Team:Chiba/Experiments:Reporter

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[[Image:Chiba-U.gif]]
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!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba/Team|The Team]]
!align="center"|[[Team:Chiba/Team|The Team]]
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==Reporter==
==Reporter==
-
本プロジェクトではある一定時間がたった時に、遺伝子発現が起こるタイマーを作ることが目的
+
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.
-
レシーバーの遺伝子発現から確認までのタイムラグができるだけ少ない出力が必要
+
We imagined three kinds of output:
-
そこで候補となったのは、以下の3種類
 
-
*Fluorescent Protein
 
-
*Bioluminescence(luciferase)
 
-
*Cell staining (LacZ and X-gal assay)
 
 +
* Fluorescent Protein
 +
* Bioluminescence(luciferase)
 +
* Cell staining (LacZ and X-gal assay)
-
蛍光たんぱくとβ-gal(lacZ)を実験で使用した。
 
 +
and tested Fluorescent Protein and β-gal(lacZ).
-
蛍光たんぱくとしてGFP、maturationの早いVenus YFP[http://www.nature.com/nbt/journal/v20/n1/full/nbt0102-87.html|(1)],目視での確認が容易なmCherry[http://www.ncbi.nlm.nih.gov/pubmed/15558047|(2)],を選んだ。
+
We selected GFP, Venus YFP, and mCherry. The latter two were chosen because Venus YFP matures quickly[[Team:Chiba/Reference#Output|(1)]],and we can easibly recognize mCherry coloration[[Team:Chiba/Reference#Output|(2)]].
-
Luciferaseは化学発光であるために、発現の確認には暗所でなければならない。使用条件が限られてしまうため候補から外した
+
 
 +
We used fluorescent proteins and &beta;-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==
==Experiment==
-
[[Image:Reporter method Chiba.jpg|rifht|thumb|'''Fig. '''レポーターのタイムレスポンスの実験方法]]
+
 
 +
 
 +
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
*Fluorescent Protein
Line 38: Line 50:
::[[Image:PGFPuv chiba.gif]]
::[[Image:PGFPuv chiba.gif]]
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
-
::[[Image:Reporter 9002 chiba.gif]]
+
::[[Image:High-Copy-Receiver Chiba.gif]]
:*Venus YFP
:*Venus YFP
::*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K084003 BBa_K084003]
::*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K084003 BBa_K084003]
Line 51: Line 63:
::[[Image:PLac-lacZ-Chiba.gif]]
::[[Image:PLac-lacZ-Chiba.gif]]
-
*Equipment
+
*Fluorescent Protein
-
:shaking incubator
+
:*GFP 
-
::Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37&deg;C) 
+
::*pGFPuv
-
:46-well plate(deep well)
+
::[[Image:PGFPuv chiba.gif]]
-
:96-well plate(deep well)  
+
::*[http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
-
:Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
+
::[[Image:High-Copy-Receiver Chiba.gif]]
-
:Beckman Allegratm X-12R Centrifuga(Beckman Coulter)
+
:*Venus YFP
 +
::*[http://partsregistry.org/wiki/index.php?title=Part:BBa_K084003 BBa_K084003]
 +
::[[Image:K084003 chiba.gif]]
 +
::*pLac-Venus YFP
 +
::[[Image:pLac_Venus_Chiba.gif]]
 +
:*mCherry
 +
::*pLac-mCherry
 +
::[[Image:PLac_mChe_Chiba.gif]]
 +
*β-gal (X-gal assay)
 +
:*pUC19(plac-LacZα)
 +
::[[Image:PLac-lacZ-Chiba.gif]]
 +
 
 +
==Result & Discussion==
 +
'''Result'''
 +
'''LacZ'''
 +
[[Image:LacZ chiba.gif|thumb|right|'''Table  1''' Dependent on X-gal concentration.]]
 +
* 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.
 +
 
 +
 
 +
 
 +
'''Fluorescent protein'''
 +
[[Image:Reporter chiba.gif|thumb|right|'''Table  2''' Result of Time chase.]]
 +
* 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.
 +
<br clear=all>
 +
[[Image:Soliduv Chiba.gif|thumb|right|'''Photo  1''' Emission of fluorescent protein]]
 +
 
 +
'''Discussion'''
 +
Discussion
 +
 
 +
 
 +
The purpose of this project is to alter the time required for expression.
 +
 
 +
* By increasing the concentration of X-gal, the time required for
 +
expression to be observable decreased, but this still takes longer
 +
than the time required when using fluorescent proteins.
 +
 
 +
 
 +
*Using β-gal, which uses X-gal as substrate, the time required for
 +
output may increase because the substrate concentration decreases with
 +
time.
 +
 
 +
For the above reasons, fluorescence proteins are more suited for our purposes.
 +
 
 +
Of the fluorescent proteins, GFP and YFP fluorescence was observable
 +
at earlier stages.
 +
 
 +
 
 +
* But to the naked eye, GFP could be observed more easily.
 +
 
 +
Therefore, we chose GFP as our output reporter.
 +
 
 +
<br clear=all>
===Method===
===Method===
 +
[[Image:Reporter method Chiba.jpg|rifht|thumb|'''Fig.  1'''      Experimental method for response measuring of reporters]]
Strain:XL10G Kan<sup>R</sup>
Strain:XL10G Kan<sup>R</sup>
*Agar plate experiment
*Agar plate experiment
Line 65: Line 135:
##Picked and cultured the following plate stocks in 2mL of LB:
##Picked and cultured the following plate stocks in 2mL of LB:
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
-
###LB-Amp, (BBa_T9002, BBa_K084003)
+
###LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
##Cultured at 37°C for 12h.
##Cultured at 37°C for 12h.
#Spread on plate
#Spread on plate
##Spread on new plate
##Spread on new plate
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
-
###LB-Amp, (BBa_T9002, BBa_K084003)
+
###LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
##Cultured at 37°C for 12h.
##Cultured at 37°C for 12h.
#Colony lift
#Colony lift
##Colony lift to inducible agar plate (containing IPTG or AHL)
##Colony lift to inducible agar plate (containing IPTG or AHL)
###LB-Amp+0.2 mM IPTG agar plate, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
###LB-Amp+0.2 mM IPTG agar plate, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
-
###LB-Amp+100 nM AHL agar plate, (BBa_T9002, BBa_K084003)
+
###LB-Amp+100 nM AHL agar plate, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
##Incubate at 37 °C
##Incubate at 37 °C
#Check expression every 30 min.
#Check expression every 30 min.
Line 83: Line 153:
##Picked and cultured the following plate in 2mL of LB:
##Picked and cultured the following plate in 2mL of LB:
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
-
###LB-Amp, (BBa_T9002, BBa_K084003)
+
###LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
##Cultured at 37°C for 12h.
##Cultured at 37°C for 12h.
#Culture
#Culture
##Dilute pre-cultures and add to new LB medium.
##Dilute pre-cultures and add to new LB medium.
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
-
###LB-Amp, (BBa_T9002, BBa_K084003)
+
###LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
-
##Cultured at 37°C for 4~5 h
+
##Cultured at 37°C for about 6 h
#Wash
#Wash
##Transfer 10mL each of the culture to 50mL centrifuge tubes.
##Transfer 10mL each of the culture to 50mL centrifuge tubes.
Line 99: Line 169:
##Dispens each culture into 48-well deep well or 96-well deep well
##Dispens each culture into 48-well deep well or 96-well deep well
##Add IPTG fainal conc. 0.2 mM (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
##Add IPTG fainal conc. 0.2 mM (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
-
##Add AHL fainal conc. 100nM (BBa_T9002, BBa_K084003)
+
##Add AHL fainal conc. 100nM ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
#Measure fluorescence intensity every 1 h.
#Measure fluorescence intensity every 1 h.
 +
*Equipment
 +
:shaking incubator
 +
::Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37&deg;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)
-
*固体編
+
{| style="color:white;" cellpadding="3" cellspacing="3" border="0" width="100%" align="center" class="menu" |
-
固体培地(pLac:0.2%グルコース入り)にまく<br>
+
-
コロニーを新しい固体培地(pLac:IPTG, plux:AHL, LacZ:IPTG&X-gal入り)にコロニーリフトする,計測開始<br>
+
-
37&deg;Cのしんとう培養器で培養<br>
+
-
30分ごとに目視で確認できるか観察<br>
+
-
 
+
-
*液体編
+
-
プレ培<br>
+
-
コロニーを固体培地からピックし,LB培養液2ml(pLacであれば0.2%グルコース入り)で12時間培養(37&deg;C)<br>
+
-
本培<br>
+
-
OD値を計測して、菌数を合わせるため培養液を1/200に希釈する。<br>
+
-
OD=1.00になるまで37&deg;Cで培養<br>
+
-
生理食塩水で洗浄(2.0min 8,000rpm)x2<br>
+
-
46穴または96穴deep wellにM9(2ml)<br>
+
-
IPTG(最終濃度0.1mM)またはAHL(最終濃度100nM)を加えて、計測開始<br>
+
-
30分ごとに蛍光強度、OD値を計測、また目視で確認できるか観察<br>
+
-
 
+
-
==Result & Discussion==
+
-
'''Result'''
+
-
'''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はX-galの濃度によって目視での確認時間が変わった
+
-
*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
+
-
::-->8時間後目視で確認できた
+
-
 
+
-
'''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
+
-
*目視で一番見やさはmCherry,GFP,YFPの順であった
+
-
*固体培地のほうが目視で確認しやすい
+
-
*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
+
-
*GFP,YFPのほうがmCherryより確認できた時間が早かった
+
-
 
+
-
 
+
-
<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]]
+
-
 
+
-
'''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。
+
-
<br clear=all>
+
-
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
+
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba|Home]]
!align="center"|[[Team:Chiba/Team|The Team]]
!align="center"|[[Team:Chiba/Team|The Team]]

Latest revision as of 12:43, 16 December 2008

Chiba-U.gif


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
PGFPuv chiba.gif
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
High-Copy-Receiver Chiba.gif
  • Venus YFP
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K084003 BBa_K084003]
K084003 chiba.gif
  • pLac-Venus YFP
PLac Venus Chiba.gif
  • mCherry
  • pLac-mCherry
PLac mChe Chiba.gif
  • β-gal (X-gal assay)
  • pUC19(plac-LacZα)
PLac-lacZ-Chiba.gif
  • Fluorescent Protein
  • GFP
  • pGFPuv
PGFPuv chiba.gif
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002]
High-Copy-Receiver Chiba.gif
  • Venus YFP
  • [http://partsregistry.org/wiki/index.php?title=Part:BBa_K084003 BBa_K084003]
K084003 chiba.gif
  • pLac-Venus YFP
PLac Venus Chiba.gif
  • mCherry
  • pLac-mCherry
PLac mChe Chiba.gif
  • β-gal (X-gal assay)
  • pUC19(plac-LacZα)
PLac-lacZ-Chiba.gif

Result & Discussion

Result LacZ

Table 1 Dependent on X-gal concentration.
  • 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.


Fluorescent protein

Table 2 Result of Time chase.
  • 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.


Photo 1 Emission of fluorescent protein

Discussion Discussion


The purpose of this project is to alter the time required for expression.

  • By increasing the concentration of X-gal, the time required for

expression to be observable decreased, but this still takes longer than the time required when using fluorescent proteins.


  • Using β-gal, which uses X-gal as substrate, the time required for

output may increase because the substrate concentration decreases with time.

For the above reasons, fluorescence proteins are more suited for our purposes.

Of the fluorescent proteins, GFP and YFP fluorescence was observable at earlier stages.


  • But to the naked eye, GFP could be observed more easily.

Therefore, we chose GFP as our output reporter.


Method

Fig. 1 Experimental method for response measuring of reporters

Strain:XL10G KanR

  • Agar plate experiment
  1. 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, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Cultured at 37°C for 12h.
  2. Spread on plate
    1. Spread on new plate
      1. LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
      2. LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Cultured at 37°C for 12h.
  3. 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, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Incubate at 37 °C
  4. Check expression every 30 min.
  • Liquid medium experiment
  1. 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, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Cultured at 37°C for 12h.
  2. 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, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
    2. Cultured at 37°C for about 6 h
  3. 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.
  4. 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 ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
  5. 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)