Team:Chiba/Experiments:Reporter

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Reporter

Our delayed switch has to turn on or off without variable time lag after the preset time has passed. Therefore we need the output module, which announces quickly that a particular concentration of the signaling molcules has been reached.

We imagined three kinds of output:

Fluorescent Protein
Bioluminescence(luciferase)
Cell staining　(LacZ and X-gal assay)

and tested Fluorescent Protein and β-gal(lacZ).

We selected GFP, Venus YFP, and mCherry. The latter two were chosen because Venus YFP matures quickly

[1],and we can easibly recognize mCherry coloration[2].

We used fluorescent proteins and β-gal(lacZ) as a reporter.

We were able to recognize chemical luminescence of luciferase only at dark place devoid of light. Therefore the conditions of use are limited, and we did not select Luciferase as our reporter.

Experiment

Measure the time course form adding inducer (IPTG or AHL) to confirm the change of color to analysis the gene expression.

Evaluate following gene:

Fluorescent Protein

GFP

pGFPuv

BBa_T9002

Venus YFP

BBa_K084003

pLac-Venus YFP

mCherry

pLac-mCherry

β-gal (X-gal assay)

pUC19(plac-LacZα)

Fluorescent Protein

GFP

pGFPuv

BBa_T9002

Venus YFP

BBa_K084003

pLac-Venus YFP

mCherry

pLac-mCherry

β-gal (X-gal assay)

pUC19(plac-LacZα)

Result & Discussion

Result LacZ

Table. X-gal濃度を変化させ、染色の様子を測定した結果

 * LacZ, which is not fluorescent, was observed under the naked eye.
  * Time required for observation of LacZ staining depended on X-gal

concentration.

  * We were not able to observe stainig at a liquid state within 4 hours.

      -->Staining was observed at the 8 hour point.蛍光たんぱく

Table. それぞれのレポーター遺伝子の評価をした結果

Similar results were obtained for liquid and solid cultures.

  * To the naked eye, fluorescence was observed most easily in the

order mCherry, GFP, and YFP.

  * The solid state cultures were easier to observe to the naked eye.
  * AHL induction took a shorter time than IPTG for fluorescence to

be observable.

  * GFP and YFP showed observable fluorescence earlier than mCherry.

FigUVを当てた固体培地上のレポーター

Discussion

このプロジェクトでは、発現するまでの時間を変えることが目的である

X-galの濃度を高くすると発現を確認できるまでの時間は短くなったが、蛍光たんぱくで発現を確認できるまでの時間より遅い

X-galを基質とするβ-galでは、時間がたつごとに基質濃度が下がり、出力までの時間が長くなってしまうことも考えられる

以上の2つの理由から蛍光タンパク質が適している

またGFP,YFPの発現時間までのタイムラグが一番少なかった

目視で確認した際、GFPのほうがYFPよりも目視で確認するのが容易であった

以上の理由から、出力はGFPとする。

Method

Fig. レポーターのタイムレスポンスの実験方法

Strain:XL10G Kan^R

Agar plate experiment

Pre-culture
1. Picked and cultured the following plate stocks in 2mL of LB:
  1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
  2. LB-Amp, (BBa_T9002, BBa_K084003)
2. Cultured at 37°C for 12h.
Spread on plate
1. Spread on new plate
  1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
  2. LB-Amp, (BBa_T9002, BBa_K084003)
2. Cultured at 37°C for 12h.
Colony lift
1. Colony lift to inducible agar plate (containing IPTG or AHL)
  1. LB-Amp+0.2 mM IPTG agar plate, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
  2. LB-Amp+100 nM AHL agar plate, (BBa_T9002, BBa_K084003)
2. Incubate at 37 °C
Check expression every 30 min.

Liquid medium experiment

Pre-culture
1. Picked and cultured the following plate in 2mL of LB:
  1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
  2. LB-Amp, (BBa_T9002, BBa_K084003)
2. Cultured at 37°C for 12h.
Culture
1. Dilute pre-cultures and add to new LB medium.
  1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
  2. LB-Amp, (BBa_T9002, BBa_K084003)
2. Cultured at 37°C for about 6 h
Wash
1. Transfer 10mL each of the culture to 50mL centrifuge tubes.
2. Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
3. Add physiological saline and resuspention
4. Repeated wash twice.
5. Add M9 minimal medium.
Mix
1. Dispens each culture into 48-well deep well or 96-well deep well
2. Add IPTG fainal conc. 0.2 mM (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
3. Add AHL fainal conc. 100nM (BBa_T9002, BBa_K084003)
Measure fluorescence intensity every 1 h.

Equipment

shaking incubator

Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37°C)

46-well plate(deep well)

96-well plate(deep well)

Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)

Beckman Allegratm X-12R Centrifuga(Beckman Coulter)

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@@ Line 92: / Line 92: @@
 '''LacZ'''
 [[Image:LacZ chiba.gif|thumb|right|'''Table.''' X-gal濃度を変化させ、染色の様子を測定した結果]]
-*蛍光たんぱくではないLacZは目視のみで発現を確認した
+  * LacZ, which is not fluorescent, was observed under the naked eye.
-*LacZはX-galの濃度によって目視での確認時間が変わった
+   * Time required for observation of LacZ staining depended on X-gal
-*液体で4時間以内に目視で確認することはできなかった
+concentration.
-::-->8時間後目視で確認できた
+   * We were not able to observe stainig at a liquid state within 4 hours.
-'''蛍光たんぱく'''
+       -->Staining was observed at the 8 hour point.'''蛍光たんぱく'''
 [[Image:Reporter chiba.gif|thumb|right|'''Table.''' それぞれのレポーター遺伝子の評価をした結果]]
-*液体、固体培地において似たような結果になった
+* Similar results were obtained for liquid and solid cultures.
-*目視で一番見やさはmCherry,GFP,YFPの順であった
+   * To the naked eye, fluorescence was observed most easily in the
-*固体培地のほうが目視で確認しやすい
+order mCherry, GFP, and YFP.
-*AHLでのインダクションのほうがIPTGより確認できた時間が早かった
+   * The solid state cultures were easier to observe to the naked eye.
-*GFP,YFPのほうがmCherryより確認できた時間が早かった
+   * AHL induction took a shorter time than IPTG for fluorescence to
+be observable.
+   * GFP and YFP showed observable fluorescence earlier than mCherry.
 <br clear=all>
 [[Image:Soliduv Chiba.gif|thumb|right|'''Fig'''UVを当てた固体培地上のレポーター]]