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

Figure 5

Expression of amyH gene in J7-2-1 via the integrative plasmid pUC19-HM1752-amyH. The plasmid pUC19-HM1752-amyH containing amyH was transformed into strain J7-2-1. Twenty-nine transformants were picked out randomly and cultured. Amylase activity in these transformants was detected on an MGM plate supplied with 2% (w/v) soluble starch. (detailed process is discussed in Materials and Methods). The plate was flooded with 0.3% I₂/0.6% KI solution after incubation at 42°C for 7–15 days. If the transformants had amylase activity, a clear zone indicating starch hydrolysis would be observed. The results were recorded and pictures were taken. (a) (1) Negative control, J7-2-1 transformed with salt solution without DNA. (2) J7-2-1 transformed with integrative plasmid pUC19-HM1752-amyH. (b) MGM plates containing soluble starch are used to detect the expression of amyH gene in J7-2-1 by integrative plasmid pUC19-HM1752-amyH. P. Positive control. N1: negative control Haloferax volcanii DS52. N2: negative control J7-2-1. N3: negative control J7-2-1 transformed with J7-2 Genome. N4: negative control J7-2-1 transformed with KM1752 fragment. 1–29: samples from transformants of J7-2-1 transformed with integrative plasmid pUC19-HM1752-amyH.

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