Transformation of the Cyanobacterium Leptolyngbya boryana by Electroporation

Materials and Reagents

1. Bottle top filter system (0.22 µm) (Corning, catalog number: 430624 )
   
2. Microcentrifuge tubes (1.5 ml) (Ina-optika corporation, BIO-BIK, catalog number: ST-0150F ) or its equivalent, sterilized by autoclave (121 ºC, 20 min)
   
3. Micropipettes tips (121 ºC, 20 min)
   
4. Sterile syringe filter (Millex-GV Syringe Filter Unit, 0.22 µm), used for filter sterilization of solutions of antibiotics and glucose (Thermo Fisher Scientific, Millipore, catalog number: SLGV033RV ) or its equivalent
   
5. Petri dish [sterile Petri dishes (90 mm x 15 mm)] (ASONE Corporation, catalog number: 1-7484-01-30 ) or its equivalent
   
6. Pulse cuvettes (Gene Pulser cuvette, 0.1 cm) (Bio-Rad Laboratories, AbD Serotec^®, catalog number: 1652089 )
   
7. Hybond N+ filter (disc 82 mm diameter) (GE Healthcare, Amersham, catalog number: RPN82B ), sterilized by autoclave (121 ºC, 20 min).
   
8. Leptolyngbya boryana (wild type or dg5) (grown on a BG-11 agar plate supplemented with 20 mM HEPES-KOH, pH 7.5 and 30 mM glucose)
   Note: The dg5 strain was isolated from the wild type as a natural mutant that grows much faster heterotrophically in the dark than wild type (Fujita et al., 1996). Recently we identified that the mutation responsible for the dg5 phenotype is one adenine insertion causing a frameshift in the cytM gene encoding cytochrome c_M (Hiraide et al., 2015).
   
9. Sterilized water (150 ml)
   Note: Water is purified by RiOs Essential Water Purification System (Merck Millipore Corporation) or WEX system (Yamato). Distilled water can be also used. Autoclave (121 ºC, 20 min) and chill on ice before use.
   
10. NaNO₃ (Nacalai tesque, catalog number: 31617-35 )
    
11. 1 M K₂HPO₄ solution (Wako Pure Chemical Industries, Siyaku, catalog number: 164-04295 )
    
12. 7.5% (w/v) MgSO₄^.7H₂O (Nacalai tesque, catalog number: 21003-75 )
    
13. 3.6% (w/v) CaCl₂^.2H₂O (Nacalai tesque, catalog number: 06731-05 )
    
14. 2.0% (w/v) Na₂CO₃ (Nacalai tesque, catalog number: 31311-25 )
    
15. Citric acid (Wako Pure Chemical Industries, Siyaku, catalog number: 038-05521 )
    
16. Ammonium iron (III) citrate, brown (Wako Pure Chemical Industries, Siyaku, catalog number: 092-00802 )
    
17. Ethylenediamine-N, N, N', N'-tetraacetic acid, disodium salt, dihydrate (EDTA-Na₂) (Dojindo, catalog number: 345-01865 )
    
18. H₃BO₄ (Wako Pure Chemical Industries, Siyaku, catalog number: 021-02195 )
    
19. MnCl₂^.4H₂O (Nacalai tesque, catalog number: 21211-45 )
    
20. ZnSO₄^.7H₂O (Wako Pure Chemical Industries, Siyaku, catalog number: 268-00405 )
    
21. Na₂MoO₄^.2H₂O (Wako Pure Chemical Industries, Siyaku, catalog number: 197-02485 )
    
22. CuSO₄^.5H₂O (Wako Pure Chemical Industries, Siyaku, catalog number: 033-04415 )
    
23. Co(NO₃)₂^.6H₂O (Wako Pure Chemical Industries, Siyaku, catalog number: 031-03752 )
    
24. 1 M Glucose (filter sterilized) (Wako Pure Chemical Industries, Siyaku, catalog number: 049-31165 )
    
25. 50 mg/ml kanamycin sulfate (filter sterilized) (Wako Pure Chemical Industries, Siyaku, catalog number: 111-00344 )
    
26. 50 mg/ml chloramphenicol (dissolve in ethanol) (Nacalai tesque, catalog number: 08027-72 )
    
27. 10 mg/ml streptomycin sulfate (filter sterilized) (Meiji Seika Pharma, catalog number: 4987222665643 )
    
28. 50 mg/ml erythromycin (filter sterilized) (Wako Pure Chemical Industries, Siyaku, catalog number: 054-05101 )
    
29. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (Nacalai tesque, catalog number: 17514-15 )
    
30. Plasmid preparation kit (GenElute HP Plasmid Midiprep Kit) (Sigma-Aldrich, catalog number: NA0200 )
    
31. Linearized plasmid solution (> 2 µg µl^–1)
    Note: The concentration of plasmid is determined by a spectrophotometer before the enzyme digestion.
    
32. Bacto Agar [1.5% (w/v)] (BD, catalog number: 214010 )
    
33. Ferric citrate solution (see Recipes)
    
34. Trace metal A₅+Co solution (1 L) (see Recipes)
    
35. 2x BG-11 solution (1 L) (see Recipes)
    
36. BG-11 agar plates (supplemented with 20 mM HEPES-KOH, pH 7.5, with or without appropriate antibiotics, Table 1) solidified with Bacto Agar [1.5% (w/v)] (BD, catalog number: 214010) (see Recipes)
    

Equipment

1. Forceps
   
2. Micropipettes ( P-1000 , P-200 and P-20 , or their equivalents) (Gilson)
   
3. Clean bench
   
4. Autoclave (TOMY DIGITAL BIOLOGY CO., model: SX-500 or its equivalent)
   
5. Gene pulser Xcell (Bio-Rad Laboratories, AbD Serotec^®, catalog numbers: 1652666 , 1652668 and 1652669 )
   
6. Spectrophotometer (Shimadzu, model: UV-1700 or GE Healthcare, model: NanoVue Plus or its equivalent)
   
7. Aspirator (with a vacuum pump)
   
8. Growth cabinet (with appropriate fluorescence light bulbs)
   

Procedure