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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/transformation|Transformation]]'''
| + | ===感度差による応答時間の差=== |
| - | ::*BBa_C0070(2007) [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_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
| + | |
| - | ::*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]
| + | |
| | | | |
| | + | === 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。 |
| | | | |
| - | :--->(20/8)[[Team:Chiba/protocol/DNA Purification/sigma|Mini prep]]
| + | *AHLsおよびその合成酵素 |
| - | | + | <table width="500" border="1" cellpadding="0" cellspacing="0" bordercolor="#000000" center> |
| - | | + | |
| - | | + | |
| - | *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
| + | |
| - | | + | |
| - | | + | |
| - | :--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
| + | |
| - | {|align="justify"
| + | |
| - | |[[Image:Chiba-0818.JPG]]
| + | |
| - | |
| + | |
| - | ::*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]]'''
| + | |
| - | <table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000"> | + | |
| | <tr> | | <tr> |
| - | <td width="257">Sample No</td> | + | <td width="200">Strain</td> |
| - | <td>①</td><td>②</td><td>③</td><td>④</td> | + | <td width="200">Signal Molecule</td> |
| | + | <td width="150">Enzyme<td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| - | <td>Sample DNA</td> | + | <td>P.aeruginosa</td> |
| - | <td>3</td><td>3</td><td>3</td><td>3</td> | + | <td>C4-HSL</td> |
| | + | <td>RhlI</td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| - | <td>Loading Dye</td> | + | <td>V. fisheri</td> |
| - | <td>2</td><td>2</td><td>2</td><td>2</td> | + | <td>C6-3-oxo-HSL</td> |
| | + | <td>LuxI</td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| - | <td>dH2O</td> | + | <td>A.tumefaciens</td> |
| - | <td>7</td><td>7</td><td>7</td><td>7</td> | + | <td>C8-3-oxo-HSL</td> |
| | + | <td>TraI</td> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| - | <td>TOTAL</td> | + | <td>P.aeruginosa</td> |
| - | <td>12</td><td>12</td><td>12</td><td>12</td> | + | <td>C10-3-oxo-HSL</td> |
| | + | <td>VanI</td> |
| | </tr> | | </tr> |
| - | </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-0820-2.JPG]]
| |
| - | |
| |
| - | *BBa_C0170(2007) [http://partsregistry.org/Part:BBa_C0170] -> OK
| |
| - | *BBa_C0170(2006) [http://partsregistry.org/Part:BBa_C0170] -> OK
| |
| - | *BBa_C0178(2007) [http://partsregistry.org/Part:BBa_C0178] -> OK
| |
| - | *BBa_C0178(2006) [http://partsregistry.org/Part:BBa_C0178] -> OK
| |
| - | |}
| |
| | | | |
| - | {|align="justify"
| |
| - | |[[Image:Chiba-0820-3.JPG]]
| |
| - | |
| |
| - | ::*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]]
| |
| - | |
| |
| - | ::*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
| |
| - |
| |
| - | |}
| |
| - |
| |
| - |
| |
| - |
| |
| - | :--->'''[[Team:Chiba/protocol/digestion|Digestion]]'''
| |
| - | ::*BBa_C0070(2007) [http://partsregistry.org/Part:BBa_C0070]
| |
| - | ::*BBa_C0070(2006) [http://partsregistry.org/Part:BBa_C0070]
| |
| - | ::*BBa_C0078(2007) [http://partsregistry.org/Part:BBa_C0078]
| |
| - | ::*BBa_C0078(2006) [http://partsregistry.org/Part:BBa_C0078]
| |
| - |
| |
| - |
| |
| - |
| |
| - | *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]
| |
| - |
| |
| - |
| |
| - | :--->'''[[Team:Chiba/protocol/gelcheck|Gel Check]]'''
| |
| - | {|align="justify"
| |
| - | |[[Image:Chiba-0821.JPG]]
| |
| - | |
| |
| - | From the left;<br>
| |
| - | ::*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]
| |
| - |
| |
| - |
| |
| - |
| |
| - | *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]
| |
| - |
| |
| - |
| |
| - | :--->'''[[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> | | <tr> |
| - | <td width="257">Sample No</td> | + | <td>P.aeruginosa</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> | + | <td>C12-3-oxo-HSL</td> |
| | + | <td>LasI</td> |
| | </tr> | | </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> | | <tr> |
| - | <td width="257">Sample No</td> | + | <td>R.leguminosarum</td> |
| - | <td>①</td><td>②</td><td>③</td><td>④</td>
| + | <td>C14-3-hydroxy-HSL</td> |
| - | </tr>
| + | <td>CinI</td> |
| - | <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> | | </tr> |
| | </table> | | </table> |
| | + | [http://partsregistry.org/Part:BBa_F2620:Specificity AHLsについて] |
| | | | |
| - | :--->[[Team:Chiba/protocol/gelcheck|Gel Check]]
| + | ==実験== |
| - | <table width="315" border="2" cellpadding="0" cellspacing="0" bordercolor="#000000">
| + | ===Construction(From BioBrick)=== |
| - | <tr>
| + | :*Sender(Autoinducer synthase) |
| - | <td width="257">Sample No</td>
| + | ::LuxI,LasI,CinI,RhlI(lacプロモーター) |
| - | <td>①</td><td>②</td><td>③</td><td>④</td>
| + | :*Receiver |
| - | </tr>
| + | ::LuxR,LasR,CinR,RhlR |
| - | <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
| + | |
| - | |}
| + | |
| - | | + | |
| - | '''[[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-0829-3.JPG]]
| + | |
| - | |
| + | |
| - | From left;<br>
| + | |
| - | |}
| + | |
| - | {|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>
| + | |
| - | |}
| + | |
| - | | + | |
| - | *30 August, 2008
| + | |
| - | | + | |
| - | ==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]] |
| | |} | | |} |
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。
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