Natural Transformation and GFP Expression in the Filamentous Cyanobacterium Phormidium lacuna

Abstract

Source: Weber, N., et al., Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna. J. Vis. Exp. (2022)

This video describes a procedure for performing natural transformation in filamentous cyanobacteria using plasmid DNA to achieve stable GFP expression and antibiotic resistance through homologous recombination.

Protocol

1. Natural transformation and GFP expression

NOTE: Transformation is based on a plasmid vector propagated in Escherichia coli (E. coli); pGEM-T or pUC19 may be used as backbone vectors. Examples for vectors for superfolder green fluorescent protein (sfGFP) expression are described in the representative results section.

1. Perform all steps using sterile material under sterile laboratory conditions (clean bench, sterile glassware).
2. Inoculate 2 x 50 mL of f/2 liquid medium in two 250 mL flasks with 2 x 1 mL of Phormidium lacuna (P. lacuna) filaments from a running culture. Cultivate....

Results

Figure 1. Phormidium lacuna filaments 5 weeks aft.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacto Agar	OttoNorwald	214010
Fluorescence microscope ApoTome	Zeiss
Fluorescence microscope Axio Imager 2	Zeiss
Kanamycin	Sigma-Aldrich	25389-94-0
Microscope DM750	Zeiss
Midi prep plasmid extraction kit NucleoBond Xtra Midi kit	Macherey-NAGEL GmbH & Co. KG	REF740410.50
Light source, fluorescent tube L18W/954 daylight	OSRAM		Cultivation of cyanobacteria
Light source, LED panel XL 6500K 140 W	Bloom Star	N/A	Cultivation of cyanobacteria
Petri dishes polystyrole, 100 mm x 20 mm	Merck	P5606-400EA
Petri dishes polystyrole, 60 mm x 15 mm	Merck	P5481-500EA
Pipetman 100-1,000 µL	Gilson	SKU: FA10006M
Pipetman 10-100 µL	Gilson	SKU: FA10004M
Plastic pipettes 10 mL, sterile	Greiner	607107
Shaker Unimax 2010	Heidolph Instruments		For cultivation
Plastic tube, sterile, 50 mL	Greiner	227261
Ultraturrax Silent Crusher M	Heidolph Instruments		Homogenizer
F/2+ liquid medium	f/2-medium, with 10 times increased NaNO₃ and NaH₂PO₄ (0.88 mM NaNO₃, 36 µM NaH₂PO₄
F/2+-agar	3 % (w/v) bacto agar, artificial seawater, 0.1 % (v/v) trace element solution, 0.05 % (v/v) vitamin solution ,8.8 mM NaNO₃, 0.36 mM NaH₂PO₄