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Biofilms function as a platform to sustain the whole system in vitro. Biofilm-anchored nanorods can efficiently convert photons to electrons, which transfer to engineered strain producing FeFe hydrogenase gene cluster, thereby achieving high-efficiency in biohydrogen production. In addition, a brilliant traits, the intrinsic adherence of biofilms towards various interfaces, allows us to grow biofilms on easy-separation micro-beads. Based on those merits, biofilm stand out by facilitating recyclable usage of the biofilm-anchored NRs and endowing this whole system with recyclability. <p></p>

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However, we view it through different lenses to transform this ill impact into merits.  We envision to establish the Solar Hunter system on E.Coli’s biofilm. Biofilm can substantially increase the resistance of bacteria to adverse conditions like acid or oxidative stress and form a stable and balanced system. These traits make them easily grow with low cost and elevate bacteria’s adaptability. So it will be a good practice to applied to industry environment.  

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What’s more, biofilm can automatically assemble extracellularly 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 without losing biological activity of peptide and self-assemble function of CsgA. 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[1].

 

<img src=" https://static.igem.org/mediawiki/parts/e/e3/Shanghaitechchina_biofilm1.png" width="60%">

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               For the reasons above, biofilms become our best candidate to engineer and would be equipped with some additional functions we want. Here, we conceive the semiconductor-enzyme system linked to the E.Coli’s biofilm, whose subunits are engineered respectively with PolyHistidine tags and SpyTag and SpyCatcher system from FbaB protein to provide binding sites for inorganic nanomaterials and enzymes. <p></p>

<h4><b>Plan 1. Strains secrected CsgA-his or His-CsgA-SpyCatcher-(Histag) biofilms for binding nanorods + Strain producing hydrogenase HydA</b></h4>

<h4><b>device: CsgA-Histag/His-CsgA-SpyCatcher-(Histag)</b></h4>

We learned that inorganic nanomaterials can template on biofilm by utilizing Co/Ni-NTA- -Histag coordination bond. Therefore, we want leverage synthetic biological engineering to program CsgA biofilm from E.coli with Histag to endow its capability to bind with nanomaterials (i.e. quantum dots, nanorods), form a bio-abiotic interface platform and produce electrons. Through this approach, we want to realize producing hydrogen by attach nanorods onto biofilms. The electrons from nanorods excited by sunlight can transfer into engineered hydrogenase-producing strain through mediator solution and accepted by hydrogenases which are not secreted. Since anaerobic hydrogenase will not be exposed to oxygen directly in this way, we view it as a practical and promising way to conduct in lab and consequently realize biohydrogen. <p></p>

<img src="https://static.igem.org/mediawiki/parts/7/78/Shanghaitechchina_plan_1_biofilm.png" width="55%">

<h4><b>device: His-CsgA-SpyCatcher-(Histag)</b></h4>

<img src="https://static.igem.org/mediawiki/parts/a/a5/Shanghaitechchina_plan_2_biofilm.png" width="55%">

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         We tested and proved that all the strains we constructed work well:<p></p>

1. Strains with engineered CsgA subunits :

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

3. Large protein SpyCatcher on CsgA subunits are also able to be secreted by transporter machinery and successfully form nanofibers. We also prove the biological function of SpyCatcher after appending on CsgA subunits, thus provide potential for our second plan mentioned above.

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        <h1 align="center">Construction and Characterization</h1>

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         <h4 class="bg"><b>Congo Red</b></h4>

Congo Red dye is a classic method to detect amyloid protein [2]. 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 [3]. 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>

<h4 class="bg"><b>Crystal Violet Assay</b></h4>

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

 

        <h4 class="bg"><b>TEM</b></h4>

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%">

<p style="text-align:center"><b>Fig 2. </b> TEM device at the National Center for Protein Science Shanghai.</p>

        <h4 class="bg"><b>Quantum Dots Binding Assay</b></h4>

Mechanisms of Quantum dots binding assay have been introduced in detail in Quantum Dots part. We utilizing Co/Ni-NTA-Metal-Histag coordination chemistry and fluorescence emission traits of Quantum Dots (QDs) to bind with the histidine in Histags on our biofilm and thus characterize its formation. The whole linkage is performed by forming firm coordinate bonds. They could be applied to quick detection of biofilm expression of 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). <p></p>

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

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>

<img src="https://static.igem.org/mediawiki/parts/f/f2/Shanghaitechchina_Histag%2BQDs.png" style="width:40%;">

<p style="text-align:center"><b>Fig 5.</b> Fluorescence test of CsgA-His binding with nanomaterials</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>

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<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.

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Finally, transmission electron microscopy(TEM) visualize the microscopic binding effect of CsgA-Histag fused biofilm with CdS nanorods in comparison with image of pure nanofiber composed by CsgA-Histag and one without inducer. From the first picture, it shows biofilm areas are densely covered by CdS nanorods. As can be clearly seen from the second figure, with inducer, there’s distinct nanofibers outside the bacteria contrast to the third picture in which E.coli are not induced. Thus we ultimately confirm the viability of bio-abiotic hybrid system.<p></p>

<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>

<b>PARTS：<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2132001">BBa_K2132001</a></b><p></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>

In constructing the sequence, we simply used Gibson Assembly to assemble the clips of CsgA, SpyCatcher, Histag and the plasmid backbone together at one single reaction. For more details and the experiment data, please download the pdf here(此处设置超链接).<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">

 

 

<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%;">

<p style="text-align:center"><b>Fig 9.</b> Congo Red Assay of His-CsgA-SpyCatcher-Histag</p>

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<b class="bg">His-CsgA-SpyCatcher-Histag</b><p></p>

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>

<b  class="tc">His-CsgA-SpyCatcher</b><p></p>

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

 

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         <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>

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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%;">

Crystal Violet Assay of the peptide fusion mutants library transformed into ΔCsgA strains. Firstly, we apply 0.1% crystal violet solution to all mutants to gain quantitative data about their relative expression and secretion performance. The result was read and exported by BioTek CYTATION5. CsgA-His mutant stands out as the highest peak in absorbance after washing. (See protocal ) The reason why His-CsgA-SpyCatcher-Histag and His-CsgA-SpyCatcher mutant doesn’t perform as well as CsgA-His might be ascribed to size hindrance of SpyCatcher, which impedes the transport process of CsgA mutant subunits from inner area to extracellular environment by CsgG outermenbrane exporter, whose pore size is around 2 nm. Yet, the differences between induced CsgA-His, His-CsgA-SpyCatcher-Histag, His-CsgA-SpyCatcher mutant and negative control exhibit a success extracellular biofilm production in all our constructed and modified strains. <p></p>

Nanoparticle binding assay of all constructs library. CdSeS/ZnS core/shell QDs which emit red fluorescence under 365nm UV light are templated by CsgA-His mutant incubated in M63 solution. We added equivalent amount of QDs solution into M63 medium with mutant E.coli which have been cultured for approximately 72h and all mutants were induced by aTc. After 1h incubation, we apply PBS washing 3 times to wash away the unbinding quantum dots. Pictures demonstrate CsgA-His were produced by three mutants we constructed and QDs are templated on biofilms .<p></p>

 

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[5] Z. Botyanszki, P. K. R. Tay, P. Q. Nguyen, M. G. Nussbaumer, N. S. Joshi, Engineered catalytic biofilms: Site‐specific enzyme immobilization onto E. coli curli nanofibers. Biotechnology and bioengineering 112, 2016-2024 (2015).<p></p>

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