Team:Chiba/jk/β

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(18 August, 2008P)
 
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-
'''β班の実験ノート'''
+
<html><link rel="stylesheet" href="https://2008.igem.org/wiki/index.php?title=User:Maiko/chiba.css&action=raw&ctype=text/css" type="text/css" /></html>
 +
>[[Team:Chiba/Internal|ノートに戻る]]
-
==Week 1==
 
-
===17 August, 2008===
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
::*BBa_R0040(2008) [http://partsregistry.org/Part:BBa_R0040] -> No colonies on plates
 
-
::*BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070] -> No colonies on plates
 
-
::*BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
 
-
::*BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] -> No colonies on plates
 
-
::*BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] -> No colonies on plates
 
-
::*BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079] -> No colonies on plates
 
 +
==送受信班の実験ノート==
 +
*[[Team:Chiba/jk/β/week 1|week 1]]
 +
*[[Team:Chiba/jk/β/week 2|week 2]]
 +
*[[Team:Chiba/jk/β/week 3|week 3]]
 +
*[[Team:Chiba/jk/β/week 4|week 4]]
 +
*[[Team:Chiba/jk/β/week 5|week 5]]
 +
*[[Team:Chiba/jk/β/week 6|week 6]]
-
===18 August, 2008===  
+
==モジュール解説==
-
:'''[[Team:Chiba/protocol/PCR|PCR]]'''
+
===感度差による応答時間の差===
-
::*BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070]
+
任意の順番と、delay-timeをもった遺伝子発現を実現するために、バクテリア間の細胞間通信、クオラムセンシングを利用する。クオラムセンシングでは、応答閾値を超えるAHLが蓄積されたとき初めて、目的遺伝子の発現がおこる。AHL情報がじゅうぶんゆっくり蓄積するとき、ひとつのAHL送信装置に対して、感度の異なる複数の受信装置によってさまざまな出力装置を独立に起動すれば、感度の高いAHL 受信機ほど先に起動する。
-
::*BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076]
+
-
::*BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077]
+
-
::*BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078]
+
-
::*BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079]
+
-
::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]・・・control
+
 +
=== Controlling the Delay time of the AHL signaling ===
 +
LuxIのつくるAHLに対して、感度差による応答時間差は、2つの方法で実現することができる。
 +
#LuxR/ Pluxの感度変異体を用いる場合<br>LuxRの変異体を用いれば、pluxがonになるAHL濃度が<1nMから~10nMまで変化する。[http://authors.library.caltech.edu/5553/ C. H. Collins.''et al.:''Mol.Microbiol.2005.'''55'''(3).712–723]
 +
#クロストークを使う場合
 +
:AHL=Acyl-homoserine Lactoneには、そのアシル基が異なる種類がある。luxIが合成するAHLは、3-oxo-hyxanoyl-Homoserine Lactone (=OHHL)であり、3-oxo-dodecanoyl-homoserine Lactone(=ODHL)や、butyryl-homoserine Lactone(=HHL)が存在する。それぞれ、合成酵素はLasI、RhlI(ともにシュードモナス由来)と呼ばれる。このほかのAHL分子と、その合成酵素を以下に示した。これらのAHL分子は、アシル基のみが異なるため、一定の割合でLuxRと相互作用する(cross-talkする)。たとえば、OdHLとLuxRとのcross-talk(LasI-LuxR)は、OHHLとLuxRの場合(LuxI-LuxR)の1/20である。AHL分子の蓄積速度が一定ならば,luxI-LuxR/Pluxよりも20倍遅くスイッチが入ることとなる。
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
+
*AHLsおよびその合成酵素
 +
<table width="500" border="1" cellpadding="0" cellspacing="0" bordercolor="#000000" center>
<tr>
<tr>
-
<td width="257">DNA Template</td>
+
<td width="200">Strain</td>
-
<td>1</td>
+
<td width="200">Signal Molecule</td>
 +
<td width="150">Enzyme<td>
</tr>
</tr>
<tr>
<tr>
-
<td>dNTP mix</td>
+
<td>P.aeruginosa</td>
-
<td>10</td>
+
<td>C4-HSL</td>
 +
<td>RhlI</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Foward Primer</td>
+
<td>V. fisheri</td>
-
<td>5</td>
+
<td>C6-3-oxo-HSL</td>
 +
<td>LuxI</td>
</tr>
</tr>
<tr>
<tr>
-
<td>Reverse Primer</td>
+
<td>A.tumefaciens</td>
-
<td>5</td>
+
<td>C8-3-oxo-HSL</td>
 +
<td>TraI</td>
</tr>
</tr>
<tr>
<tr>
-
<td>DNA polymerase</td>
+
<td>P.aeruginosa</td>
-
<td>1</td>
+
<td>C10-3-oxo-HSL</td>
 +
<td>VanI</td>
</tr>
</tr>
-
<tr>
 
-
<td>Thermopol Buffer</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>68</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>100μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0818.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
<tr>
<tr>
-
<td width="257">Sample DNA</td>
+
<td>P.aeruginosa</td>
-
<td>3</td>
+
<td>C12-3-oxo-HSL</td>
 +
<td>LasI</td>
</tr>
</tr>
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
::*BBa_C0070(2008) [http://partsregistry.org/Part:BBa_C0070] ->None
 
-
::*BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] ->None
 
-
::*BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] ->None
 
-
::*BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] ->None
 
-
::*BBa_C0079(2008) [http://partsregistry.org/Part:BBa_C0079] ->None
 
-
::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]・・・control ->OK
 
-
|}
 
-
 
-
 
-
 
-
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
compitent cell : XL10G
 
-
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
 
-
::*BBa_C0076(2006) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates(19/8)
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]
 
-
 
-
 
-
:--->(20/8)[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]
 
-
 
-
===19 August, 2008===
 
-
:'''[[Team:Chiba/protocol/PCR|PCR]]'''
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_S3154(2007) [http://partsregistry.org/Part:BBa_S3154]
 
-
::*BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154]
 
-
::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]・・・control
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
<tr>
<tr>
-
<td width="257">DNA Template</td>
+
<td>R.leguminosarum</td>
-
<td>1</td>
+
<td>C14-3-hydroxy-HSL</td>
-
</tr>
+
<td>CinI</td>
-
<tr>
+
-
<td>dNTP mix</td>
+
-
<td>10</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Foward Primer</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Reverse Primer</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>DNA polymerase VENT</td>
+
-
<td>1</td>
+
-
</tr>
+
-
<tr>
+
-
<td>Thermopol Buffer</td>
+
-
<td>5</td>
+
-
</tr>
+
-
<tr>
+
-
<td>dH2O</td>
+
-
<td>28</td>
+
-
</tr>
+
-
<tr>
+
-
<td>TOTAL</td>
+
-
<td>50μL</td>
+
</tr>
</tr>
</table>
</table>
 +
[http://partsregistry.org/Part:BBa_F2620:Specificity AHLsについて]
 +
==実験==
 +
===Construction(From BioBrick)===
 +
:*Sender(Autoinducer synthase)
 +
::LuxI,LasI,CinI,RhlI(lacプロモーター)
 +
:*Receiver
 +
::LuxR,LasR,CinR,RhlR
-
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0819.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026] ->None
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026] ->None
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030] ->None
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030] ->None
 
-
::*BBa_S3154(2007) [http://partsregistry.org/Part:BBa_S3154] ->None
 
-
::*BBa_S3154(2006) [http://partsregistry.org/Part:BBa_S3154] ->None
 
-
::*BBa_F2620(2007) [http://partsregistry.org/Part:BBa_F2620]・・・control  ->OK
 
-
|}
 
-
 
-
 
-
 
-
===20 August, 2008===
 
-
:(18/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820-3.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
|}
 
-
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820-4.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>24μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> OK
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070] -> OK
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070] -> OK
 
-
|}
 
-
 
-
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;<br>
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK<br>
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK<br>
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK<br>
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK<br>
 
-
|}
 
-
 
-
 
-
--->'''[Team:Chiba/protocol/digestion|Digestion test]]'''
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170]</td>
 
-
<td>3</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170]</td>
 
-
<td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178]</td>
 
-
<td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>3</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178]</td>
 
-
<td>-</td><td>-</td><td>-</td><td>3</td><td>-</td><td>-</td><td>-</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>-</td><td>-</td><td>-</td><td>-</td><td>0.1</td><td>0.1</td><td>0.1</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer EcoRⅠ</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4.9</td><td>4.9</td><td>4.9</td><td>4.9</td><td>4.8</td><td>4.8</td><td>4.8</td><td>4.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820-2.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
:From left;
 
-
:(Single Digestion 1~4)
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK
 
-
:(Double Digestion 1~4)
 
-
::*BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK
 
-
::*BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK
 
-
::*BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK
 
-
::*BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK
 
-
|}
 
-
 
-
 
-
 
-
 
-
===21 August, 2008===
 
-
:(20/8)--->'''[[Team:Chiba/protocol/PCR|PCR]]'''
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076]
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">DNA Template</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dNTP mix</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Foward Primer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Reverse Primer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>DNA polymerase TAQ</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Thermopol Buffer</td>
 
-
<td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>28</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>50μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:95℃,5min -> ( 95℃,1min -> 52℃,1min -> 72℃,1min )・・・25cycles -> 72℃,10min -> 6℃
 
-
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
{|align="justify"
 
-
|[[Image:Chiba-0821.JPG]]
 
-
|
 
-
:<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12μL</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:From left;
 
-
::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078] -> None
 
-
::*BBa_C0076(2007) [http://partsregistry.org/Part:BBa_C0076] -> None
 
-
::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070] -> OK
 
-
::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070] -> OK
 
-
|}
 
-
 
-
 
-
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
 
-
Compitentcell : XL10G
 
-
 
-
 
-
--->(23/8)'''[[Team:Chiba/protocol/digestion|Digestion]]'''
 
-
 
-
 
-
 
-
===22 August, 2008===
 
-
:'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
::*BBa_J13002(2007) [http://partsregistry.org/Part:BBa_J13002]
 
-
::*BBa_J13002(2006) [http://partsregistry.org/Part:BBa_J13002]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
::*BBa_J04500(2006) [http://partsregistry.org/Part:BBa_J04500]
 
-
:(Locate & 2 Punch ~iyama ver.~)
 
-
::*BBa_C0076(2008) [http://partsregistry.org/Part:BBa_C0076] -> No colonies on plates
 
-
::*BBa_C0077(2008) [http://partsregistry.org/Part:BBa_C0077] -> No colonies on plates
 
-
::*BBa_C0078(2008) [http://partsregistry.org/Part:BBa_C0078] -> No colonies on plates
 
-
 
-
 
-
 
-
 
-
===23 August, 2008===
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
 
-
 
-
:(21/8)--->'''[[Team:Chiba/protocol/digestion|Digestion]]'''
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
 
-
 
-
:--->[[Team:Chiba/protocol/gelcheck|Gel Check]]</table>
 
-
{|align="justify"
 
-
|[[Image:Chiba-0820.JPG]]
 
-
|
 
-
From the left;<br>
 
-
①BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK<br>
 
-
②BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK<br>
 
-
③BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK<br>
 
-
④BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0823-1.JPG]]
 
-
|
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
|}
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0823-2.JPG]]
 
-
|
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
::*M
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03154(2006) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9026(2006) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0823-3.JPG]]
 
-
|
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Single Digestion
 
-
::*BBa_J04500(2007) [http://partsregistry.org/Part:BBa_J04500]Double Digestion
 
-
|}
 
-
 
-
==Week 2==
 
-
*24 August, 2008
 
-
:(22/8)--->[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]
 
-
::*BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
 
-
::*BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
 
-
::*BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::*BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
 
-
 
-
:--->[[Team:Chiba/protocol/digestion|Digestion]]
 
-
::*BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
 
-
::*BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
 
-
::*BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::*BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
 
-
 
-
:--->[[Team:Chiba/protocol/gelcheck|Gel Check]]
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td><td>④</td><td>⑤</td><td>⑥</td><td>⑦</td><td>⑧</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>6</td><td>6</td><td>6</td><td>6</td><td>6</td><td>6</td><td>6</td>
 
-
</tr>
 
-
</table>
 
-
{|align="justify"
 
-
|[[Image:Chiba-0824-1.JPG]]
 
-
|
 
-
From the left;
 
-
::⑤BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::⑥BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
 
-
::⑦BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
 
-
::⑧BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
 
-
(↓single digestuion)
 
-
::⑤BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::⑥BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
 
-
::⑦BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
 
-
::⑧BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
 
-
|}
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>⑤</td><td>⑥</td><td>⑦</td><td>⑧</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>9</td><td>9</td><td>9</td><td>9</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
{|align="justify"
 
-
|[[Image:Chiba-0824-2.JPG]]
 
-
|
 
-
From the left;
 
-
(single digestuion)
 
-
::⑤BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::⑥BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2006)
 
-
::⑦BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2007)
 
-
::⑧BBa_J13002[http://partsregistry.org/Part:BBa_J13002](2006)
 
-
|}
 
-
 
-
*25 August, 2008
 
-
:(24/8)--->[[Team:Chiba/protocol/digestion|Digestion]]
 
-
::①Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::②Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
::③Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
::④BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007)
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td><td>④</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>5</td><td>5</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.5</td><td>o.5</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td>
 
-
<td>0.5</td><td>0.5</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 1</td>
 
-
<td>1</td><td>1</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>-</td><td>-</td><td>1</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>1</td><td>1</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>2</td><td>2</td><td>7.5</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>1</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:--->[[Team:Chiba/protocol/gelcheck|Gel Check]]
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td><td>④</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td><td>2</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>-</td><td>-</td><td>-</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td><td>12</td><td>6</td>
 
-
</tr>
 
-
</table>
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-1.JPG]]
 
-
|
 
-
From right;
 
-
::①Double Digestion:BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007) -> OK
 
-
::②Double Digesiton:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
 
-
::③Single Digestion:BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> ?(low)
 
-
::④BBa_R0010[http://partsregistry.org/Part:BBa_R0010](2007) -> OK(low)
 
-
|}
 
-
 
-
 
-
--->[[Team:Chiba/protocol/digestion|Digestion]]
 
-
::①BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
 
-
::②BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
 
-
::③BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>33ng/μl×60μl</td><td>33ng/μl×60μl</td><td>100ng/μl×20μl</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td>
 
-
<td>2</td><td>2</td><td>-</td>
 
-
</tr>]
 
-
<td>SpeⅠ</td>
 
-
<td>-</td><td>-</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 2</td>
 
-
<td>-</td><td>-</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>10</td><td>10</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>10</td><td>10</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>16</td><td>16</td><td>16</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>100</td><td>100</td><td>52</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>30</td><td>30</td><td>30</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>6</td><td>6</td><td>6</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>36</td><td>36</td><td>36</td>
 
-
</tr>
 
-
</table>
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-2.JPG]]
 
-
|
 
-
::①BBa_C0178[http://partsregistry.org/Part:BBa_C0178]
 
-
:::Insert:609bp -> OK
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-3.JPG]]
 
-
|
 
-
::②BBa_C0170[http://partsregistry.org/Part:BBa_C0170]
 
-
:::Insert:609bp -> OK
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-4.JPG]]
 
-
|
 
-
::③BBa_J04500[http://partsregistry.org/Part:BBa_J04500](2007)
 
-
::: -> OK
 
-
|}
 
-
 
-
 
-
*26 August, 2008
 
-
(8/25)--->'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
 
-
:::①5μl
 
-
:::②5μl
 
-
:::③27μl
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>H2O</td>
 
-
<td>8</td><td>8</td><td>8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0825-5.JPG]]
 
-
|
 
-
From left;
 
-
::①insert:C0178 -> OK
 
-
::②insert:C0170 -> OK?
 
-
::③Vector:J04500 -> OK
 
-
|}
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/ligation/dephosphorylation|Dephosphorylation]]'''
 
-
:::③
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
 
-
:::③->9μl
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA ③</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td></tr>
 
-
</table>
 
-
{|align="justify"
 
-
|[[Image:Chiba-0826.JPG]]
 
-
|
 
-
③ -> OK ・・・30ng?
 
-
|}
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/ligation/ligation|Ligation]]'''
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>(1)</td><td>(2)</td><td>(3)</td><td>(4)</td><td>(5)</td>
 
-
</tr>
 
-
<tr>
 
-
<td>insert①C0178</td>
 
-
<td>1</td><td>-</td><td>-</td><td>1</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>insert②C0170</td>
 
-
<td>-</td><td>1</td><td>-</td><td>-</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>vector③J04500</td>
 
-
<td>-</td><td>-</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>]
 
-
<td>ligase</td>
 
-
<td>1</td><td>1</td><td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer</td>
 
-
<td>2</td><td>2</td><td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>6</td><td>6</td><td>5</td><td>4</td><td>4</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
::(1)insert①C0178
 
-
::(2)insert②C0170
 
-
::(3)vector③J04500
 
-
::(4)insert①C0178+vector③J04500
 
-
::(5)insert②C0170+vector③J04500
 
-
 
-
 
-
 
-
*27 August, 2008
 
-
[[Team:Chiba/protocol/digestion|Digestion Test]]
 
-
::Sample DNA : BBa_R0010[http://partsregistry.org/Part:BBa_R0010]
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>-</td><td>-</td><td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>-</td><td>0.5</td><td>0.5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>-</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>-</td><td>-</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>-</td><td>7.5</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>1</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①</td><td>②</td><td>③</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>5</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>H2O</td>
 
-
<td>4</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>6</td><td>6</td>
 
-
</tr>
 
-
</table>
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0827.JPG]]
 
-
|
 
-
From left;<br>
 
-
:①BBa_R0010
 
-
:②Single Digestion
 
-
:③Double Digestion
 
-
|}
 
-
 
-
 
-
 
-
:(26/8)--->'''[[Team:Chiba/protocol/PCR|PCR]]'''
 
-
::Colony PCR of 5 colonies from ligation plates (26/8-(4),(5)) and one from control plate(BBa_F2620[http://partsregistry.org/Part:BBa_F2620](2007)).
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>(4)-1~5</td><td>(5)-1~5</td><td>(F2620)</td>
 
-
</tr>
 
-
<tr>
 
-
<td>insert①C0178</td>
 
-
<td>1</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>insert②C0170</td>
 
-
<td>-</td><td>1</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>vector③J04500</td>
 
-
<td>-</td><td>-</td><td>2</td>
 
-
</tr>]
 
-
<td>ligase</td>
 
-
<td>1</td><td>1</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer</td>
 
-
<td>2</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>6</td><td>6</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td></tr>
 
-
</table>
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0827-2.JPG]]
 
-
|
 
-
From left;<br>
 
-
:1~5  insert:C0170 + vector:J04500  -> OK
 
-
:6    F2620・・Positive Control  -> OK
 
-
:7~11 insert:C0178 + vector:J04500 -> OK
 
-
|}
 
-
 
-
 
-
 
-
 
-
:--->(28/8)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''[[Team:Chiba/protocol/digestion|Digestion Test]]
 
-
 
-
 
-
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
comptent cell : XL10G
 
-
::*BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
 
-
::*BBa_C0062(2006) [http://partsregistry.org/Part:BBa_C0062] -> no colony on plates
 
-
::*BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
 
-
::*BBa_C0179(2006) [http://partsregistry.org/Part:BBa_C0179]
 
-
::*BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
 
-
::*BBa_S03156(2006) [http://partsregistry.org/Part:BBa_S03156]
 
-
::*BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
 
-
::*BBa_S03158(2006) [http://partsregistry.org/Part:BBa_S03158]
 
-
::*BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
 
-
::*BBa_S0316(2006) [http://partsregistry.org/Part:BBa_S03160]
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
 
-
*28 August, 2008]
 
-
(27/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]
 
-
::①insert:C0170 + vector:J04500
 
-
::②insert:C0178 + vector:J04500
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
 
-
 
-
:--->(29/8)'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>①②</td><td>Single Digesiton:①②</td><td>Double Digesiton:①②</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>10</td><td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>1</td><td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>H2O</td>
 
-
<td>4</td><td>-</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>6</td><td>12</td><td>12</td>
 
-
</tr>
 
-
</table>
 
-
{|align="justify"
 
-
|[[Image:Chiba-0828.JPG]]
 
-
|
 
-
From left;<br>
 
-
:① -> OK
 
-
:② -> OK
 
-
:Single Digestion:① -> OK
 
-
:Single Digestion:② -> OK
 
-
:Double Digestion:① -> OK
 
-
:Double Digestion:② -> OK
 
-
|}
 
-
 
-
*29 August, 2008
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
 
-
::①BBa_R0010(2007) [http://partsregistry.org/Part:BBa_R0010]
 
-
::②BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
 
-
::③BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
 
-
::④BBa_C0179(2006) [http://partsregistry.org/Part:BBa_C0179]
 
-
::⑤BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
 
-
::⑥BBa_S03156(2006) [http://partsregistry.org/Part:BBa_S03156]
 
-
::⑦BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
 
-
::⑧BBa_S03158(2006) [http://partsregistry.org/Part:BBa_S03158]
 
-
::⑨BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
 
-
::⑩BBa_S03160(2006) [http://partsregistry.org/Part:BBa_S03160]
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>S①~⑩</td><td>w①</td><td>w②~⑩</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>1</td><td>4</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>-</td><td>0.075</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>EcoRⅠ</td>
 
-
<td>0.1</td><td>0.075</td><td>0.1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>1</td><td>0.75</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>-</td><td>0.75</td><td>1</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>7.9</td><td>4.35</td><td>5.8</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>10</td><td>10</td><td>10</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>10</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td></tr>
 
-
</table>
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829.JPG]]
 
-
|
 
-
From left;<br>
 
-
①~⑤,S①⑤,W①~⑤
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-2.JPG]]
 
-
|
 
-
From left;<br>
 
-
⑥~⑩,S⑥~⑩,W⑥~⑩
 
-
|}
 
-
 
-
 
-
(27/8)--->
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-3.JPG]]
 
-
|
 
-
R0010<br>
 
-
|}
 
-
 
-
 
-
'''[[Team:Chiba/protocol/digestion|Digestion]]'''
 
-
::①BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::②BBa_I9030(2007) [http://partsregistry.org/Part:BBa_I9030]
 
-
::③BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::④BBa_R0010(2006) [http://partsregistry.org/Part:BBa_R0010]
 
-
 
-
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample No</td>
 
-
<td>S①~③</td><td>④</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Sample DNA</td>
 
-
<td>100ng/μL×20</td><td>200ng/μL×30</td>
 
-
</tr>
 
-
<tr>
 
-
<td>PstⅠ</td>
 
-
<td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>XbaⅠ</td>
 
-
<td>2</td><td>-</td>
 
-
</tr>
 
-
<tr>
 
-
<td>SpeⅠ</td>
 
-
<td>-</td><td>2</td></tr>
 
-
<tr>
 
-
<td>Buffer 2</td>
 
-
<td>-</td><td>5</td></tr>
 
-
<tr>
 
-
<tr>
 
-
<td>Buffer 3</td>
 
-
<td>5</td><td>-</td></tr>
 
-
<tr>
 
-
<td>BSA</td>
 
-
<td>5</td><td>5</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>16</td><td>6</td></tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>50</td><td>50</td>
 
-
</tr>
 
-
</table>
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
<table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
 
-
<tr>
 
-
<td width="257">Sample DNA</td>
 
-
<td>10</td><td>3</td>
 
-
</tr>
 
-
<tr>
 
-
<td>Loading Dye</td>
 
-
<td>2</td><td>2</td>
 
-
</tr>
 
-
<tr>
 
-
<td>dH2O</td>
 
-
<td>-</td><td>7</td>
 
-
</tr>
 
-
<tr>
 
-
<td>TOTAL</td>
 
-
<td>12</td></tr>
 
-
</table>
 
-
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-4.JPG]]
 
-
|
 
-
From left;<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-5.JPG]]
 
-
|
 
-
From left;<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-6.JPG]]
 
-
|
 
-
From left;<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0829-7.JPG]]
 
-
|
 
-
From left;<br>
 
-
|}
 
-
 
-
 
-
)--->'''[[Team:Chiba/protocol/ligation/gelex|Gel Extract]]'''
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
 
-
:::=>1.5μL/2colum
 
-
 
-
:--->'''SAP'''
 
-
  ④
 
-
 
-
:--->'''[[Team:Chiba/protocol/DNA Purification/zymocleam|Zymo Clean]]'''
 
-
 
-
:---> miss!
 
-
 
-
 
-
*30 August, 2008
 
-
 
-
'''[[Team:Chiba/protocol/transformation|Transformation]]'''
 
-
comptent cell : XL10G
 
-
::*BBa_I9026(2007) [http://partsregistry.org/Part:BBa_I9026]
 
-
::*BBa_I9030(2006) [http://partsregistry.org/Part:BBa_I9030]
 
-
::*BBa_S03154(2007) [http://partsregistry.org/Part:BBa_S03154]
 
-
::*BBa_S03156(2007) [http://partsregistry.org/Part:BBa_S03156]
 
-
::*BBa_S03158(2007) [http://partsregistry.org/Part:BBa_S03158]
 
-
::*BBa_S03160(2007) [http://partsregistry.org/Part:BBa_S03160]
 
-
::*BBa_C0062(2007) [http://partsregistry.org/Part:BBa_C0062]
 
-
::*BBa_C0179(2007) [http://partsregistry.org/Part:BBa_C0179]
 
-
 
-
:--->(31/8)'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
 
-
 
-
(29/8)--->'''[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]'''
 
-
::①insert:C0170 + vector:J04500
 
-
::②insert:C0178 + vector:J04500
 
-
 
-
 
-
:--->'''[[Team:Chiba/protocol/digestion|Digestion Test]]'''
 
-
 
-
:--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
 
-
 
-
 
-
 
-
{|align="justify"
 
-
|[[Image:Chiba-0830.JPG]]
 
-
|
 
-
From left;<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0830-2.JPG]]
 
-
|
 
-
From left;<br>
 
-
|}
 
-
{|align="justify"
 
-
|[[Image:Chiba-0830-3.JPG]]
 
-
|
 
-
From left;<br>
 
-
|}
 
-
 
-
==Week 3==
 
-
*31 August, 2008
 
-
 
-
*1 September, 2008
 
-
 
-
*2 September, 2008
 
-
 
-
*3 September, 2008
 
-
 
-
*4 September, 2008
 
-
 
-
*5 September, 2008
 
-
 
-
*6 September, 2008
 
-
 
-
 
-
==Week 4==
 
-
*7 September, 2008
 
-
 
-
*8 September, 2008
 
-
*9 September, 2008
+
===Cross-Talk Check===
 +
#LuxR vs LuxI('''OK'''),LasI('''OK'''),CinI('''No'''),RhlI('''OK''')
 +
#LuxI vs LuxR,LasR,CinR,RhlR-->発現を確認できず
-
*10 September, 2008
+
===time-delay test===
 +
[[Team:Chiba/protocol/phenotype/timedelay/j|time-delay protocol]]
 +
==連絡==
 +
===pptに載せたいもの===
 +
====作ったもの====
 +
*絵で、わかりやすく。~Isごとに色を変える。
 +
====time-delay実験結果の整理(25/9)====
 +
*実験操作を分かりやすく書く
 +
*比較するもの
 +
**LuxI,LasI,RhlI
 +
**TAG有/無
 +
**温度
 +
**菌数比
 +
**株
 +
*ポイント
 +
#正しく書きだす
 +
#推論を立てる(帰納的に、結論を急がないこと)
 +
====固体培地spot実験(28/9)====
 +
前日
 +
*IPTG入りLB-Agar-amp(20ml)に、T9002を2ml(10<sup>3</sup>-10<sup>5</sup> cells/2ml)撒く-->37℃,O/N
 +
*~Is(5種類)をLB-Amp,2ml培養
 +
当日
 +
*T9002がコロニーを形成したプレートの中心に、~Isの培養液を1μl,spotする。
 +
*gfpの発現が同心円状に広がっていく。その計時変化を記録する。
-
{| style="color:white;background-color:Maroon" cellpadding="3" cellspacing="3" border="1" bordercolor="white" width="100%" align="center"
+
{| style="color:white;" cellpadding="3" cellspacing="3" border="0" width="100%" align="center" class="menu" |
!align="center"|[[Team:Chiba/j|ホーム]]
!align="center"|[[Team:Chiba/j|ホーム]]
!align="center"|[[Team:Chiba/Team/j|メンバー紹介]]
!align="center"|[[Team:Chiba/Team/j|メンバー紹介]]
!align="center"|[[Team:Chiba/Project/j|プロジェクト紹介]]
!align="center"|[[Team:Chiba/Project/j|プロジェクト紹介]]
-
!align="center"|[[Team:Chiba/Parts/j|Parts Submitted to the Registry]]
+
!align="center"|[[Team:Chiba/Parts/j|作成したDNAパーツ]]
-
!align="center"|[[Team:Chiba/Modeling/j|モデリング]]
+
!align="center"|[[Team:Chiba/Internal|ノート]]
!align="center"|[[Team:Chiba/Internal|ノート]]
|}
|}

Latest revision as of 11:27, 14 December 2008

>ノートに戻る


Contents

送受信班の実験ノート

モジュール解説

感度差による応答時間の差

任意の順番と、delay-timeをもった遺伝子発現を実現するために、バクテリア間の細胞間通信、クオラムセンシングを利用する。クオラムセンシングでは、応答閾値を超えるAHLが蓄積されたとき初めて、目的遺伝子の発現がおこる。AHL情報がじゅうぶんゆっくり蓄積するとき、ひとつのAHL送信装置に対して、感度の異なる複数の受信装置によってさまざまな出力装置を独立に起動すれば、感度の高いAHL 受信機ほど先に起動する。

Controlling the Delay time of the AHL signaling

LuxIのつくるAHLに対して、感度差による応答時間差は、2つの方法で実現することができる。

  1. LuxR/ Pluxの感度変異体を用いる場合
    LuxRの変異体を用いれば、pluxがonになるAHL濃度が<1nMから~10nMまで変化する。[http://authors.library.caltech.edu/5553/ C. H. Collins.et al.:Mol.Microbiol.2005.55(3).712–723]
  2. クロストークを使う場合
AHL=Acyl-homoserine Lactoneには、そのアシル基が異なる種類がある。luxIが合成するAHLは、3-oxo-hyxanoyl-Homoserine Lactone (=OHHL)であり、3-oxo-dodecanoyl-homoserine Lactone(=ODHL)や、butyryl-homoserine Lactone(=HHL)が存在する。それぞれ、合成酵素はLasI、RhlI(ともにシュードモナス由来)と呼ばれる。このほかのAHL分子と、その合成酵素を以下に示した。これらのAHL分子は、アシル基のみが異なるため、一定の割合でLuxRと相互作用する(cross-talkする)。たとえば、OdHLとLuxRとのcross-talk(LasI-LuxR)は、OHHLとLuxRの場合(LuxI-LuxR)の1/20である。AHL分子の蓄積速度が一定ならば,luxI-LuxR/Pluxよりも20倍遅くスイッチが入ることとなる。
  • AHLsおよびその合成酵素
Strain Signal Molecule Enzyme
P.aeruginosa C4-HSL RhlI
V. fisheri C6-3-oxo-HSL LuxI
A.tumefaciens C8-3-oxo-HSL TraI
P.aeruginosa C10-3-oxo-HSL VanI
P.aeruginosa C12-3-oxo-HSL LasI
R.leguminosarum C14-3-hydroxy-HSL CinI

[http://partsregistry.org/Part:BBa_F2620:Specificity AHLsについて]

実験

Construction(From BioBrick)

  • Sender(Autoinducer synthase)
LuxI,LasI,CinI,RhlI(lacプロモーター)
  • Receiver
LuxR,LasR,CinR,RhlR


Cross-Talk Check

  1. LuxR vs LuxI(OK),LasI(OK),CinI(No),RhlI(OK)
  2. LuxI vs LuxR,LasR,CinR,RhlR-->発現を確認できず

time-delay test

time-delay protocol

連絡

pptに載せたいもの

作ったもの

  • 絵で、わかりやすく。~Isごとに色を変える。

time-delay実験結果の整理(25/9)

  • 実験操作を分かりやすく書く
  • 比較するもの
    • LuxI,LasI,RhlI
    • TAG有/無
    • 温度
    • 菌数比
  • ポイント
  1. 正しく書きだす
  2. 推論を立てる(帰納的に、結論を急がないこと)

固体培地spot実験(28/9)

前日

  • IPTG入りLB-Agar-amp(20ml)に、T9002を2ml(103-105 cells/2ml)撒く-->37℃,O/N
  • ~Is(5種類)をLB-Amp,2ml培養

当日

  • T9002がコロニーを形成したプレートの中心に、~Isの培養液を1μl,spotする。
  • gfpの発現が同心円状に広がっていく。その計時変化を記録する。