Transcript FliC-System

Flagellin,FliC
Flagella
Flagellum
Graduate School of Frontier Biosciences, Osaka University
ナノ生体科学講座 プロトニックナノマシン研究室（難波研究室）
http://www.fbs.osaka-u.ac.jp/labo/09a.html
Flagellin,FliC
Salmonella typhimurium
dispensable
indispensable
プロトニックナノマシンプロジェクト, ERATO
研究成果 (1)
http://www.npn.jst.go.jp/index_jp.html
FliC-His
FliC-His
His-Tag
His-tag
Beads adsorption
Beads
・Four of cobalt’s six coordination sites are occupied.
・The imidazole rings of histidine residues are able to occupy the
two remaining coordinate sites, resulting in protein binding.
Beads adsorption
RM培地
5mL
Add dynabeads
Adsorption cells
30℃
IPTG5μL
Magnetic separation
Remove supernatant
Elution
Get a Micrograph
inculate
Colony count
Containing eluted
histidine-tagged protein
Beads adsorption
・Colony Count
2000
1500
1000
500
0
・Micrograph
Beads-Co
No plasmid
FliC-His
Co2+
Ni2+
FliC-His
No Metal
No metal
Toxicity Check
Trip
Medium
5mL
30℃
IPTG
5μL
RM
Medium
5mL
30℃
IPTG
5μL
Add Cocl2
0M,1μM,
10μM,
100μM,
1mM
inculate
Micro
-graph
Toxicity Check
・Toxicity Check
200000
180000
160000
140000
120000
100000
80000
60000
40000
20000
0
・Get a Micrograph
1μM
10μM
100μM
1mM
Ni2+
Co2+
Micrograph
Sender aggregation
RM培地
5mL
30℃
IPTG5μL
顕微鏡
Add Ni2+
Co2+
(Stationary)
Sender aggregation
・
・
・
・
顕微鏡写真
Receiver aggregation
RM培地
5mL
30℃
IPTG5μL
顕微鏡
Add Ni2+
Co2+
(Stationary)
Receiver aggregation
・
・
・
・
顕微鏡写真
Phenotype check
転写
M ①
④ ⑧ pET-49b
⇒FliC-Hisの発現が確認された。