Archaea / 2015 / Article / Fig 2

Research Article

Isolation and Molecular Identification of Auxotrophic Mutants to Develop a Genetic Manipulation System for the Haloarchaeon Natrinema sp. J7-2

Figure 2

Morphological changes to the cells during the transformation process. As there was no transformation protocol available for the genus of Natrinema, we used the transformation process used for Hfx. volcanii. All procedures were performed at room temperature. We picked out a single colony of J7-2 and cultivated it in liquid MGM media at 42°C. Selected cultures of J7-2 were obtained and treated with spheroplasting solution for 15 min, while some were not treated to provide a control. Cell morphology was observed and recorded under the microscope. The results are shown in (a) and (b). During the recovery process, three different regeneration solutions were tested to determine whether the cell morphology could be recovered to short rods and which medium was more suitable for Natrinema: H-2 medium, 18% MGM and regeneration solution. We modified the protocol slightly. The regeneration time was changed from 20–30 min at RT to 2 to 5 hours at 37°C and the transformation solutions were also changed from 1 M NaCl to 2 M NaCl. The regeneration results were also observed and recorded under microscope. All recovered results are shown in (c), (d), and (e). (a) Cells of J7-2 grown in MGM medium before spheroplasting solution treatment. (b) Cells of J7-2 treated with spheroplasting solution. (c) Cells of J7-2 regenerated with H-2 medium treatment. (d) Cells of J7-2 regenerated with 18% MGM medium treatment. (e) Cells of J7-2 regenerated with regeneration solution treatment.

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