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.
Keywords: Cyanobacteria, Transformation, Electroporation, Targeted gene disruption, Shuttle vector
Materials and Reagents
Equipment
Procedure
Representative data
Representative results in targeted gene disruption are shown in Table 2. In most cases almost all transformants appeared on selective plates were double recombinants. A procedure of repeated inoculations of transformants to segregate cells in which all wild-type copies are completely replaced with the mutant copies is required in Synechocystis sp. PCC 6803, but this process is not needed in L. boryana (Note 8). Table 2. Examples of electroporation of L. boryana 1WT, wild type 2Total DNA amount in the cell suspension for pulse application 3Number of colonies. Single or double recombinant was confirmed by Southern blot analysis or PCR Figure 4. Typical appearance of transformants on a selective plate. Two tiny green colonies (red arrows) appeared 14 days after pulse application.
Notes
Recipes
Acknowledgments
This protocol was adapted from the previously published studies, Hiraide et al. (2015), Tsujimoto et al. (2014) and Fujita et al. (1992). The original protocol was described in Fujita et al. (1992), and we modified it as described here. We thank Yasuhiro Takahashi, Tomohiro Matsumura, Toshiharu Hase, and Hiroshi Matsubara for initial works on establishment of this transformation system. We thank Douglas K. Walton, Carl E. Bauer and Peter Wolk for donating plasmids pPBH201, pJRD215 and pRL425, respectively. We thank Chie Tomatsu for providing technical help. This work was supported by the Japan Society for the Promotion of Science (JSPS) (Grants-in-Aid for Scientific Research Nos. 05740481, 06740601, 07740617, 08836006, 11740445, 23370020, 23000007, 26660084, 15H04387 and 15H01397), Precursory Research for Embryonic Science and Technology (PRESTO) and the Advanced Low Carbon Technology Research and Development Program (ALCA).
References
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