Team:IISc-Bangalore/Experiments
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SEM
DLS
Chitosan Flocculation
Biotin-Streptavidin Interaction
SpyCatcher-SpyTag Binding
Experimental Data
Visual Analysis
Flotation Spectrophotometry
Chitosan
Double replicates of four different concentrations of chitosan were used with gas vesicles (30ul stock) and the resulting solutions were diluted to 2ml to perform a flotation spectrophotometry assay.
| Tube Label | Effective gas vesicle concentration (ng/μl) |
Effective chitosan concentration (ng/μl) |
Remarks |
|---|---|---|---|
| 1 | 15 | 0 | Control tube |
| 2A | 15 | 5 | First replicate |
| 2B | 15 | 5 | Second replicate |
| 3A | 15 | 50 | First replicate |
| 3B | 15 | 50 | Second replicate |
| 4A | 15 | 500 | First replicate |
| 4B | 15 | 500 | Second replicate |
| 5A | 15 | 5000 | First replicate |
| 5B | 15 | 5000 | Second replicate |
The data from the spectrophotometer assays for chitosan can be found here.
An analysis of the data is given in the results section
Electron microscopy
Multiple dilutions of pure gas vesicles suspended in PBS were imaged under a Scanning Electron Microscope after applying a 10nm gold sputter. In the images, gas vesicles can be seen as translucent polygon shaped particles. Note that some lysed gas vesicle membranes are also seen in the image owing to the drying step during the sample preparation that precedes electron microscopy. Air drying can be carried out over a longer period of time to reduce the number of such events. Three dilutions were prepared for microscopy, out of these the 0.01ug/ul samples gave the best results.
Dynamic Light Scattering
Gas vesicle suspensions prepared as in the spectrophotometry assay were used to perform Dynamic light scattering. Three replicates of each concentration were run through the machine thrice. It was noted that the average particle size decreased after every run indicating the particles were either sedimenting or floating up.
The data can be accessed here.
The theory behind dynamic light scattering becomes quite simple if the implications of Einstein's brownian motion hypothesis are well known. Smaller particles tend to get a stronger "kick" when a solvent particle hits them. What the system actually detects are the correlations that persist in the scattered intensities at consequent time intervals. A large correlation implies that the particle hasn't moved much in the interval and hence is larger.
The actual values obtained from the system are those of the translation diffusion coefficient, to which the software applies the famous Einstein relation (see Mathematical model) giving the hydrodynamic diameter, \[ d_{H}=\frac{kT}{3 \pi \eta D} \]where dH is the hydrodynamic diameter and D the translation diffusion coefficient.
