Oct 04, 2021

Public workspaceCyanobacteria Yolk-Shell Preparation V.2

DOI
dx.doi.org/10.17504/protocols.io.byr5pv86
  • 1iGEM MADRID_UCM 2021
Jorge Fernández Méndez
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DOI: dx.doi.org/10.17504/protocols.io.byr5pv86
Protocol Citation: Jorge Fernández Méndez, Celia Martin Morales 2021. Cyanobacteria Yolk-Shell Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.byr5pv86Version created by Jorge Fernández Méndez
Manuscript citation:
L. Wang et al., "Single-cell yolk-shell nanoencapsulation for long-term viability with size-dependent permeability and molecular recognition", National Science Review, vol. 8, no. 4, 2020. Available: 10.1093/nsr/nwaa097 [Accessed 26 September 2021].
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: October 04, 2021
Last Modified: October 04, 2021
Protocol Integer ID: 53789
Keywords: 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


Materials
  • Fresh Cyanobacterial Culture
  • ReagentLUDOX® TMA colloidal silicaSigma AldrichCatalog #420859
  • Protamine Sulfate (Biochemistry Grade)
  • PBS x1 Buffer pH = 7.4
  • BG-11 Media (HEPES 10 mM pH = 8)


Cells Pretreatment
Cells Pretreatment
Centrifigation2800 rpm, 00:10:00
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.
10m
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.
Take Amount1000 µL 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 , Go togo to step #2 and add more sterile PBS till reaching the required OD720 value.

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 Duration00:05:00 . (No agitation required)


5m

Centrifigation2800 rpm, 00:10:00 Centrifugue the cells and discard the supernatant.

IMPORTANT! Write the final volume of cell suspension in the tube before discarding the supernatant.

10m
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
Yolk-Shell Self Assembly
22m
22m
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 =

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 forDuration00:20:00 with mild agitation using a rocker shaker at minimum speed.

20m
Storing Yolk-Shell
Storing Yolk-Shell
10m
10m
Centrifigation3600 rpm, 00:04:00 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).

4m
Repeat the wash step Go togo to step #9 , 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.