Team:Chiba/Experiments:copy number

From 2008.igem.org

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

We used a pMB1 ori high copy vector containing BBa_T9002 and a low copy vector containing BBa_T9002 with a P15A as ori.

We determined the effects of altering the plasmid copy number by comparing the above situations.

Experiments used the following genetic circuits:

In addition, a Chloramphenicol resistance marker-containing empty plamid was transformed to the strain containing BBa_T9002, and a Ampicillin resistance marker-containing empty vector was transformed into the low copy variant, both resulting in a double transformation.

Method

1. 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).
2. Inoculated them independently in liquid media. Incubated at 37°C 12h.
3. Inoculated again in Fresh liquid media upto about OD600=2 at 37°C
4. Washed sender and receivers.
5. Mixed them. (Sender:Receiver=1000μL:1000μL)
6. Incubated at 30°C.
7. 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

1. 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。
2. 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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