Generation of Antibiotic-Marked Mutants in Cyanobacteria via Homologous Recombination

Abstract

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

The video demonstrates a method for generating antibiotic-marked mutants in cyanobacteria. The protocol begins with culturing cyanobacteria, followed by centrifugation and washing steps that preserve surface pili. A plasmid carrying an antibiotic resistance gene flanked by homologous sequences is then introduced. During incubation, the plasmid is internalized via pili and replaces the target gene through homologous recombination. Antibiotic selection and restreaking are used to isolate and purify the mutant strain.

Protocol

1. Preparation of Culture Media
   1. Prepare Blue-Green medium 11 (BG11 medium) according to Castenholz (1988).
      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....

Results

Figure 1: Plasmid construction for the generation of marked and unmarked knockouts, .......

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
K₂HPO₄	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
Glycerol	Sigma	G5516
DMSO	Sigma	D8418