Team:ShanghaitechChina/Proof
Detailed Proof
a. We, first, need to obtain a suitable nanomaterial, CdS, that absorbs solar energy and convert it into electrons. The detailed synthesis and characterization data is shown in our webpage Nanomaterials Session for further information. Some key data are reproduced below.
Synthesis and Characterization
Results
Synthesis and Characterization of CdS Nanorods
We synthesized CdS nanorods following a procedure adapted from a previously published protocol[1]. The synthesis procedure mainly contains two steps: synthesis of CdS seeds, followed by growth of CdS nanorods using CdS nanoparticles as nuclei.
Figure 3. Solutions of CdS nanoparticle seeds in TOP (left), CdS NRs in toluene (right)
Characterization of UV-Vis was performed to calculate the concentration of CdS seed in TOP solution and CdS nanorod in toluene solution. Also, PL spectrum of CdS NRs in toluene was collected to investigate the emission attribute of the nanorod. TEM image of the nanorods was acquired to study the shape and size distribution.
Figure 4. Result of CdS NRs ligand exchange experiments.
A ligand exchange experiment was performed and the result is shown in Figure 4
Figure 5. UV-Vis spectra of CdS seeds in TOP (A) and CdS nanorods in toluene (B);. Photoluminescence Spectrum of CdS nanorods in toluene (C).
The concentration of CdS seeds and CdS NR products were determined by using the UV-Vis spectrometer (Fig. 5 A, B). The peak shown in PL spectrum (Fig. 5 C) matches the absorption peak of the UV-Vis spectra, which thus proves the synthesis of CdS NRs.
Figure 6. TEM images of CdS NRs (A) and size distribution of CdS NRs (B). Note: 100 NRs in total were measured to determine the size distribution. The NRs thus measured have an average diameter of 3.93±0.57nm and average length of 66.81±6.74nm.
TEM confirms that synthesized products show nanorod feature, with an average diameter of 3.93±0.57nm and average length of 66.81±6.74nm.
b. To allow easy recycling of precious semiconductor nanomaterials, we utilized engineered biofilms to anchor nanomaterials via metal coordination chemistry. Please refer to Biofilm Session for details of the successful construction and characterization of engineered biofilms that allow firm binding of nanomaterials. Key data are reproduced below.
First, the simplest design, CsgA-HisTag.
3.Quantum dots fluorescence test: successful binding test of Histag with nanomaterials (CdSeS/CdSe/ZnS core/shell quantum dots)
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.
Fig 5. Fluorescence test of CsgA-His binding with nanomaterials
4. TEM: visualization of binding test
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
Fig 6.Representative TEM images of biotemplated CdS 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.
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 is 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.
Fig 7.Representative TEM images of biotemplated CdS nanorods on CsgA-His.
Second, The complex design with extra function of binding SpyTag-linked enzymes in addition to its nanomaterial-binding through HisTag. This is realized with our Part BBa_K2132001 under the promoter of tetO.
2. Quantum dots fluorescence test: successful binding test of Histag with nanomaterials
Then comes to the next part: we want to check if SpyCatcher protein will be too large to cause steric hindrance effect on Histag peptide. The best approach to verify is the fluorescence assay of binding with nanomaterials.
Fig 9. Congo Red Assay of His-CsgA-SpyCatcher
His-CsgA-SpyCatcher-Histag
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.
Fig 10. Quantum dots templating assay on His-CsgA-SpyCatcher-Histag biofilm.
3. TEM: visualization of binding test
TEM further characterize the biofilm expressed by strains secreted His-CsgA-SpyCatcher-Histag (HSCH). The distinct nanofiber network manifests the large biofilm expression.
Fig 12. aTc induced secretion of His-CsgA-SpyCatcher-Histag visualized by TEM. Without the presence of inducer, there’s no nanofiber formation around scattered bacteria.
CsgA-His can interface with different inorganic materials since they form the coordinate bonds with the same ligand, Co-NTA, on nanomaterials. Here we use to AuNPs in place of quantum dots and nanomaterials to characterize the validity of Histags on CsgA fused amyloid protein and meanwhile prove the versatility of our biofilm-based platform. As the figures shown, we confirm the feasibility of our newly constructed biobricks to template inorganic material and thus form bio-abiotic hybrid system.
Fig 13. After aTc induced, biofilm secreted by His-CsgA-SpyCatcher-Histag organizes AuNP around the cells. In contrast with the one without inducer, where nothing was on the smooth outermembrane of bacteria.
