<h2>b. To allow easy recycling of precious semiconductor nanomaterials, we utilized engineered biofilms to anchor nanomaterials via metal coordination chemistry. Please refer to <a href="https://2016.igem.org/Team:ShanghaitechChina/Biofilm"><b>Biofilm Session</b></a> for details of the successful construction and characterization of engineered biofilms that allow firm binding of nanomaterials. Key data are reproduced below. </h2><p></p></div>  

 

<h2> First, the simplest design, CsgA-HisTag.</h2>

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>

<p style="text-align:center">

<b>Fig 6.</b>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.

</p>

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.<p></p>

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

</center>

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

 

<h2> 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. </h2>

 

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. <p></p>