Cyanobacteria Yolk-Shell Preparation
Jorge Fernández Méndez, Celia Martin Morales
Cyanobacteria
Encapsulation
Immobilization
Yolk-Shell
Nanomaterials
Biohybrids
Photosynthesis
4C_Fuels
iGEM
Abstract
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.
Yolk-Shell structures can be easily synthetized via self-assebly of colloidal suspensions of silica nanoparticles as described in this protocol
Steps
Cells Pretreatment
2800rpm
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.
In sterility, discard the supernatant and add 1/10th of the initial volume with sterile PBS buffer. th 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.
Take 1000µL sample from the resuspended cells and measure the Optical Density at 720 nm using a Spectrophotometer. OD720 should be 1.3 720 should be 1.3. If OD720 is greater than 1.3 720 is greater than 1.3 , and add more sterile PBS till reaching the required OD720 value. 720value.
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 0h 5m 0s . (No agitation required)
2800rpm Centrifugue the cells and discard the supernatant.
IMPORTANT! Write the final volume of cell suspension in the tube before discarding the supernatant.
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 = Ludox-TMA =
NOTE. 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.
Incubate the suspension for0h 20m 0s with mild agitation using a rocker shaker at minimum speed.
Storing Yolk-Shell
3600rpm 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).
Repeat the wash step , and resuspend the cells with fresh BG-11 media to a final OD720 = 1. 720 = 1.
In this conditions yolk shell can be stored at room temperature and mild illumination for long periods of time.
