Team:Chiba/Sender experiments/Senders(XL10Gold) T9002(JW1908)

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Results

Reaction temparature:37°C

  • Sender culture：500μL,Receiver：500μL
Fig.1　　
E.coli strain,Senders:XL10Gold,BBa_T9002:JW1908,37°C
Fig.2　　
E.coli strain,Senders:XL10Gold,BBa_T9002:JW1908,37°C
Fig.3　　
E.coli strain,Senders:XL10Gold,BBa_T9002:JW1908,37°C


  • result

Cultures containing cells transformed with LuxI gene(BBa_K084012)and cells transformed with RhlI gene(BBa_K084008) express gfp and fluorescence intensity was reached to 500.Cultures containing cells transformed with LasI gene(BBa_K084007) weakly expressed gfp,and fluorescence intensity was not reached to a significant level.

  • Discussion:Maximum fluorescence intensity was vary,however,time before gfp expression doesn't differ.
  • We concluded that LasI gene(BBa_K084007) was not work well at this condition.

middle

  • Results

Fluorescence intensity of the culture containing cells transformed with LuxI gene(BBa_K084012) was lower than that of the cultures containing the cells transformed with LVA LuxI-LVA gene(BBa_K084014).The difference of maximum fluorescence intesity between the two cultures was 200.

  • Discussion
  • The rate of AHL synthesis decreased by the degradation of autoinducer synthase,however,the length of time before gfp expression is same.


  • right
  • Result

Cultures containing cells transformed with RhlI gene(BBa_K084008) and RhlI+LVA(BBa_K084009) draw the same transfer curve.

  • Discussion
  • We concluded that there was no effect of LVA-tag on time before gfp expression.


Fig.4　　
E.coli strain,Senders:XL10Gold,BBa_T9002:JW1908,37°C
Fig.5　　
E.coli strain,Senders:XL10Gold,BBa_T9002:JW1908,37°C



Reaction temparature:30°C

  • Sender culture：500μL, Receiver culture：500μL
Fig.6　　
E.coli strain,BBa_K084007:XL10Gold,BBa_T9002:JW1908,30°C
Fig.7　　
E.coli strain,BBa_K084007:XL10Gold,BBa_T9002:JW1908,30°C


  • 左
    • Result 　
      • 蛍光強度はRhl,LuxI+LVA,LasIの順に高かった。
      • LuxI,RhlIはLasIよりも約2時間、蛍光強度200に達するまでの時間が早かった。
    • 考察
      • 30°CではRhlIの活性のほうがLuxIより高い。もしくは、LVAの効果が出ている。その場合、右のグラフ

　　　のLVA自体に問題があると考えられる。


  • 右
    • Result
      • RhlI+LVAとRhlIによるGFPの発現量を比べた場合、蛍光強度はほぼ同じ値だった
    • 考察
      • LVAが働いていないか、働いていたとしてもこの条件だとAHLシンターゼの合成速度のほうがLVAによるLuxIの分解速度よりも

　　大きいと考えられる

Reaction temparature:25°C

Sender culture：500μL,Receiver culture：500μL

Fig.8
E.coli strain,Senders:XL10Gold,BBa_T9002:JW1908,25°C,Receiver cells/Sender cells = 1.
Fig.9
　E.coli strain,Senders:XL10Gold,BBa_T9002:JW1908,25°C,Receiver cells/Sender cells = 1.


  • Left:
    • result
      • 蛍光強度が,ネガコンとほとんどおなじだった。
      • LasIにおける最終形高強度はLuxI,RhlIの場合の約1/2であった。
    • Discussion
      • 蛍光強度が極端に下がったのは30°C,37°Cで行った実験と比べ温度が低いため活性が落ちたためと考えられる。
      • またそれだけではなく静置して行ったためSenderとReceiverがよく混ざらず、その結果GFPの発現量が大幅に減少した可能性も考えられる。
  • Right:
    • Result
      • The fluoroscence intensity by RhlI with LVAtag is less than RhlI without LVAtag.
    • Discussion
      • LVAtag is effective in this condition.


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