Table 1.
List of PCR primers.
Table 2.
Summary of NT protocol for Phormidium lacuna.
Fig 1.
Growth of Phormidium lacuna on selection medium.
(A-C) Filaments of Phormidium lacuna HE10DO after transformation with pFN_7_37_kanR during or after selection on agar plates. (A, B) Two weeks after transformation (A. 70 μg/ml Km; B. 120 μg/ml Km); (C) 4 weeks after transformation, selection on 120 μg/ml Km. In A brownish (red arrows) and greenish (white arrows) filaments can be distinguished, which represent non-transformed and transformed lineages, respectively; in B an area of the agar plate with a high frequency of disintegrated filaments was chosen, the intact filaments are transformed and alive; in C all filaments are alive and transformed. Bundles of parallel filaments are characteristic for growth of Phormidium lacuna on agar medium, the growth pattern is comparable to that of wild type on Km-free agar medium. Scale bars 100 μm. (D) Kanamycin resistance of Phormidium lacuna HE10JO wild type and pFN_7_37_kanR transformants (liquid cultures 1 week after inoculation). Wild type cannot proliferate at 50 μg/ml Km or above, while pFN_7_37_kanR transformants can grow up to 14 mg/ml Km.
Table 3.
Transformation of Phormidium lacuna HE10DO by electroporation and natural transformation.
Fig 2.
Validation of Phormidium lacuna HE10DO transformants by PCR.
(A) Plasmid map of pFN_7_37_kanR, red—homologous sequences of the sc_7_37 locus, blue—kanamycin resistance cassette kanR, yellow—origin of replication (f1—bacteriophage origin, other—pUC origin for E. coli), purple—ampicillin resistance cassette; (B) Integration site of pFN_7_37_kanR and primer binding sites. red—homologous sequences encoded on the vector, pale red—Phormidium lacuna chromosome, blue—kanamycin resistance gene. Primer pair: F25/F28 covering whole insertion site; (C) agarose gel for the PCR with the primer pair that covers the full insert for Phormidium lacuna HE10DO WT and pFN_7_37_kanR transformants. Transformants T1a and T1b were cultivated after selection on agar plate for one cultivation period (7 days) in f/2+ liquid medium with 250 μg/ml Km and one period in f/2+ with 1000 μg/ml Km. T2a and T2b were cultivated only for one period at 250 μg/ml Km. M: 1kb DNA ladder (NEB, USA). Integration of the kanR cassette into the genome of Phormidium lacuna is indicated by the larger PCR product (2560 bp), the native sequence in indicated by the small PCR product (1213 bp).
Fig 3.
DAPI assay to determine DNA concentrations in Phormidium lacuna HE10DO extracts.
(A, B) excitation spectra from 300 nm to 400 nm, fluorescence emission were measured at 470 nm. (A) spectra of DAPI (black) and DAPI with calf thymus DNA (red); excitation maxima are indicated by arrows; (B) spectra of Phormidium lacuna extract (thick line, black), extract after addition of DAPI (thick line, red) and after DNAse addition, measured at intervals as given in the legend (thin lines, various colors), after 30 min, fluorescence was constant (thick line, brown), excitation maxima of extract and extract+DAPI are indicated; (C) calibration curve, fluorescence of DAPI with calf thymus DNA as used for calculation of DNA concentrations, correlation coefficient 0.998; (D) number of chromosome copies of Phormidium lacuna calculated according to the methods section and plotted against cell densities, correlation coefficient and slope of a linear regression are 0.3 and -41, respectively, the number of cells is 22 Mio / OD750 nm.
Fig 4.
Detection of wild type and recombinant chromosomes in kanamycin resistant Phormidium lacuna HE10DO pFN_7_37_kanR transformants with PCR.
(A-D) Following cultivation in 0.1 mg/ml Km until 2 weeks after transformation, the sample was divided and subcultivated one to four times on different Km concentrations (A, 0 mg/ml; B, 0.1 mg/ml, C, 0.98 mg/ml; D, 8.3 mg/ml). Primers: F25, F28. PCR product length: native– 1213 bp, recombinant– 2560 bp. Marker: 1 kb DNA ladder (NEB, USA).
Table 4.
Prediction of cyanobacteria that are potentially naturally competent.