Chitosan Flocculation

Chitosan is a linear polymer composed of randomly distributed β-(1→4)-linked D-glucosamine and N-acetyl-D-glucosamine subunits. The amine group in chitosan has a pKa of 6.5 leading to a protonation at slightly acidic pH and making it a bioadhesive which readily binds to negatively charged surfaces. We speculated that the use of chitosan might help in flocculation of these vesicles knowing that their membranes are highly negatively charged.

Biotin-Streptavidin Interaction

NHS-Biotin is a biotinylating reagent that reacts with primary amines (eg. lysines) in a peptide chain attaching a small spacer separated biotin moiety to them. The presence of a tetravalent biotin binding molecule (avidin/streptavidin) made us propose a biotin-streptavidin mediated strategy for increasing the effective hydrodynamic radius of these vesicles. It is interesting to note that the hydrophobicity of the internally exposed part of GvpA is what keeps the gases from diffusing out and leads to the formation of the gas filled cavity.

The tertravalent nature of Streptavidin was key for its selection for aggreegation of gas vesicles. The binding of Streptavidin to biotin is one of the strongest covalent bond interactions known in nature. Avidin is another most notable biotin binding protein. It even has a higher affinity for biotin, but streptavidin is a better biotin-conjugate binder. That is, streptavidin has higher affinity to biotin when the biotin is conjugated to another molecule, which is the case we deal with. Also, significant non-specefic binding can be prevented by using streptavidin.

SpyCatcher-SpyTag Binding

Anabaena flos-aquae

SEM

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.

DLS

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 for chitosan flocculation can be accessed here.

To assay biotin-streptavidin aggregation, the gas vesicles were centrifuged at 500rpm overnight (9 hours) and the top layer was resuspended in 1mL PBS. This was done to remove the excess biotin that might bind with the streptavidin. Different concentrations of Streptavidin were then added to these suspensions and they were analysed using a DLS machine

The data for biotin-streptavidin aggregation is given 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,

where d_H is the hydrodynamic diameter and D the translation diffusion coefficient.

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.

The data from the spectrophotometer assays for chitosan can be found here.

An analysis of the data is given in the results section

Verification of presence of Gas Vesicles

The easiest way to assay presence of gas vesicles is their disappearance under high pressure under a microscope. This was observed even during normal experiments. Fully filled micro-centrifuge tubes containing dilute gas vesicle suspensions lost their faint opalescence when the tube was closed (this did lead to a loss of samples). A more strict assay was done using DLS (See Dynamic Light Scattering) and SEM Imaging to pinpoint the exact size of the nano-particles. It was found that these gas vesicles have an effective hydrodynamic radius of around 230nm. This estimate was particularly valuable in the development of our model.

Typical H. salinarum gas vesicles measure 300 nm in length and around 200 nm in diameter. A. flos-aquae vesicles are slightly larger in size but the culturing conditions required for the algae make them harder to extract. All the gas vesicles used in our flocculation experiments were extracted from H. Salinarum and were stripped of GvpC by using 6M urea lysis method. It becomes necessary for us to show that such a particle at room temperature is not a very potent floater and the steady state distribution is not good enough to allow considerable separation between the solution and the protein phase.(See "Analytical solution at steady state")