Biofilms are ubiquitous as they can be found both in human and some extreme environments. They can be formed on inert surfaces of devices and equipments, which will be hard to clean and cause dysfunction of the device.

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1) CsgA-Histag  2) His-CsgA-SpyCatcher-Histag  3) His-CsgA-SpyCatcher

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<img src=" https://static.igem.org/mediawiki/parts/6/68/Shanghaitechchina_dye.png " width="55%">

In order to visualize the formation and different appearance of biofilm nanowire network, we utilize transmission electron microscope to directly look into the microscopic world. TEM can visualize nano-structure with the maximal resolution of 0.2nm which is beyond the range of optical microscope.  

<img src=" https://static.igem.org/mediawiki/parts/2/26/Shanghaitechchina_TEM_device.png" width="55%">

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[1], containing 10 g/l of casmino acids, 1 g/l of yeast extract and 20 g/l of agar, supplemented with 34 μg /ml of chloromycetin,  5 μg/ ml of Congo Red and 5 μg/ ml of Brilliant Blue. (Details in protocol 链接) Red staining indicates amyloid production.<p></p>

<p style="text-align:center"><b>Fig 3.</b>Congo red assay of CsgA-Histag on YESCA plates</p>

The figures shown above point out that the CsgA-Histag mutant induced by 0.25 μg ml-1 of aTc produced amyloid structures which are dyed red by CR in comparison to the negative control after 72h culture at 30℃. This assay indicates the success in expression of the self-assembly curli fibers. <p></p>

<h4 ><b>2. Crystal Violet Assay： Quantification test of biofilm </b></h4>

Further, we use crystal violet assay to simply obtain quantitative information about the relative density of cells and biofilms adhering to multi-wells cluster dishes. As illustrated in pictures, CsgA-Histag mutant distinguishes itself in absorbance after applying standard crystal violet staining procedures (See protocal) in comparison to strain ΔCsgA and 30% acetic acid negative control. There’s certain amount of background absorption of strain ΔCsgA because the dye can stain the remaining E.coli adhering to the well. This difference between induced strains secreted CsgA-Histag and ΔCsgA manifest a distinct extracellular biofilm production in the modified strain. <p></p>

<p style="text-align:center"><b>Fig 4.</b>Crystal violet assay of CsgA-Histag.</p>

<h4 ><b>3.Quantum dots fluorescence test: successful binding test of Histag with nanomaterials (CdSeS/CdSe/ZnS core/shell quantum dots)</b></h4>

After confirming that our parts success in biofilm expression, we are going to test the effect of binding between CsgA-Histag mutant and inorganic nanoparticles. We apply suspended QDs solution into M63 medium which has cultured biofilm for 72h. After 1h 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 QDs are attached with biofilms, thus show bright fluorescence. Therefore, we ensure the stable coordinate bonds between CsgA-Histag mutant and QDs can manage to prevent QDs from being taken away by liquid flow. The picture was snapped by ChemiDoc MP,BioRad, false colored.<p></p>

Since biofilm nanofibers are thin and inconspicuous against the background, we harness CdSe QDs binding to highlight the biofilm area. The first image illustrates biofilm areas which are densely covered by QDs after induced for 72h and incubated, compared to the second image which is not incubated with nanoparticles CdSe. The third one is a negative control without inducer, bacteria scattered without forming biofilm<p></p>

<img src="https://static.igem.org/mediawiki/parts/f/f0/Shanghaitechchina_CsgAHistag%2BQD.png" style="width:100%;">

<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 densely attached to biofilm.

<img src="https://static.igem.org/mediawiki/parts/e/e1/Shanghaitechchina_CsgAHistag%2Bnanorods.png" style="width:100%;">

<p style="text-align:center"><b>Fig 7.</b>Representative TEM images of biotemplated  CdS nanorods on CsgA-His. </p>

In light of the immunization platform of biofilm for enzymes, we need some tags acting like glues or stickers that could be connected to the tags on the enzyme. The SpyCatcher and SpyTag system seem like a good choice for us. The SpyCatcher on the biofilm will mildly bind the SpyTag on the enzyme. Note that there is no the other way around, given that the huge size (138 amino acids) may impair the normal function of some delicate enzyme, hydrogenase in our case. For more details for the principles of SpyCatcher and SpyTag and our motivation on this system, see <a href="#p5">Linkage System</a>.  On top of the linkage to the enzyme, we would like to equip the biofilm the ability to bind nanorods and quantum dots. This goal makes the construction of His-CsgA-SpyCatcher-Histag or His-CsgA-SpyCatcher necessary. The two sequences are submitted as our first two original parts. See webpage of the parts here: <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132001">BBa_K2132001</a><p></p>

Since the sequence is actually a fusion protein, we identify each unit individually in characterization.<p></p>

<h4><b>1. Congo Red：Successful biofilm secretion and expression</b></h4>

After CR dye, the figure indicates that the His-CsgA-SpyCatcher-Histag mutant induced by 0.25 μg ml-1 of aTc and cultured for 72h at 30℃ 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>

<img src="https://static.igem.org/mediawiki/parts/0/05/Shanghaitechchina_HISCsgASpyCatcher_CR.png" style="width:60%;align:center">

After 72h culture, we scratched the biofilm down from the well and apply 25 μg/ ml 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>

<h4><b>2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials</b></h4>

<img src="https://static.igem.org/mediawiki/parts/c/c8/Shanghaitechchina_hisCsgASpyCatcherHistag_CR.png" style="width:100%;">

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 Histags on it is not blocked by SpyCatcher protein. What is more, it is firmly attached with inorganic materials (i.e.quantum dots) through ligand. From this assay, we assure that the SpyCatcher will not impose negative effect on the binding between nanomaterial and biofilm. The picture was snapped by ChemiDoc MP, BioRad, false colored.<p></p>

<img src="https://static.igem.org/mediawiki/parts/5/56/Shanghaitechchina_hisCsgASpyCatcherHistag%2BQD.png" style="width:60%;">

<p style="text-align:center"><b>Fig 10.</b> Quantum dots templating assay on His-CsgA-SpyCatcher-Histag biofilm.</p>

<img src="https://static.igem.org/mediawiki/parts/4/45/Shanghaitechchina_hisCsgASpyCatcher%2BQD.png" style="width:60%;">

TEM further characterize the biofilm expressed by strains secreted His-CsgA-SpyCatcher-Histag (HSCH) and His-CsgA-SpyCatcher (HSC) respectively. The distinct nanofiber network manifests the large biofilm expression.<p></p>

<img src="https://static.igem.org/mediawiki/parts/d/d5/Shanghaitechchina_hsch.png" style="width:60%;align:center">

<img src="https://static.igem.org/mediawiki/parts/e/ec/Shanghaitechchina_Au.png" style="width:60%;align:center">

<p style="text-align:center"><b>Fig 13.</b> After aTc induced, biofilm secreted by His-CsgA-SpyCatcher-Histag and His-CsgA-SpyCatcher mutants organize AuNP around the cells. In contrast with the third one without inducer, there’s nothing templating on the seemingly smooth outermembrane of bacteria.</p>

         <h1 align="center">Linkage System</h1>

As figure illustrated, his-CsgA-SpyCatcher-his mutant incubated with mcherry-SpyTag show a clear biofilm-associated mcherry fluorescence signal, which indicating the accurate conformation and function of the SpyTag and SpyCatcher linkage system. The third figure is merged by the first and second figures of each sample are snapped respectively under green laser field with 558 nm wavelength and bright field of fluorescence microscopy, Zeiss Axio Imager Z2. As for controls, strains secreted CsgA–histag and ΔCsgA both are unable to specifically attach to SpyTag thus no distinct localization highlight of red fluorescence on E.coli. That to a large extent prove the specificity of our desired linkage between SpyTag and SpyCatcher system. <p></p>

<p style="text-align:center"><b>Fig 14. </b>The first figures of each sample are snapped under green laser of 558 nm wavelength and mcherry-SpyTags emit red fluorescence. The second figures of each sample are snapped under bright field of fluorescence microscopy and we can clearly see a group of bacteria.. The third figures are merged by the first and second ones. All photos are taken by Zeiss Axio Imager Z2.</p>

We cultured all E.coli mutants in multi-wells with increasing inducer gradient. The result demonstrated in accordance that 0.25 μg/ ml 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 of aTc just reach the optimal point.<p></p>

<img src="https://static.igem.org/mediawiki/parts/2/23/Shanghaitechchina_inducer_concentration.png" style="width:100%;">

<a href="#p5">Linkage System</a>