1. Protein induction method

1. 1. Protein induction method • One colony inoculated in 10㎖ LB (add ampicillin) medium and incubated in a shaking incubator overnight. • 4 ㎖ X 2 incubated cell was inoculated in 400 ㎖ X 2 LB (add ampicillin) medium and incubated in a shaking incubator of 37℃ for 3 hours. • 0.3 mM and 0.5 mMIPTG in each LB (add ampicillin) medium and incubated in a shaking incubator of 18℃ for overnight. • Each 200 ㎖ LB (add ampicillin) medium was harvested. ( Total 200 ㎖ 씩 4개 ) • Each of harvested cells were suspended in 3 ㎖ and 6 ㎖ 50 mM potassium phosphate 0.5M NaCl [ pH 8.0 ] buffer. • Broke the cells with a sonicator. • Centrifugation at 15000 x g for 10 min and 30 min at 4℃. • Proceeded SDS PAGE after Crude extract gained.

2. Lane 1 and 10 : Protein marker 4 ㎕ Lane 2 and6 : 0.1 mM IPTG and cultivated at 18℃ for overnight and suspended 3 ㎖ phosphate buffer and centrifugation 10 min and 30 min → 20 ㎕ + SDS dye 4 ㎕ Lane 3 and7 : 0.1 mM IPTG and cultivated at 18℃ for overnight and suspended 6 ㎖ phosphate buffe and centrifugation 10 min and 30 min → 20 ㎕ + SDS dye 4 ㎕ Lane 4 and8 : 0.3 mM IPTG and cultivated at 18℃ for overnight and suspended 3 ㎖ phosphate buffe and centrifugation 10 min and 30 min → 20 ㎕ + SDS dye 4 ㎕ Lane 5 and9 : 0.3 mM IPTG and cultivated at 18℃ for overnight and suspended 6 ㎖ phosphate buffer and centrifugation 10 min and 30 min → 20 ㎕ + SDS dye 4 ㎕ 2. CbbR Protein induction Protein size : 33 kDa 1 2 3 4 5 6 7 8 9 10 198 ( kDa ) ( kDa ) 187 127 115 90.5 80 61.5 52 48.2 42 37.8 27 20 20 14 18.5 9 10

3. 3. Protein induction method • One colony inoculated in 10㎖ LB (add ampicillin) medium and incubated in a shaking incubator overnight. • 4 ㎖ X 2 incubated cell was inoculated in 400 ㎖ X 2 LB (add ampicillin) medium and incubated in a shaking incubator of 37℃ for 3 hours. • 0.1 mM and 0.3 mMIPTG in each LB (add ampicillin) medium and incubated in a shaking incubator of 30℃ for 6 hours. • Each 400 ㎖ LB (add ampicillin) medium was harvested. • Each of harvested cells were suspended in 6 ㎖ 50 mM potassium phosphate 0.5M NaCl [ pH 8.0 ] buffer. • Broke the cells with a sonicator. • Centrifugation at 15000 x g for 10 min and 30 min at 4℃. • Proceeded SDS PAGE after Crude extract gained.

4. Lane 1: Protein marker 4 ㎕ Lane 2 and 5 : Cultivate at 37℃ for 3 hours and suspended 6 ㎖ phosphate buffer and centrifugation 10 min and 30 min ( pProEX-HTa vector) → 20 ㎕ + SDS dye 4 ㎕ Lane 3and 6 : 0.1 mM IPTG and cultivated at 30℃ for 6 hours and suspended 6 ㎖ phosphate buffer and centrifugation 10 min and 30 min → 20 ㎕ + SDS dye 4 ㎕ Lane 4 and 7 : 0.3 mM IPTG and Cultivated at 30℃ for 6 hours and suspended 6 ㎖ phosphate buffer and centrifugation 10 min and 30 min → 20 ㎕ + SDS dye 4 ㎕ 4. CbbR Protein induction Protein size : 33 kDa 1 2 3 4 5 6 7 ( kDa ) 198 115 90.5 61.5 46.2 37.8 20 18.5 9

5. 5. Next experiments • React CbbR protein induction • EMSA (Electrophoretic mobility shift assay)