In order to test the effect of binding between CsgA-Histag mutant and inorganic nanoparticles, 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 and 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. 7：Binding test between CsgA-his and Quantum dots. The image was snaped by ChemiDoc MP,BioRad, false colored.
Comparison test of Quantum dots Binding between CsgA-his and CsgA
In order to prove the effect of binding between CsgA-Histag mutant and inorganic nanoparticles is distinct, we apply same amount of suspended CdSeS/ZnS QDs solution into M63 medium which has cultured biofilm for 72h. After 1h incubation, we used PBS washing 2 times. The picture verify out postulation: On the left, CsgA-Histag mutant were induced and its biofilm bind with QDS. CsgA biofilm cannot bind with QDs thus its red fluorescence is a lot weaker.
Fig. 8：Comparison test of Quantum dots Binding between CsgA-his and CsgA.
CdS nanorods Templating
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. Amazingly, after incubation with CdS nanorods , the biofilm areas are densely templated by CdS nanorods and we can easily confirm the expression of biofilm.
Fig. 9：Representative TEM images of biotemplated CdS nanorods on CsgA-His. After applied inducer, CsgA-His mutant constructed and expressed to form biofilm composed by CsgA-His subunits. Incubation with nanorods for 1h, nanomaterials are densely attached to biofilm.
2.Parts Collection Two (Hydrogenase gene clusters)
We utilize [FeFe] Hydrogenases originally from the bacterium Clostridium acetobutylicum (coding sequence: hydA, BBa_K2132004 & BBa_K2132005) to accept electrons and therefor enable catalytic production of hydrogen in our project. Synthesis of heterologous [FeFe] hydrogenase in E. coli requires co-expression of HydE (coding sequence: hydE, BBa_K2132006), HydF (coding sequence: hydF, BBa_K2132007), and HydG (coding sequence: hydG, BBa_K2132008).
In this collection, we sub-cloned the coding sequence into the pSB1C3 individually, with two appended tags at the N-terminus (His-tag to faciliate purification and TEV site as cleavable site for Histag cutting off). We have confirmed that the presence of the two tags won’t disrupt expression and normal functionalites of HydA.
➤ HydA with SpyCatcher, Histag and TEV site (E. coli) - BBa_K2132004
➤ HydA with SpyTag, Histag and TEV site (E. coli) - BBa_K2132005
➤ HydE with Histag and TEV site (E. coli) - BBa_K2132006
➤ HydF with Histag and TEV site (E. coli) - BBa_K2132007
➤ HydG with Histag and TEV site (E. coli) - BBa_K2132008
★Optimization of this collection
The original sequences of hydrogenase were found in www.genome.jp. With the help of OptimumGene™, We used the following parameters to optimize our gene sequences without changing their amino acids sequence: Codon usage bias, GC content, CpG dinucleotides content, mRNA secondary structure, Cryptic splicing sites, Premature PolyA sites, Internal chi sites and ribosomal binding sites, Negative CpG islands, RNA instability motif (ARE), Repeat sequences (direct repeat, reverse repeat, and Dyad repeat).
