Measurement

We established a new system to qualitatively and quantitatively measure protein expression level extracellularly by utilizing Co/Ni-NTA-Metal-Histag coordination chemistry and fluorescence emission traits of Quantum Dots (QDs) in our project. We demonstrated the validity of the approach for measurement of biofilm density of E. coli curli system (comprising CsgA-His and His-CsgA-SpyCatcher-Histag, respectively) and think highly of this characterization for its general application in other biofilm systems. More broadly, they could be applied to fast detection of protein expression of any His-tagged proteins with naked eye under UV light owing to the photoluminescence of QDs, and accurate concentration measurement under fluorescence spectrum (A detailed protocol for repeatable measurements is included in our Wikipage). The advantages include simple His-tags commonly used in bio-lab and convenient detection of nanomaterial fluorescence without worrying about secretion of fused proteins with large fluorescence proteins/domains.

Experimental Part

Quantum dots fluorescence test

No. 1 CsgA-Histag + CdSeS/CdSe/ZnS core/shell quantum dots

In order to have a fast detection of the biofilm formation and as well as test the binding effect between CsgA-Histag mutant and inorganic nanoparticles, we conducted the following experiments according to the same protocol (Details in our Protocol: Quantum Binding Assay). We apply same amount of suspended QDs solution into M63 medium which has cultured biofilm for 72h. After 30-min incubation, we used PBS to mildly wash the well, and the result was consistent with our anticipation: On the left, CsgA-Histag mutant were induced and thus secreted biofilm, and firmly attached with QDS, showing bright fluorescence. Therefore, we ensure the secretion and formation of biofilm expressed by engineered strains and prove the success of parts construction. The picture was snapped by ChemiDoc MP, BioRad, false colored.

**Fig 1.** Fluorescence test of CsgA-Histag binding with QDs

No. 2 His-CsgA-SpyCatcher-Histag + CdSeS/CdSe/ZnS core/shell quantum dots

When it comes to the strain secreted His-CsgA-SpyCatcher-Histag, we want to check if SpyCatcher protein will be too large to cause steric hindrance effect on Histag peptide. The best approach to verify Histag’s function is the fluorescence assay of binding with nanomaterials.

After applying the same steps as introduced above, the bottom of left well show a large area of bright fluorescence, manifesting His-CsgA-SpyCatcher-Histag mutant secreted biofilms under the control of inducer and Histag on it is not blocked. What is more, it is firmly attached with QDs after washing. From this assay, we are confident to introduce this binding test as a quick way to detect the formation of biofilm as well as examine the functional validation of Histags. The picture was snapped by ChemiDoc MP, BioRad, false colored.

**Fig 2.** Fluorescence test of His-CsgA-SpyCatcher-Histag binding with QDs

No. 3 His-CsgA-SpyCatcher + CdSeS/CdSe/ZnS core/shell quantum dots

Using the same approach, we also conducted binding assay of Histag-CsgA-SpyCatcher with QDs to characterize the expression of biofilm and the visual result shows vividly that Histag-CsgA-SpyCatcher can bind successfully with the QDs with the existence of inducer aTc. The picture was snapped by BioRad ChemiDoc MP, false colored.

**Fig 3.** Fluorescence test of His-CsgA-SpyCatcher binding with QDs

No. 4 A library of engineered CsgA mutants + CdSeS/ZnS core/shell quantum dots

We try an another kind of quantum dots to confirm that this binding assay can be generally applied to any nanoparticles with Ni-NTA ligand and all constructs library. Pictures below demonstrate Histags were produced by three mutants we constructed and QDs are successfully templated on biofilms.

**Fig 4.** Nanomaterial binding test. Images were shot by BioRad ChemiDoc MP, false colored

What’s more, the fluorescence can be viewed with naked eye under UV light owing to the photoluminescence of QDs. Here we demonstrated an amazingly beautiful picture taken by iPhone. Undoubtly, this approach of characterization shows itself of convenience. (BTW, it’s a real feast to eyes.)

**Fig 5.** Nanomaterial binding test. Images were shot by iPhone 5s under 365nm UV light, Tanon UV-100

Additional Part

TEM: visualization of binding test

As for biofilm characterization, transmission electron microscopy is frequently to be used to visualize the nanofiber network. However, we found it really difficult to find out whether biofilm is well self-assemble extracellularly due to its thin and inconspicuous attributes against the background. The following are examples which we cannot identify the presence of biofilm after induced for 72h.

Based on our own suffering experience in search of biofilm, we think highly the way of utilizing QDs to colocalize biofilm. After incubation with QDs, the biofilm areas are densely templated by QDs and we can easily confirm the expression of biofilm.

**Fig 7.** Representative TEM images of biotemplated CdSe quantum dots on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with QDs for 1h, nanomaterials are attached to biofilm.

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+<b>Fig 6. </b> Inconspicuous Biofilm.
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-<b>Fig 6. </b> Representative TEM images of biotemplated CdSe quantum dots on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with QDs for 1h, nanomaterials are attached to biofilm.
+<b>Fig 7. </b> Representative TEM images of biotemplated CdSe quantum dots on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with QDs for 1h, nanomaterials are attached to biofilm.
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