Long-Term Cryopreservation of Cyanobacterial Strains Using Glycerol or Dimethyl Sulfoxide

Abstract

Source: Lea-Smith, D. J., et al. Generation of Marked and Markerless Mutants in Model Cyanobacterial Species. J. Vis. Exp. (2016)

This video demonstrates the cryopreservation of cyanobacterial strains by combining concentrated cell suspensions with glycerol or DMSO and storing them at low temperatures, enabling long-term maintenance of viable cultures for photosynthesis and genetic studies.

Protocol

1. Preparation of Culture Media

1. Prepare BG11 medium
   1. Prepare stock solutions of 100x BG11, trace elements, and iron stock (Table 1).
   2. Prepare separate solutions of phosphate stock, sodium carbonate (Na₂CO₃) stock N-[Tris(hydroxymethyl)methyl] -2-aminoethanesulfonic acid (TES) buffer, and sodium bicarbonate (NaHCO₃) (Table 1).
   3. Autoclave the phosphate and Na₂CO₃ stocks. Filter-sterilize TES buffer and NaHCO₃ with 0.2 µm filters.
   4. Prepare BG11 by combining 976 ml of water, 10 ml of 100x BG11, 1 ml of trace elements, and 1 ml of iron stock, and autoclave the solution. After this solution has cooled to room temperature, add 1 ml of phosphate stock, 1 ml of Na₂CO₃ stock, and 10 ml of NaHCO₃.
   5. For BG11 solid medium, add 15 g of agar and 700 ml of water to one flask. To the second flask, add 3 g of sodium thiosulfate (Na₂S₂O₃), 226 ml of water, 10 ml of 100x BG11, 1 ml of trace elements, and 1 ml of iron stock. Autoclave both solutions. After these solutions have cooled to room temperature, combine them and add 1 ml of phosphate stock, 1 ml of Na₂CO₃ stock, 10 ml of TES buffer, and 10 ml of NaHCO₃.
      NOTE: Solutions are prepared separately to avoid precipitation of certain salts.
2. For selection on sucrose, prepare a 50% (w/v) sucrose solution. Filter sterilize the solution with 0.2 µm filters and add to BG11 (100 ml of 50% sucrose to 900 ml of BG11) to produce BG11/5% sucrose plates.
   NOTE: Do not add NaHCO₃ to BG11/5% sucrose agar plates. Add Na₂CO₃ as normal.
3. For culturing of Synechococcus, add 10 ml of 1 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) and 1 ml of vitamin B12 (Table 1) to 1 L of BG11 medium.
   NOTE: Transformation of strains cultured in commercially available BG11 media is significantly less efficient than in the BG11 media recipes described here and therefore is not recommended.

2. Long-term Storage of Strains

1. Set up a fresh culture of the strain by inoculating a loop full of cells into 30-50 ml of BG11 medium. Grow the culture for 3-4 days to OD_750nm = 0.4 to 0.7.
2. Wash cells once with BG11 and resuspend in ~2 ml of BG11.
3. Add 0.8 ml of concentrated cells to one tube. Then add 0.2 ml of 80% filter-sterilized glycerol.
4. Optional: Add 0.93 ml of concentrated cells to another tube. Add 0.07 ml of DMSO to this tube.
   CAUTION: DMSO is toxic and should be handled with appropriate protection.
5. Store both tubes at -80 °C. To revive strains, remove the tube and scrape off some cells with a blunt toothpick onto an agar plate without antibiotics. Streak out as normal using a sterile loop.

Table 1: Solutions used in this study

Stock solution recipes
Chemical	Amount (g)
100x BG11 (per L)
NaNO3	149.6
MgSO4.7H2O	7.49
CaCl2.2H2O	3.6
Citric acid	0.6
Add 1.12 ml 0.25 M Na2EDTA, pH 8.0
0.25 M Na2EDTA, pH 8.0 (per 100 ml)
Na2EDTA	9.3
Trace elements (per 100 ml)
H3BO3	0.286
MnCl2.4H2O	0.181
ZnSO4.7H2O	0.022
Na2MoO4.2H2O	0.039
CuSO4.5H2O	0.008
Co(NO3)2.6H2O	0.005
Iron stock (per 100 ml)
Ferric ammonium citrate	1.11
Phosphate stock (per 100 ml)
K2HPO4	3.05
Na2CO3 stock (per 100 ml)
Na2CO3	2
TES buffer, pH 8.2 (per 100 ml)
TES	22.9
NaHCO3 stock (per 100 ml)
NaHCO3	8.4
HEPES, pH 8.2 (per 500 ml)
HEPES	119.15
Vitamin B12 (Per 50 ml)
Cyanocobalamin	0.02
Luria Bertani media (Per 500 ml)
Luria Bertani broth	12.5
1 M MgCl2 (Per 100 ml)
MgCl2.6H2O	20.33
Solution A (Per 200 ml)
MnCl2.4H2O	0.395
CaCl2.2H2O	1.47
2-(N-Morpholino)ethanesulfonic acid hydrate, 4-Morpholineethanesulfonic acid (MES)	0.4265
Solution A + glycerol
10 ml solution A
1.5 ml glycerol

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaNO₃	Sigma	S5506
MgSO₄·7H₂O	Sigma	230391
CaCl₂	Sigma	C1016
Citric acid	Sigma	C0759
Na₂EDTA	Fisher	EDT002
H₃BO₃	Sigma	339067
MnCl₂·4H₂O	Sigma	M3634
ZnSO₄·7H₂O	Sigma	Z4750
Na₂MoO₄·2H₂O	Sigma	331058
CuSO₄·5H₂O	Sigma	209198
Co(NO₃)₂·6H₂O	Sigma	239267
Ferric ammonium citrate	Sigma	F5879
K₂HPO	Sigma	P3786
Na₂CO₃	Fisher	SODC001
TES	Sigma	T1375
NaHCO₃	Fisher	SODH001
HEPES	Sigma	H3375
Cyanocobalamin	Sigma	47869
Na₂S₂O₃	Sigma	72049
Bacto agar	BD	214010
Sucrose	Fisher	SUC001
Petri dish 90 mm triple vented	Greiner	633185
0.2 µm filters	Sartorius	16534
100 ml conical flasks	Pyrex	CON004
Parafilm M 100 mm x 38 m	Bemis	FIL003
Phusion high fidelity DNA polymerase	Phusion	F-530
Agarose	Melford	MB1200
DNA purification kit	MoBio	12100-300
Restriction endonucleases	NEB
T4 ligase	Thermo Scientific	EL0011
Luria Bertani broth	Invitrogen	12795-027
MES	Sigma	M8250
Kanamycin sulfate	Sigma	60615
Ampicillin	Sigma	A9518
GeneJET plasmid miniprep kit	Thermo Scientific	K0503
14 ml round-bottom tube	BD falcon	352059
GoTaq G2 Flexi DNA polymerase	Promega	M7805
425-600 µm glass beads	Sigma	G8772
Glycerol	Sigma	G5516
DMSO	Sigma	D8418
Fluorescent bulbs	Gro-Lux	69
HT multitron photobioreactor	Infors