, February 24). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. PNAS. </li>

<li>Neel S. Joshi, P. Q. (2014, September 17). Programmable biofilm-based materials from engineered curli nanofibres. nature communications. </li>

<li> Soares, M. J. (n.d.). Crystal Violet Assay. Retrieved from KU MEDICAL CENTER: http://www2.kumc.edu/soalab/LabLinks/protocols/cvassay.htm</li>

<li>Zsofia Botyanszki, 1. P. (2015, May 20). Engineered Catalytic Biofilms: Site-Specific Enzyme Immobilization onto E. coli Curli Nanofibers. Biotechnology and Bioengineering. </li>

<li>Puchtler, H. S. (1962). On the binding of Congo red by amyloid. Cytochem. </li>

</ul>