TITLE: Cyanobacteria Yolk-Shell Preparation
AUTHORS: Jorge  Fernández Méndez, Celia Martin Morales

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Cyanobacteria as well as other photosynthetic microorganisms can be encapsulated within biohybrid nanomaterials. Cyanobacterial Yolk-shell are biohybrid nanostructures with a size of 1 to 6 µm, where the cell is encapsulated within a non-contacting silica nanocapsule. Yolk-shell encapsulation provides long-term cell viability, higher resistance against harsh environments and superior photosynthetic activiy.  In addition, engineering the colloidal packing allows tunable  shell-pore diameter for size-dependent permeability and introduction of  new functionalities for specific molecular recognition. </div><br/><div class = "text-block">Yolk-Shell structures can be easily synthetized via self-assebly of colloidal suspensions of silica nanoparticles as described in this protocol</div><br/><br/></div>

[STEPS]
?. [Cells Pretreatment]
In sterility, centrifugue a suffcient  ammount of exponentially growing cyanobacteria culture (20 to 100 mL) . OD720 should be between 0.5 to 1. It is recommended to split the culture in two different sterile centrifugue tubes.
Centrifuge: 2800 33, 10 min
?. [Cells Pretreatment]
In sterility,  discard the supernatant and add  1/10th of the initial volume with sterile PBS buffer.  Resuspend the cell pellet gently, gently flicking the tube or slowly pipetting up and down with a wide open pipette tip.
?. [Cells Pretreatment]
Take  sample from the resuspended cells and measure the Optical Density at 720 nm using a Spectrophotometer. OD720 should be 1.3. If OD720 is greater than 1.3 ,  and add more sterile PBS till reaching the required  OD720 value.
1000 µl
?. [Cells Pretreatment]
In Sterility, add 1/50 part (v/v) of the available resuspended cells volume of 5% protamine sulfate in sterile PBS. It is recommended to flick the tube up and down a couple of times and incubate the cells with protamine sulfate for  . (No agitation required)
?. [Cells Pretreatment]
Centrifugue the cells and discard the supernatant.IMPORTANT! Write the final volume of cell suspension in the tube before discarding the supernatant.
Centrifuge: 2800 33, 10 min
?. [Cells Pretreatment]
After centrifugation, resuspend the cell pellet in the same volume of cell suspension formerly used. Final OD720 should still be around 1.3
?. [Yolk-Shell Self Assembly]
Add the necessary ammount of Ludox(R) TMA colloidal suspension to reach a final concentration of 1 mg/mL in the cell suspension.VLudox-TMA  = VLudox (μL) = (VcellSuspension (mL)/418)* 1000NOTE. Ludox TMA has a ρ = 1.23 g/mL and it's a colloidal suspension at 34% wt , then each mL of the stock will carry 418 mg of silica nanoparticles. To know the required ammount of Ludox, just divide your cell suspension volume by 418.
?. [Yolk-Shell Self Assembly]
Incubate the suspension for with mild agitation using a rocker shaker at minimum speed.
?. [Storing Yolk-Shell]
After incubation, centrifugue the former suspension and discard the supernatant.  Wash the yolk-shell pellet with fresh BG-11 media using the same ammount of volume used during incubation (Other media could also be used for marine cyanobacteria strains).
Centrifuge: 3600 33, 4 min
?. [Storing Yolk-Shell]
Repeat the wash step , and resuspend the cells with fresh BG-11 media to a final OD720 = 1. In this conditions yolk shell can be stored at room temperature and mild illumination for long periods of time.