Team:Chiba/Calendar-Home/2 September 2008

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==Laboratory work==
==Laboratory work==
===Team:Input===
===Team:Input===
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UV照射実験
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UV irradiation test
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Incubated cultures (from Glycerol Stocks) with 2ml of LB-Ampicillin Medium for 12 hours at 37 degrees.
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#Incubated cultures (from Glycerol Stocks) with 2ml of LB-Ampicillin Medium for 12 hours at 37 degrees.
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<BR>
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#Pre-incubated was plated so as to produce about 1000 colonies.(Ptet:1,Ptet-RFP:1,PrecA-RFP:3)
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ODを測定して10<sup>2</sup>~10<sup>3</sup>のコロニーができるようにプレートに撒く。Ptet×1(コントロール用),Ptet-RFP×1(コントロール用),Prec-RFP×3(コントロール用×1,UV照射用×2)
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#We put　Negative Control　of　Prec-RFP in a dark place.
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*新たなAmpプレート8枚(2.5cm,6.5cmのそれぞれ30sec,1min,30min,1h用)に、撒いて37&deg;C,12hたったコントロール用のPtet,Ptet-RFPのコロニーをニトロセルロースでうつしてはる。
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#To 8 new Amp plates (2.5cm or 6.5cm apart from UV source for 30sec,1min, 30min, and 1h each) cultures grown at 37&deg;C for 12h were transfered using nitrocellulose filters.
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*PrecA-RFPのプレートにUVを照射する。UVからの距離は2.5cmと,6.5cmの2パターンで実験する。Prec-RFPのコントロール用は暗所に置いておく。
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#These are controls with Ptet and Ptet-RFP colonies.
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*UV照射後30sec,1min,30min,1h経過したらそれぞれ（2.5cmでUV当てたもの、6.5cmでUV当てたもの、コントロール用）のプレートからニトロセルロースでコロニーをうつしとり、あらかじめコントロールをはっておいたAmpプレートにはりつけた。
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#PrecA-RFP plates were exposed to UV(254nm) at a distance 2.5cm or 6.5cm apart.
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*それぞれのプレートでUVが照射されてからどのくらいの時間でRFPが発現するのかを調べるためにUV照射してから、0min,10min,30min,1h,2h,3h,4h,5h,6h後にスキャナーで取り込んで色の変化を見る。
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#A Negative Control　of　Prec-RFP was placed in a dark place.
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#After UV exposure for 30sec, 1min, 30min, or 1h (each with either 2.5 or 6.5cm from the UV source or without light exposure), we transfered using nitrocellulose colonies from these plates to Amp control plates.
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#We then tested the amount of time required for RFP expression after UV exposure. To do this, we scanned in the exposed plates after the various exposure times and observed the color change.
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*結果
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result
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::PrecA-RFPにUVを当てたものはいづれもRFPが目で確認できなかった。
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We were not able to visually observe RFP fluorescence from  plates containing PrecA-FRP.
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#Incubated cultures(from Glycerol Stocks) with 2mL of LB-Ampicillin Medium for 12 hours at 37 degrees.
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OD:#UV⊕:5.21,UV⊖(negative control):5.38
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#moved the cultures to small plates and started UV-irradiation.(wavelength:254nm,distance from the UV lamp to the cultures were 7.5cm.Put the negartive control in adark place.
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#covered the plates with polyethylene wrap.
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#after irradiation(Sample:0min,10min,30min,1h,2h,4h,6h,8h)(Negative Control:0h,1h,4h,8h),agitate the culture and
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よく攪拌してから、20μl採取。
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#diluted each cultures with LB-ampicillin medium 10<sup>4</sup>-fold and 10<sup>5</sup>-fold (volume/volume).
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#incoculated 20μl of the cultures and incubated for 12 hours at 37 degrees.
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#counted the CFU(determined viable cell count).
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===Team:Communication===
===Team:Communication===

Latest revision as of 03:10, 30 October 2008

>Home | Notebook

1 September 2008 <|> 3 September 2008

Contents

Laboratory work

Team:Input

UV irradiation test

  1. Incubated cultures (from Glycerol Stocks) with 2ml of LB-Ampicillin Medium for 12 hours at 37 degrees.
  2. Pre-incubated was plated so as to produce about 1000 colonies.(Ptet:1,Ptet-RFP:1,PrecA-RFP:3)
  3. We put　Negative Control　of　Prec-RFP in a dark place.
  4. To 8 new Amp plates (2.5cm or 6.5cm apart from UV source for 30sec,1min, 30min, and 1h each) cultures grown at 37°C for 12h were transfered using nitrocellulose filters.
  5. These are controls with Ptet and Ptet-RFP colonies.
  6. PrecA-RFP plates were exposed to UV(254nm) at a distance 2.5cm or 6.5cm apart.
  7. A Negative Control　of　Prec-RFP was placed in a dark place.
  8. After UV exposure for 30sec, 1min, 30min, or 1h (each with either 2.5 or 6.5cm from the UV source or without light exposure), we transfered using nitrocellulose colonies from these plates to Amp control plates.
  9. We then tested the amount of time required for RFP expression after UV exposure. To do this, we scanned in the exposed plates after the various exposure times and observed the color change.

result

We were not able to visually observe RFP fluorescence from plates containing PrecA-FRP.

Team:Communication

(31/8)--->Gel Check
Chiba-0902.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6μl
From left;
[http://partsregistry.org/Part:BBa_I9026 BBa_I9026] -> OK, 100/μL
[http://partsregistry.org/Part:BBa_I9030 BBa_I9030] -> OK, 50ng/μL
[http://partsregistry.org/Part:BBa_S03154 BBa_S03154] -> OK, 30ng/μL (too low for the ligation:1/9 )


(1/9)---> Colony PCR
Colony PCR of 8 colonies from ligation plates (1/9:(1)[http://partsregistry.org/Part:BBa_K084009 BBa_K084009](R1~R8),(2)[http://partsregistry.org/Part:BBa_K084010 BBa_K084010](C1~C8)) and one from control plate([http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2007)).
DNA Template 1
dNTP mix 5
Foward Primer 0.3
Reverse Primer 0.3
DNA polymerase TAQ 0.5
Thermopol Buffer 3
dH2O 20.5
TOTAL 30μL


95°C,5min -> ( 95°C,1min -> 52°C,1min -> 72°C,1min )･･･25cycles -> 72°C,10min -> 6°C


--->Gel Check

Chiba-0902-2.JPG
Sample DNA 1
Loading Dye 1
dH2O 4
TOTAL 6μl
From left;
  • Plac+RBS+RhlI+LVA
R1 -> OK
R2 -> Bad
R3~R7 -> OK
R8 -> Bad
Chiba-0902-3.JPG
From left;
  • Plac+RBS+CinI+LVA
C1,C2 -> OK
C3 -> Bad
C4~C6 -> OK
Chiba-0902-4.JPG
From left;
  • Plac+RBS+CinI+LVA
C7,C8 -> OK
  • [http://partsregistry.org/Part:BBa_F2620 BBa_F2620](2007):Positive control -> OK


--->(3/9)Mini prep



(1/9)--->Liquid Culture

Cultured the following cells (2mL LB-Amp, at 37°C, 7 hours)
from transformed plates:
  • [http://partsregistry.org/Part:BBa_K084007 BBa_K084007](Plac+RBS+LasI, Competent Cells : JW1908)
  • [http://partsregistry.org/Part:BBa_K084008 BBa_K084008](Plac+RBS+RhlI, Competent Cells : JW1908)
  • [http://partsregistry.org/Part:BBa_T9002 BBa_T9002](Ptet+RBS+LuxR+GFP, Competent Cells : JW1908)
from Glycerol Stock:
  • [http://partsregistry.org/Part:BBa_S03623 BBa_S03623](Ptet+RBS+LuxI, Competent Cells : JW1908)

--->(3/9)Phenotype test


Transformation

Competent cells : XL10G 30μL
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2007)
  • [http://partsregistry.org/Part:BBa_C0161 BBa_C0161](2006)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2007)
  • [http://partsregistry.org/Part:BBa_C0261 BBa_C0261](2006)

--->(4/9)Mini prep

Team:Output

TIME RESPONCE (Solid)

Colony PCR

  • [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]+[http://partsregistry.org/Part:BBa_J52008 BBa_J52008]
Sample No. 1
culture 1
Fwd primer 1.5
Rev primer 1.5
Thermo pol Buffer 3
dNTP mix 3
Taq DNA pol (NEB) 0.2(1　unit)
dH2O 19.8
TOTAL 30μl


-->95°C 5 min -->(95°C 1min -->50°C　30sec -->72°C　1min)x25 -->72°C　10min


Mini prep

  • [http://partsregistry.org/Part:BBa_R0079 BBa_R0079]
  • [http://partsregistry.org/Part:BBa_R0071 BBa_R0071]
  • [http://partsregistry.org/Part:BBa_R0077 BBa_R0077]
  • [http://partsregistry.org/Part:BBa_R0078 BBa_R0078]
  • [http://partsregistry.org/Part:BBa_R0062 BBa_R0062]
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