Team:ShanghaitechChina/Description
Improve the characterization
>Contribution:
- Biobrick: BBa_K1583003
- Group: ShanghaitechChina
- Author: Lechen Qian, Shijie Gu
- Summary: We created new way to characterize this biobrick which was originally designed and characterized by iGEM15_TU_Delft. We utilize NTA-Metal-Histag coordination chemistry and fluorescence emission traits of Quantum Dots (QDs) in our project to improve the characterization. We demonstrated the validity of the approach for measurement of biofilm composed by CsgA-His density of E. coli curli system and think highly of this characterization for its general application in other biofilm systems. Also, we utilized TEM to help us scrutinize the binding effect in microscopic world.
>Improvement:
Quantum dots binding test
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 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 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.
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 followed by the same procedure mentioned above. After 1h incubation, we used PBS washing 2 times. The picture verify our postulation: On the left, CsgA-Histag mutant were induced and its biofilm bind with QDS. CsgA biofilm without Histag cannot bind with QDs thus its red fluorescence is much weaker.
CdS nanorods Templating
As for biofilm characterization, transmission electron microscopy is frequently to be used to visualize the nanofiber network. However, TEM is not very efficient to visualize soft matter due to the less dense of elections produced on soft matter even after negative staining. Amazingly, after incubation with CdS nanorods , the biofilm areas are densely templated by better conductive materials such as CdS nanorods and we can easily confirm the expression of biofilm.
