Transformation of the Cyanobacterium Leptolyngbya boryana by Electroporation

Abstract

Leptolyngbya boryana (L. boryana) (formerly Plectonema boryanum) is a versatile, filamentous cyanobacterium that has the ability to fix nitrogen under microoxic conditions and to grow heterotrophically with glucose in the dark, providing an excellent system to investigate photosynthesis, nitrogen fixation, and their regulatory mechanisms. While L. boryana is not naturally transformable different from the unicellular cyanobacterium Synechocystis sp. PCC 6803, it can be transformed by electroporation. Here we describe the transformation of L.boryana by electroporation to isolate mutants in which a targeted gene is disrupted. Materials and Reagents1. 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 cM (Hiraide et al., 2015).

Identification of a cis‐acting element in nitrogen fixation genes recognized by CnfR in the nonheterocystous nitrogen‐fixing cyanobacterium Leptolyngbya boryana

“…Synechocystis 6803 (strain YF, Aoki et al ., ) was used as the WT. BG‐11 agar plates solidified with 1.5% Bacto Agar (BD Biosciences, Franklin Lakes, NJ, USA) and adjusted to pH 7.5 and 8.2 with 20 mM HEPES‐KOH were used for L. boryana and Synechocystis 6803, respectively (Tsujimoto et al ., ). In BG‐11 (nitrate‐replete medium), 15 mM KNO 3 was present as the sole nitrogen source, whereas the nitrate‐depleted medium BG‐11 0 did not contain any available nitrogen compounds.…”

Cyanobacteria as Chassis for Industrial Biotechnology: Progress and Prospects

“…Over the past decade, Synechocystis 6803 metabolism has been modeled [69,70,71,72,73,74,75,76,77,78,79] to understand and improve cyanobacterial biofuel production. In 2009, the first genome-scale model of Synechocystis 6803 to predict the effect of biofuel production was produced [80].…”

“…Since the discovery of cyanobacterial transformation in the 1970s, many techniques such as electroporation and ultrasonic treatment have been developed and applied to several cyanobacterial strains [70,73,74]. Electroporation has been used in the transformation of Anabaena sp., Synechococcus sp., Synechocystis sp., Fremylla diplosiphon and Plectonema boryanum , [70,73,74].…”

“…Electroporation has been used in the transformation of Anabaena sp., Synechococcus sp., Synechocystis sp., Fremylla diplosiphon and Plectonema boryanum , [70,73,74]. Ultrasonic transformation has been employed in the transformation of Synechocystis sp.…”

“…Ultrasonic transformation has been employed in the transformation of Synechocystis sp. [70,73,74]. It was noted that, in Synechocystis 6803, exogenous DNA is integrated into the genome following natural transformation by means of a well-established double homologous recombination system [72,73].…”