-Visualization-

-Plasmid extraction

Promoter - suppressed expression system(Am)

-Reining

Above samples

-Make competent cells

F+ and RP4

Cultured to OD600:0.6

Harvested

Suspended with 0.1 M CaCl2

Incubated on ice for 10 min

Harvested and abandoned supernatant

Suspended 0.1 M CaCl2

-Transformation

Promoter - suppressed expression system for F+

oriT - GFP for RP4

@colony PCR oriTr-GFP-oriTr

@preculture oriTr-GFP-oriTr

-Targeting-

Colony PCR

Colony PCR J23100-P0140(J61002)-pSB1C3,I13522-pSB1C3 and AHL inducible Device①②(pSB1C3)-E0240.But failure.

electrophoresis

Anti-killer Device⑥(pSB1C3),Anti-killer Device⑦(pSB1C3) yesterday emzimed and find binds.

preculture

J23100-P0140(J61002) Master Plate No.9,I13522(pSB1C3) Master Plate No.2,AHL inducible Device①-E0240 Master Plate No.1,AHL inducible Device②-E0240 Master Plate No.2