Team:Osaka/Tests

From 2011.igem.org

(Difference between revisions)
(Test)
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== Test ==
== Test ==
=== Cell survival assay ===
=== Cell survival assay ===
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まくパーツをLB3mlでプレ培養18h。照射するエネルギー量に応じて希釈濃度を変える。
 
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プレートに50μlまいて乾燥させてからUV照射。
 
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可視光を当てないようにアルミ箔で包んでincubate 37℃
 
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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℃.
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=== SOS promoter assay ===
=== SOS promoter assay ===
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・リコピンアッセイ
 
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3mlのLBでプレ培養。それを40mlのLBに100μl加えてincubate 20h。OD600測定しUV照射。2h振盪培養してOD600測定。
 
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遠心沈殿で上澄を捨て、水1mlを加えてvortex。もう一度遠心沈殿で上澄捨てる。アセトン500μl加えて 55℃ vortex 15min  
 
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アセトン100%でブランク  
 
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A474測定
 

Revision as of 05:28, 5 October 2011


Test

Cell survival assay

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


SOS promoter assay

Results

Cell viability

グラフ


recA gene could induce high cell viability. RecA protein has key role of SOS response.

This result revealed that the cell inserted recA gene can get tolerance against DNA damage.

pprM gene could also bring high tolerance to inserted cells. discussion 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? 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)