Construction of Expression Shuttle Vectors for the Haloarchaeon Natrinema sp. J7 Based on Its Chromosomal Origins of Replication

Figure 6

Amylase expression in J7-F cells using pFJ1, pFJ4, and pFJ6. (a) The pFJ1-A, pFJ4-A, and pFJ6-A plasmids were transformed into J7-F cells. Three random transformants were transferred to 5 mL of Halo-2 medium, grown to exponential phase, and 2 μL of each culture was spotted onto 18% MGM plates supplemented with 2% (w/v) soluble starch. After five days, iodine solution was added to the plates, and halos formed immediately around the selected transformants indicating that amylase was successfully expressed. 1-1: J7-F transformed with pFJ1 (negative control); 1-2, 1-3, and 1-4: J7-F transformants harboring pFJ1-A; 4-1: J7-F transformed with pFJ4 (negative control); 4-2, 4-3, and 4-4: J7-F transformants harboring pFJ4-A; 6-1: CJ7 cells transformed with pFJ6 (negative control); 6-2, 6-3, and 6-4: J7-F transformants harboring pFJ6-A (negative control). (b) Amylase-specific activities of supernatants collected from CJ7/pFJ1-Apro-amyH, CJ7/pFJ4-Apro-amyH, CJ7/pFJ6-Apro-amyH, and CJ7/pYCJ-Apro-amyH cultures. One unit of amylase activity was defined as the quantity of amylase required to hydrolyze 1 mg of starch in 1 h.

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