-Speeding Up-

Colony PCR of HNS(wt)+I20260. Confirmed amplifing DNA but length of fragment was incorrect.

Electrophoresis of digested HNS(wt)(XbaI+SpeI),I20260(BlnⅠ).Concentration of both HNS(wt)(XbaI+SpeI) and I20260(BlnⅠ) was low.

Preculture(pSB1C3)

-Targeting-

-Colony PCR

Did colony PCR for J23100-P0440.

Did not show bands.

-Ligation

Ligated for J23100-K317037 and J23100-P0440.

-Transformation

Transformed for J23100-K317037, J23100-P0440 and J23100 (control).

-Preculture

Spread no. 26 and no.31 for liquid medium with ampicillin from masterplate of J23100-P0440 on 5th September. After, had been incubated at 37 deg C.

-Visualization-

-Digestion

Promoter: Spe1

Suppressed expression system: Xba1 and Spe1

-Electrophoresis

Above samples

-Gel extraction

Above samples

-Electrophoresis to check

Above samples