Team:Chiba/jk/γ/Trl

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==Time Responce Liquid==
==Time Responce Liquid==
===purpose===
===purpose===
-
<br>インダクションをかけてからいち早く、確認できる出力を見つけること
 
<br>To find the earliest output gene after induction
<br>To find the earliest output gene after induction
-
===装置&試薬(apparatus&reagents)===
+
===Reporter===
-
'''装置（apparatus)'''
+
*Fluorescent Protein
-
*しんとう培養器(shaking incubator)(37℃,30℃)
+
:*GFP
-
*48 well plate(deep well)
+
::*pGFPuv
-
*Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)
+
::BBa_T9002 
-
'''試薬(reagents)'''
+
:*Venus YFP
-
*AHL(100uM,5uM,100nM)
+
::*BBa_K084003 
-
*IPTG(100nM)
+
:::*pLac-Venus YFP
-
*X-gal
+
:*mCherry
-
*M9
+
pLac-mCherry
-
*PBS
+
*β-gal (X-gal assay)
 +
:*pUC19(plac-LacZα)
-
===Protocol===
+
===Equipment===
-
*pre培養(Day,1:pre culture)
+
:shaking incubator
-
:*LB培養液(2ml)に固体培地から大腸菌をpickし３７℃で12時間培養(48穴Deep Well)
+
::Innova 4200 Benchtop or Floor-Stackable Incubator Shaker(37&deg;C)
-
:*pick colony(E.coli) from solid culture medium
+
:46-well plate(deep well)
-
:*into LB culture fluid(2 ml)  
+
:96-well plate(deep well)  
-
:*cultivate for 12hours in shaking incubator(37℃) 
+
:Fluoroskan Ascent 2.5(program:Ascent Software Version 2.6)  
-
*本培(Day,2:main culture)
+
:Beckman Allegratm X-12R Centrifuga(Beckman Coulter)
-
:*measure OD values and dilution with LB culture fluid to 0.01(OD values)
+
-
:*cultivate to 1.0(OD values)
+
-
:*wash with physiological saline (2.0min 8,000rpm)x2
+
-
:ODを計測し1/200に希釈しOD=0.01から1.0になるまで培養
+
-
:OD=1.0になったら生理食塩水でwash(2.0min 8,000rpm)x2
+
-
:M9またはPBSで培養し、IPTG,AHLを加え計測する。
+
-
*計測
+
-
:0min,30min,60min,90min,120min,,,,,240minまで蛍光リーダー、OD値計測
+
-
:また、肉眼で確認
+
-
<br>'''Result'''
+
===Method===
 +
[[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]]
 +
Strain:XL10G Kan<sup>R</sup>
 +
 
 +
*Liquid medium experiment
 +
#Pre-culture
 +
##Picked and cultured the following plate in 2mL of LB:
 +
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
 +
###LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
 +
##Cultured at 37°C for 12h.
 +
#Culture
 +
##Dilute pre-cultures and add to new LB medium.
 +
###LB-Amp+0.2 % Glucose, (pGFPuv, pLac-Venus YFP, pLac-mCherry, pUC19)
 +
###LB-Amp, ([http://partsregistry.org/Part:BBa_T9002 BBa_T9002], [http://partsregistry.org/Part:BBa_K084003 BBa_K084003])
 +
##Cultured at 37°C for  about 6 h
 +
#Wash
 +
##Transfer 10mL each of the culture to 50mL centrifuge tubes.
 +
##Centrifuged for 6min at 3600rpm,20°C and discarded the supernatant.
 +
##Add physiological saline and resuspention
 +
##Repeated wash twice.
 +
##Add M9 minimal medium.
 +
#Mix
 +
##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 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.
 +
 
 +
===Result===
*[[TEam:Chiba/jk/γ/Trl/0828|28,August,2008]]
*[[TEam:Chiba/jk/γ/Trl/0828|28,August,2008]]
*[[TEam:Chiba/jk/γ/Trl/0905|5,September,2008]]
*[[TEam:Chiba/jk/γ/Trl/0905|5,September,2008]]
*[[TEam:Chiba/jk/γ/Trl/0910|10,September,2008]]
*[[TEam:Chiba/jk/γ/Trl/0910|10,September,2008]]
*[[TEam:Chiba/jk/γ/Trl/0914|14,September,2008]]
*[[TEam:Chiba/jk/γ/Trl/0914|14,September,2008]]
 +
 +
'''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.
 +
 +
 +
 +
 +
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"

Latest revision as of 04:41, 30 October 2008

実験ログ
出力班


Contents

Time Responce Liquid

purpose


To find the earliest output gene after induction

Reporter

  • Fluorescent Protein
  • GFP
  • pGFPuv
BBa_T9002
  • Venus YFP
  • BBa_K084003
  • pLac-Venus YFP
  • mCherry

pLac-mCherry

  • β-gal (X-gal assay)
  • pUC19(plac-LacZα)

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)

Method

Fig. レポーターのタイムレスポンスの実験方法

Strain:XL10G KanR

  • 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, (BBa_T9002, 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, (BBa_T9002, 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 (BBa_T9002, BBa_K084003)
  5. Measure fluorescence intensity every 1 h.

Result

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.




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