These protocols that we developed are important to study the secretion mechanisms in cyanobacteria and their released products, namely the extracellular carbohydrate polymers and proteins. The knowledge generated will allow us to customize these products for several applications, namely biotechnological and biomedical applications. These protocols are very straightforward.
They can be tailored according to the producing organism, the user needs, and the final application of the product. The main advantage of these protocols is that they can easily be adapted to other living systems, in particular to other bacteria. These protocols are easy.
However, one may need to introduce some changes depending on the bacterial strain or the degree of purity required. To begin, cultivate the cyanobacterial strain under standard conditions. Measure growth using standard protocols.
Next, transfer the culture into dialysis membranes, and dialyze against a minimum of 10 volumes of deionized water for 24 hours with continuous stirring. Centrifuge the culture at 15, 000 times g at four degrees Celsius for 15 minutes. Transfer the supernatant to a new vial, and discard the pellet.
Centrifuge again at 20, 000 times g at four degrees Celsius for 15 minutes to remove contaminants, such as cell wall debris or lipopolysaccharides. After the centrifugation, transfer the supernatant to a glass beaker, and discard the pellet. To precipitate the polymer, add two volumes of 96%ethanol to the supernatant, and incubate the suspension at four degrees Celsius overnight.
For small or not visible amounts of precipitated polymer, centrifuge the suspension at 13, 000 times g at four degrees Celsius for 25 minutes. Gently discard the supernatant, and resuspend the pellet in one to two milliliters of autoclaved deionized water. Transfer the aqueous suspension to a vial.
For visible, large amounts of precipitated polymer, collect the precipitated polymer with sterile metal forceps to a vial. Squeeze the polymer, and discard the excess ethanol. To perform the lyophilization process otherwise known as freeze-drying, keep the vials with the precipitated polymer at minus 80 degrees Celsius overnight.
Lyophilize the polymer for at least 48 hours. Then, store the dried polymer in a desiccator at room temperature until further use. To concentrate the medium, first cultivate cyanobacteria under standard conditions.
Monitor the cyanobacterium growth using standard procedures. Then, centrifuge the cultures at 4, 000 times g at room temperature for 10 minutes. Transfer the supernatant to a flask, and discard the cell pellet.
Next, filter the decanted medium through a 0.2-micron pore size filter. To concentrate the medium approximately 500 times, use centrifugal concentrators with a nominal molecular weight cut-off of three kilodaltons to centrifuge 4, 000 times g and 15 degrees Celsius for a maximum of one hour per centrifugation round. After centrifugation, rinse the walls of the filter device sample reservoir with the concentrated sample, and transfer the content to a microcentrifuge tube.
Perform an additional washing step of the filter device sample reservoir with the autoclaved culture medium to ensure maximal exoproteome recovery. Then, store the exoproteome samples at minus 20 degrees Celsius until further use. The ethanol must be added vigorously to optimize the polymer precipitation and yield.
After centrifuging the precipitated polymer, the discard of the supernatant should be made carefully in order to avoid resuspension of the pellet that may be loosely attached on the wall of flask. If manual filtration is performed, it should be gentle to avoid breaking the filter membrane. Some precipitated polymers can only be visible after centrifugation, mainly on the walls of the flasks, such as the case of EPS from Synechocystis.
In other cases, such as Cyanothece polymer, it is possible to see the polymer clumps floating in the glass beaker just after precipitation. Some contaminations can be easily detected macroscopically after polymer lyophilization since they will alter polymer pigmentation, usually white or light brown. Orange polymers are contaminated with carotenoids or structures containing these pigments.
For example, green polymers are contaminated with cell debris and chlorophyll. Exoproteomes can vary significantly depending on the bacterial strain. See, for example, the exoproteome from a unicellular cyanobacterium and the one from a filamentous strain.
The high amount of polysaccharides in exoproteome-concentrated samples can hinder the exoproteome analysis. For example, it can delay protein separation and mask less abundant proteins. The user can characterize the polymer in terms of composition, physical/chemical properties, test polymer biological activity, or modified polymer components.
For protein identification, separation of proteins in gel and mass spectrometry can be performed. And for separation of vesicles, ultracentrifugation of the exoproteome should be performed. In our group, these protocols have already paved the way to unravel cyanobacterial secretion mechanisms or applications of the extracellular carbohydrate polymers in different fields, such as in bioremediation or as antitumor or drug delivery agents.
