"
Team:Osaka/Tests
From 2011.igem.org
| Line 4: | Line 4: | ||
== Test == | == Test == | ||
=== Cell survival assay === | === Cell survival assay === | ||
| + | ・生存率 | ||
| + | |||
| + | |||
| + | |||
As an initial screen for DNA repair protein activity , we used the UV assay. The cells were plated on respective agar plates at different dilutions, air dried, and then exposed to different doses of UV radiation. Plates were wrapped with aluminum foil and incubated in the dark. Colonyforming units were scored after 16h incubation at 37℃. | As an initial screen for DNA repair protein activity , we used the UV assay. The cells were plated on respective agar plates at different dilutions, air dried, and then exposed to different doses of UV radiation. Plates were wrapped with aluminum foil and incubated in the dark. Colonyforming units were scored after 16h incubation at 37℃. | ||
放射線耐性菌D.radioduranceのもつDNA-repair proteinであるPprI,PprA,PprM,RecAをそれぞれ導入した大腸菌にUV照射 | 放射線耐性菌D.radioduranceのもつDNA-repair proteinであるPprI,PprA,PprM,RecAをそれぞれ導入した大腸菌にUV照射 | ||
| + | |||
| + | |||
| + | ・リコピンアッセイ | ||
| + | 3mlのLBでプレ培養 | ||
| + | 40mlのLBに100μl加えてincubate 20h | ||
| + | OD600測定 | ||
| + | UV照射 | ||
| + | incubate 2h | ||
| + | OD600測定 | ||
| + | 遠心沈殿で上澄捨てる | ||
| + | 水を加えて洗う | ||
| + | 遠心沈殿で上澄捨てる | ||
| + | アセトン500μl加えて 55℃ vortex 15min | ||
| + | アセトン100%でブランク | ||
| + | A474測定 | ||
| + | |||
=== Results === | === Results === | ||
Revision as of 13:24, 4 October 2011
Test
Cell survival assay
・生存率
As an initial screen for DNA repair protein activity , we used the UV assay. The cells were plated on respective agar plates at different dilutions, air dried, and then exposed to different doses of UV radiation. Plates were wrapped with aluminum foil and incubated in the dark. Colonyforming units were scored after 16h incubation at 37℃.
放射線耐性菌D.radioduranceのもつDNA-repair proteinであるPprI,PprA,PprM,RecAをそれぞれ導入した大腸菌にUV照射
・リコピンアッセイ
3mlのLBでプレ培養
40mlのLBに100μl加えてincubate 20h
OD600測定
UV照射
incubate 2h
OD600測定
遠心沈殿で上澄捨てる
水を加えて洗う
遠心沈殿で上澄捨てる
アセトン500μl加えて 55℃ vortex 15min
アセトン100%でブランク
A474測定
Results
生存率のグラフ
PprM,RecAは生存率アップ、 I,Aは微妙な結果
PprIはRecAとPprAを誘導 PprAはRecAに依存しない修復機構をもっているらしい（変異が入った？） PprMは不明
RecA欠損株であるDH5αを用いたためPpr系の生存率は変化しなかったと思われる。 RecAを持つ株なら生存率上がってたかも。
Future work
We created some parts (PprI , PprA , PprM , RecA) but did not have time to evaluate them. Also, devices containing 2 or more DNA repair gene should have been constructed and assayed.
Reference
放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)
PprA: a protein implicated in radioresistance of Deinococcus radiodurans stimulates catalase activity in Escherichia coli, Swathi Kota et al (2006)
