Team:Chiba/Experiments:copy number

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Revision as of 03:36, 30 October 2008

Copy number of Receiver Plasmid

Design

Fig. 1 Player Balancing

In this section, we intended to create time-delay by altering the amount of luxR protein per cell.(*1) Since the amount of LuxR that receives AHL differs, we hypothesized that the time required to reach the threshold AHL concentration will differ.

To do this, we used different copy number plasmids of the Receivers. (*1)

By altering the copy number of plasmids in the receiver cells, the time required for the response is altered.
If the copy number is altered, the amount of LuxR produced by the receivers will also change.
The amount of LuxR that receives AHL will change, so the time required for AHL to reach a threshold level will be altered.

Experiment

oriがpMB1のハイコピーベクターにのったBBa_T9002と、oriがP15AのローコピーベクターにのせかえたBBa_T9002を使用した。

これらを比較することでコピーナンバーを変えることでの影響を調べた。

以下の遺伝子回路を使って、実験を行った。

Sender

Receivers

**[http://partsregistry.org/Part:BBa_S03623 BBa_S03623 (AHL autoinucer)]

- [http://partsregistry.org/Part:BBa_T9002 BBa_T9002 (Express GFP in response to AHL)] |

Low Copy Receiver

これらのほかにBBa_T9002にはChloramphenicol耐性マーカーを持つ空ベクター、Low copyバリエーションにはAmpicillin耐性マーカーの空ベクターをダブルトランスフォーメーションさせた。

Method

Transformed sender(Ptet-LuxI), high copy receiver(Ptet-LuxR-Plux-GFP-pMB1) and Medium copy receiver(Ptet-LuxR-Plux-GFP-p15A) respectively into E coli strains(JW1908).
Inoculated them independently in liquid media. Incubated at 37°C 12h.
Inoculated again in Fresh liquid media upto about OD600=2 at 37°C
Washed sender and receivers.
Mixed them. (Sender:Receiver=1000μL:1000μL)
Incubated at 30°C.
Measured intensity of green fluorescence at regular time intervals.(Fluoroskan AscentR FL＆Fluoroskan AscentR　Thermo ELECTRON CORPORATION)

Result & Discussion

Fig.3.2 Time Delay Test: Highcopy Receiver & Reporter vs.Lowcopy Receiver & Reporter

(Ptet-LuxR-plux-GFP-pMB1)の発現量に比べて(Ptet-LuxR-pLux-GFP-p15A)の発現量は大きく減少してしまった。(Fig.3.2)

この理由は

GFPがローコピーのベクターに乗っていたので発現量が落ちた。
LuxRの合成量が少なすぎる。

が考えられるが、(Ptet-luxR-p15A + plux-GFP-pMB1)と(Ptet-LuxR-pLux-GFP-pMB1)ではまったく同じTrasnfor Carveの軌跡を描いた。(Fig.3.3) このことより、p15Aのベクターに乗ったLuxRの発現量は、ハイコピーのベクターに乗ったpLuxを活性化するのに十分な量であると考えられる。したがって理由1が有力である。

Fig.3.3 Time Delay Test: Lowcopy Receiver & Highcopy Reporter vs.Highcopy Receiver-Reporter

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@@ Line 16: / Line 16: @@
 ===Design===
 [[Image:Receiver copy number chiba.jpg|left|frame|'''Fig. 1  Player Balancing''']]
-In this section, we intended to make an time-delay by differing the luxR protein's amount per cell.(*1) AHLを受け取るLuxRが変わるので応答閾値までの時間が変わると考えた。
-To do this, we used a different copy number plasmid of the Receivers. (*1)
-レシーバーのコピーナンバーを変えることで、応答までの時間を変える<br>
+In this section, we intended to create time-delay by altering the
-コピーナンバーを変えれば、レシーバーによるLuxRの合成量は変化する<br>
+amount of luxR protein per cell.(*1) Since the amount of LuxR that
-AHLを受け取るLuxRが変わるので応答閾値までの時間が変わると考えた<br>
+receives AHL differs, we hypothesized that the time required to reach
+the threshold AHL concentration will differ.
+To do this, we used different copy number plasmids of the Receivers. (*1)
+By altering the copy number of plasmids in the receiver cells, the
+time required for the response is altered.<BR>
+If the copy number is altered, the amount of LuxR produced by the
+receivers will also change.<BR>
+The amount of LuxR that receives AHL will change, so the time required
+for AHL to reach a threshold level will be altered.
 <br clear=all>