Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Summary

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

Abstract

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Introduction

Cyanobacteria are prokaryotic organisms that utilize photosynthesis as an energy source^1,2. Research is increasingly focused on cyanobacterial species. Several cyanobacteria can be transformed with DNA³. Genes can be knocked out or overexpressed in these species. However, transformation is restricted to a few species^{4,5,6,7,8,9,10,}

Protocol

1. Isolation from the natural environment

NOTE: Green algae, diatoms, filamentous cyanobacteria, and other microalgae can be isolated. The protocol can be used for any microalga species from rockpools growing under laboratory conditions. Filamentous cyanobacteria that belong to Oscillatoriales can be easily recognized by their movement and filamentous shape. The species can be identified in a semipure state by genome sequencing or 16S rRNA sequencing.

1. Transfer liquid seawater samples from marine rockpools (i.e., cavities in the rocky coast) into 50 mL flasks. For each flask, note the exact place or coordinates o....

Results

Following the above-mentioned methods, 5 different strains of P. lacuna were isolated from rockpools and sequenced (Figure 1 and Table 1). All cultures were sterile after ~1 year of subculturing except P. lacuna HE10JO. This strain is still contaminated with Marivirga atlantica, a marine bacterium. During subsequent Helgoland excursions, other filamentous cyanobacteria were isolated from rock pools, which are different from P. lacuna and n.......

Discussion

Although many strains of cyanobacteria are available from culture collections^{32,33,34,35,36}, there is still a demand for new cyanobacteria from the wild because these species are adapted to specific properties. P. lacuna was collected from rockpools and is adapted to variations of salt concentrations and temperature³⁰. Strains .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Autoclave 3870 ELV	Tuttnauer	3870 ELV
Bacto Agar	OttoNorwald	214010
BG-11 Freshwater Solution	Sigma Aldrich	C3061
BG-11 medium	Merck	73816-250ML
Boric acid	Merck	10043-35-3	H3BO3
Calcium chloride dihydrate	Carl Roth	10035-04-8	CaCl2 · 2 H2O
Cell culture flasks Cellstar with filter screw cap, sterile, 250 mL	Greiner	658190
Cell culture flasks Cellstar with filter screw cap, sterile, 50 mL	Greiner	601975
Centrifuge LYNX 4000	Thermo Scientific	75006580	and rotor
Centrifuge microstar 17	VWR International	N/A	for up to 13,000 rpm
Cetyltrimethylammonium Bromide (CTAB)	PanReac AppliChem	57-09-0	C19H42BrN
Chloroform : Isoamyl Alcohol 24 : 1	PanReac AppliChem	A1935
Cobalt(II) chloride hexahydrate	Merck	7791-13-1	CoCl2 · 6 H2O
Copper(II) sulphate pentahydrate	Merck	7758-99-8	CuSO4 · 5 H2O
D(+)-Biotin	Carl Roth	58-85-5	C10H16N2O3S
DNA ladder 1 kb	New England Biolabs	N3232
DNA ladder 100 bp	New England Biolabs	N3231
Electrical pipetting help accujet-pro S	Brand GmbH	26360	for pipetting 1-25 mL
Ethanol	VWR	64-17-5	C2H6O
Ethylenediamine tetraacetic acid disodium salt dihydrate	Carl Roth	6381-92-6	EDTA-Na2 · 2 H2O
Fluorescence microscope ApoTome	Zeiss
Fluorescence microscope Axio Imager 2	Zeiss
French Pressure Cell Press	American Instrument Company	N/A
Gel documation System Saffe Image	Invitrogen
Gelelctrophoresis system Mupid-One/-exu	ADVANCED
Glassware, different
Glycerol	Carl Roth	56-81-5	C3H8O3
Iron(III) chloride hexahydrate	Merck	10025-77-1	FeCl3 · 6 H2O
Kanamycin	Sigma-Aldrich	25389-94-0
Kanamycin sulphate	Carl Roth	25389-94-0	C18H36N4O11 · H2SO4
Lauroylsarcosine, Sodium Salt (Sarcosyl)	Sigma Aldrich	137-16-6	C15H28NO3 · Na
LB Broth (Lennox)	Carl Roth	X964.4
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, up to 1,000 µmol m-2 s-1
Magnesium chloride hexahydrate	Carl Roth	7791-18-6	MgCl2 · 6 H2O
Manganese(II) chloride tetrahydrate	Serva	13446-34-9	MnCl2 · 4 H2O
Microscope DM750	Zeiss
Midi prep plasmid extraction kit NucleoBond Xtra Midi kit	Macherey-NAGEL GmbH & Co. KG	REF740410.50
Minicomputer Raspberry Pi 4 +	Conrad Electronics	2138863-YD	for time-lapse recording
Ocular camera EC3	Leica		for continuous recording up to 30 s
Ocular camera MikrOkular Full HD	Bresser		for time-lapse recordings, coupled to Raspberry Pi minicomputer
Petri dishes polystyrole, 100 mm x 20 mm	Merck	P5606-400EA
Petri dishes polystyrole, 60 mm x 15 mm	Merck	P5481-500EA
Photometer Nanodrop ND-1000	Peqlab Biotechnologie
Photometer Uvikon XS	Goebel Instrumentelle Analytik GmbH
Pipetman 100-1,000 µL	Gilson	SKU: FA10006M
Pipetman 10-100 µL	Gilson	SKU: FA10004M
Plastic pipettes 10 mL, sterile	Greiner	607107
Plastic tube, sterile, 15 mL	Greiner	188271
Plastic tube, sterile, 50 mL	Greiner	227261
Potassium bromide	Carl Roth	7758-02-3	KBr
Potassium chloride	Carl Roth	7447-40-7	KCl
Power supply Statron 3252-1	Statron Gerätetechnik GmbH
Power supply Voltcraft PPS 16005	Conrad Electronics		for LED
Proteinase K	Promega	MC500C	from Maxwell 16 miRNA Tissue Kit AS1470
Q5 polymerase	New England Biolabs	M0491S
Sequencing kit NextSeq 500/550 v2.5	Illumina
Sequencing system NextSeq 550 SY-415-1002	Illumina
Shaker Unimax 2010	Heidolph Instruments		for cultivation
Sodium acetate	Carl Roth	127-09-3	NaCH3COO
Sodium chloride	Carl Roth	7647-14-5	NaCl
Sodium dihydrogen phosphate monohydrate	Carl Roth	10049-21-5	NaH2PO4 · H2O
Sodium fluoride	Carl Roth	7681-49-4	NaF
Sodium hydrogen carbonate	Carl Roth	144-55-8	NaHCO3
Sodium molybdate dihydrate	Serva	10102-40-6	Na2MoO4 · 2 H2O
Sodium nitrate	Merck	7631-99-4	NaNO3
Sodium sulphate	Carl Roth	7757-82-6	Na2SO4
Strontium chloride hexahydrate	Carl Roth	10025-70-4	SrCl2 · 6 H2O
Thiamine hydrochloride	Merck	67-03-8	C12H17ClN4OS · HCl
TRIS	Carl Roth	77-86-1	C4H11NO3
Ultrasonic device UP100H with sonotrode MS3	Hielscher Ultrasound Technology	UP100H
Ultraturrax Silent Crusher M	Heidolph Instruments		homogenizer
Urea	Carl Roth	57-13-6	CH4N2O
Vitamin B12	Sigma	68-19-9	C63H88CoN14O14P
Vitamin solution			0.3 µM thiamin-HCl, 2.1 nM biotin, 0.37 nM cyanocobalamin
Water Stills, Water treatment	VEOLIA water technologies	ELGA_21001
Zinc sulphate heptahydrate	Sigma	7446-20-0	ZnSO4 · 7 H2O
software, URL
gatb-minia program for DNA assembly	https://github.com/GATB/gatb-minia-pipeline	makes large scaffolds from short DNA reads, Linux based
ImageJ		software for immage processing (pixel intensities, circle diameter)
RAST annotation server	https://rast.nmpdr.org	input: genome DNA sequence, detects open reading frames, lists protein sequences and their functions
Culture media
Artificial seawater	0.41 M NaCl , 53 mM MgCl2,28 mM Na2SO4, 10 mM CaCl2 , 9 mM  KCl , 2.4 mM NaHCO3 ,0.84 mM KBr, 0.49 mM H3BO3, 90 µM SrCl2, 72 µM NaF
f/2 -liquid medium	artificial seawater, 0.1 % (v/v) trace element solution, 0.05 % (v/v) vitamin solution, 0.88 mM NaNO3, 36 µM NaH2PO4
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
f/2-agar	3 % (w/v) bacto agar, artificial seawater, 0.1 % (v/v) trace element solution, 0.05 % (v/v) vitamin solution ,0.88 mM NaNO3, 36 µM NaH2PO4
Trace element solution	0.36 mM NaH2PO4, 12 µM Na2EDTA, 39 nM CuSO4, 26 nM Na2MoO4 , 77 nM ZnSO4, 42 nM CoCl2, 0.91 µM MnCl2
Vitamin solution	0.3 µM thiamin-HCl, 2.1 nM biotin, 0.37 nM cyanocobalamin