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Team:NCTU Formosa/calendar
From 2011.igem.org
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/*content*/ | /*content*/ | ||
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</style> | </style> | ||
</head> | </head> | ||
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<body> | <body> | ||
| - | + | <div id="banner"><a href = "https://2011.igem.org/Team:NCTU_Formosa"><img src = "https://static.igem.org/mediawiki/2011/8/8c/Banner010.jpg" height="236" width="944"/></a></div> | |
| - | + | ||
<ul id="cm-nav"> | <ul id="cm-nav"> | ||
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<ul> | <ul> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/members">Members</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/members">Members</a></li> | ||
| - | <li><a href=" | + | <li><a href="http://www.nctu.edu.tw/english/index.php">School</a></li> |
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/gallery">Gallery</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/gallery">Gallery</a></li> | ||
</ul> | </ul> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_data">Data</a></li> | ||
| - | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_discussion"> | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_discussion">Modeling</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_data">Data</a></li> | ||
| - | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_discussion"> | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_discussion">Modeling</a></li> |
</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_data">Data</a></li> | ||
| - | |||
</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_data">Data</a></li> | ||
| - | |||
</ul> | </ul> | ||
</li> | </li> | ||
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_design">Design</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_design">Design</a></li> | ||
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_data">Data</a></li> | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_data">Data</a></li> | ||
| - | |||
</ul> | </ul> | ||
| - | </li> | + | </li> |
</ul> | </ul> | ||
</li> | </li> | ||
| - | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/modeling"> | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/modeling">Measurements</a></li> |
| - | <li><a | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/parts">Parts</a></li> |
| - | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/safty">Safety</a></li> | |
| - | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/humanpractice">Human Practice</a></li> | |
| - | + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/contributions">Attribution</a></li> | |
| - | + | <li><a class="arrow no-click">Notebook </a> | |
| - | + | <ul> | |
| - | + | <li><a class="arrow no-click">Protocols</a> | |
| - | + | <ul> | |
| + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol">Mutation</a></li> | ||
| + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_F">Flow Cytometry</a></li> | ||
| + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_G">GC</a></li> | ||
| + | </ul> | ||
| + | <li><a href="https://2011.igem.org/Team:NCTU_Formosa/calendar">Calendar</a></li> | ||
| + | </ul> | ||
</li> | </li> | ||
</ul> | </ul> | ||
| - | + | ||
| - | <div | + | <br><br> |
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{background-color:white;font-family:arial,sans,sans-serif;font-size:100.0%;font-weight:normal;font-style:normal;color:#000000;text-decoration:none;text-align:left;vertical-align:bottom;white-space:normal;overflow:hidden;border-right:;border-bottom:;} .tblGenFixed td.s3 {background-color:white;font-family:arial,sans,sans-serif;font-size:100.0%;font-weight:normal;font-style:normal;color:#000000;text-decoration:none;text-align:left;vertical-align:bottom;white-space:normal;overflow:hidden;border-top:1px solid #CCC;border-right:;border-bottom:;} .tblGenFixed td.s10 {background-color:white;font-family:arial,sans,sans-serif;font-size:100.0%;font-weight:normal;font-style:normal;color:#000000;text-decoration:none;text-align:left;vertical-align:bottom;white-space:normal;overflow:hidden;border-right:;border-bottom:;} .tblGenFixed td.s4 {background-color:white;font-family:arial,sans,sans-serif;font-size:100.0%;font-weight:normal;font-style:normal;color:#000000;text-decoration:none;text-align:right;vertical-align:bottom;white-space:normal;overflow:hidden;border-right:;border-bottom:;border-left:1px solid #CCC;} .tblGenFixed td.s11 {background-color:#dddddd;font-family:arial,sans,sans-serif;font-size:100.0%;font-weight:normal;font-style:normal;color:#000000;text-decoration:none;text-align:left;vertical-align:bottom;white-space:normal;overflow:hidden;border-right:;border-bottom:;} </style></head><body style='border:0px;margin:0px'><table border=0 cellpadding=0 cellspacing=0 class='tblGenFixed' id='tblMain'><tr class='rShim'><td class='rShim' style='width:0;'><td class='rShim' style='width:71px;'><td class='rShim' style='width:421px;'><td class='rShim' style='width:419px;'><td class='rShim' style='width:418px;'><td class='rShim' style='width:217px;'><tr><td class='s0'>Date<td class='s1'>G1<td class='s2'>G2<td class='s1'>G2-2<td class='s3'>G2-1</tr><tr><td class='s4'>4/26/2011<td class='s5'>colon PCR vioABCE+terminator<td class='s6'>digest Ptet ES site, Ligate CrtEBI+Ptet +1K3 overnight<td class='s7'><td ></tr><tr><td class='s4'>4/27/2011<td class='s8'>extract Plac plasmid, ligation Plac+37RBS<td class='s6'>Ptet+crt EBI transform<td class='s7'><td ></tr><tr><td class='s4'>4/28/2011<td class='s5'>Electrophoresis of vioABCE+terminator & incubate LB ; transform Plac+37RBS<td class='s6'>ligate Ptet+crtEBI+1c3(zip) and transform<td class='s7'><td ></tr><tr><td class='s4'>4/29/2011<td class='s5'>prepare plasmid of vioABCE+terminator<td class='s6'>Yesterday transform failed.ligate ptet+crtEBI+1c3 again.(non-purify 1C3)<td class='s7'><td ></tr><tr><td class='s4'>4/30/2011<td class='s7'><td class='s6'>ligate ptet+crtEBI+1c3(purify) overnight.<br/><td class='s7'><td ></tr><tr><td class='s4'>5/1/2011<td class='s5'>transform Plac+37RBS<td class='s6'>extract Ptet plasmid. Transform <br/>Ptet+crtEBI+1c3<td class='s7'><td ></tr><tr><td class='s4'>5/2/2011<td class='s5'>transform Ptet+vioABCE+terminator<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>5/3/2011<td class='s7'><td class='s6'>Digest crtEBI XP and Ptet SP <br/><td class='s7'><td ></tr><tr><td class='s4'>5/4/2011<td class='s5'> Ptet digest ES ; vioABCE+terminator digest XP ;ligation Ptet+vioABCE+terminator<td class='s6'>Ligate ptet(purify) Amp (digest SP)+crtEBI(purify)(digestXP). Ligate ptet(ES)+crtEBI(XP)+1c3(EP)<td class='s7'><td ></tr><tr><td class='s4'>5/5/2011<td class='s5'>transform Ptet+vioABCE+terminator<td class='s6'>Transform 5/4 two ligations.Ligate ptet(purify)Amp (SP)+crtEBI(purify)(XP).<td class='s7'><td ></tr><tr><td class='s4'>5/6/2011<td class='s7'><td class='s6'>Transform ptet(purify)Amp(SP)+crtEBI(purify)(XP).<td class='s7'><td ></tr><tr><td class='s4'>5/7/2011<td class='s7'><td class='s6'>incubate Ptet,CrtEBI<td class='s7'><td ></tr><tr><td class='s4'>5/8/2011<td class='s7'><td class='s6'>extract Ptet CrtEBI plasmid<td class='s7'><td ></tr><tr><td class='s4'>5/10/2011<td class='s5'>Plac+37RBS colony PCR ; prepare plasmid of Plac+37RBS <td class='s6'>Ligate Ptet+CrtEBI+1c3(original method) and Ligate Ptet+CrtEBI+1c3(purify)<td class='s7'><td ></tr><tr><td class='s4'>5/11/2011<td class='s5'>Ptet+vioABCE+terminator colony PCR ;Plac+37RBS extract plasmid<td class='s6'>Transform Ptet+CrtEBI+1c3(original method) ,Plac+CctEBIY,Plac+CrtEBI<td class='s7'><td ></tr><tr><td class='s4'>5/12/2011<td class='s5'>ligation LuxR+terminator<td class='s6'>PCR Ptet+CrtEBI+1c3<td class='s5'>CrtY point mutation PCR<td ></tr><tr><td class='s4'>5/13/2011<td class='s5'>transform LuxR+terminator<td class='s6'>Electrophoresis.PCR Ptet+CrtEBI+1c3 again<td class='s5'>CrtY self-ligation<td ></tr><tr><td class='s4'>5/14/2011<td class='s5'>ligation LuxR+terminator<td class='s6'>Electrophoresis.<td class='s5'>CrtY transform<td ></tr><tr><td class='s4'>5/15/2011<td class='s5'>transform LuxR+terminator<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>5/16/2011<td class='s5'> LuxR+terminator colony PCR<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>5/17/2011<td class='s5'> LuxR+terminator incubate LB<td class='s6'>Ptet digest sp ,crtEBI digestXP (purify both),Ligation.<br/><td class='s5'>CrtY colonyPCR<td class='s10'>Digest C0012 ES & XP; <br/>B0034 ES<br/><br/><br/></tr><tr><td class='s4'>5/18/2011<td class='s5'>prepare plasmid of LuxR+terminator ; ligation J24679+LuxR+terminator; prepare plasmid of Ptet & digest EP SP; incubateLB of k274003 <td class='s6'>Transform Ptet SP+CrtEBI XP<td class='s7'><td class='s10'>Ligase C0012+37RBS<br/>+tetR+terminator & B0034+C0012<br/>Digest B0034+GFP+terminator</tr><tr><td class='s4'>5/19/2011<td class='s5'>ligation Ptet+vioABCE+terminator ; prepare plasmid of k274003 ; transform J24679+LuxR+terminator<td class='s9'><td class='s7'><td class='s10'>transform C0012+37RBS<br/>+tetR+terminator & B0034+C0012</tr><tr><td class='s4'>5/20/2011<td class='s5'> J24679+LuxR+terminator colony PCR ; incubate LB of Ptet <td class='s9'><td class='s5'>CrtY colonyPCR<td class='s10'>PCR <br/>C0012+37RBS<br/>+tetR+terminator</tr><tr><td class='s4'>5/21/2011<td class='s5'>prepare plasmid of Ptet ; ligation and transform J24679+LuxR+terminator ; ligation and transform Ptet+vioABCE+terminator<td class='s9'><td class='s5'>digest CrtY(S) →success<td ></tr><tr><td class='s4'>5/22/2011<td class='s5'>Ptet+vioABCE+terminator colony PCR <td class='s6'>dilute plasmid RBS+crtZ from 2011plate19B-I742158<td class='s7'><td ></tr><tr><td class='s4'>5/24/2011<td class='s7'><td class='s9'><td class='s5'>digest CrtY(E,S) Terminator(E,X)<td ></tr><tr><td class='s4'>5/25/2011<td class='s5'>ligation and transform J24679+LuxR+terminator <td class='s9'><td class='s5'>ligase CrtY+Terminator<td ></tr><tr><td class='s4'>5/26/2011<td class='s5'>think of new ligation and transform protocol of J24679+LuxR+terminator <td class='s9'><td class='s5'>transform CrtY+Terminator<td ></tr><tr><td class='s4'>5/27/2011<td class='s5'>follow new protocol to ligation and transform protocol of J24679+LuxR+terminator (add 1mM ATP when ligation)<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>5/28/2011<td class='s5'>J24679+LuxR+terminator colony PCR & incubate LB<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>5/29/2011<td class='s5'>prepare plasmid of J24679+LuxR+terminator X2<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>5/30/2011<td class='s5'>prepare plasmid of J24679+LuxR+terminator X5<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>5/31/2011<td class='s5'>check J24679+LuxR+terminator plasmid<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>6/1/2011<td class='s7'><td class='s6'>transform RBS+crtZ<td class='s5'>colony PCR CrtY+Terminator<td ></tr><tr><td class='s4'>6/2/2011<td class='s7'><td class='s6'>colony PCR RBS+crtZ<br/>electrophoresis check <br/>sequencing RBS+crtZ<td class='s7'><td ></tr><tr><td class='s4'>6/3/2011<td class='s7'><td class='s9'><td class='s5'>ligase CrtY+Terminator<td ></tr><tr><td class='s4'>6/4/2011<td class='s7'><td class='s9'><td class='s5'>transform CrtY+Terminator<td ></tr><tr><td class='s4'>6/7/2011<td class='s7'><td class='s6'>sequencing result RBS+crtZ → successs<br/>incubate RBS+crtZ in LB-medium<td class='s5'>colony PCR CrtY+Terminator →success<td ></tr><tr><td class='s4'>6/8/2011<td class='s7'><td class='s6'>extract plasmid RBS+crtZ<td class='s7'><td ></tr><tr><td class='s4'>6/10/2011<td class='s7'><td class='s6'>digest RBS+crtZ(ES)(anti-A)<br/>digest terminator-J61048(XP&EX)(anti-A)<br/>digest PSB1K3(EP)(anti-K)<br/>ligate 1.RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)<br/> 2.RBS+crtZ(ES)+terminator(EX)(anti-A)<td class='s7'><td ></tr><tr><td class='s4'>6/11/2011<td class='s7'><td class='s6'>transform 1.RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)<br/> 2.RBS+crtZ(ES)+terminator(EX)(anti-A)<td class='s5'>incubate 1.37RBS 2.CrtY_Terminator 3.psb1k3 4.psb1c3<td ></tr><tr><td class='s4'>6/12/2011<td class='s7'><td class='s6'>only RBS+crtZ(ES)+terminator(EX)(anti-A) grown<br/>colony PCR above-mentioned<br/>electrophoresis check <br/>sequencing RBS+crtZ(ES)+terminator(EX)(anti-A)<br/>digest RBS+crtZ(ES)(anti-A)again<br/>digest terminator-J61048(XP)(anti-A)again<br/>digest PSB1K3(EP)(anti-K)again<br/>ligate RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)again<td class='s5'>extract plasmid digest 1.37RBS(E,S).(S,P) 2.CrtY_Terminator(X,P) 3.psb1c3(E,P) ligase 37RBS+CrtY_Terminator<td ></tr><tr><td class='s4'>6/13/2011<td class='s7'><td class='s6'>transform RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)again<td class='s5'>transform 37RBS+CrtY_Terminator<td ></tr><tr><td class='s4'>6/17/2011<td class='s7'><td class='s6'>sequencing result RBS+crtZ(ES)+terminator(EX)(anti-A) → successs<td class='s7'><td ></tr><tr><td class='s4'>6/23/2011<td class='s5'>digest 37RBS and kivd ; incubate LB of B0030,B0032,B0034<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>6/24/2011<td class='s5'>prepare plasmid of B0030,B0032,B0034 & check ; ligation 37RBS+kivd<td class='s6'>incubate RBS+crtZ+terminator(anti-A) in LB-medium<td class='s7'><td ></tr><tr><td class='s4'>6/25/2011<td class='s5'>digest ilvC,ilvD,malss,B0030,B0032,B0034<td class='s6'>extract plasmid RBS+crtZ+terminator(anti-A)<br/>digest RBS+crtZ+terminator(XP)(anti-A)<br/>digest pcI(ES&SP)(anti-A)<br/>digest PSB1K3(EP)(anti-K)<br/>ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)<br/> 2.pcI(SP)+RBS+crtZ+terminator(XP)(anti-A)<td class='s5'>colony PCR 37RBS+CrtY_Terminator→success<td ></tr><tr><td class='s4'>6/26/2011<td class='s5'>check digest product<td class='s6'>transform 1.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) 2.pcI(SP)+RBS+crtZ+terminator(XP)(anti-A)<td class='s7'><td ></tr><tr><td class='s4'>6/27/2011<td class='s7'><td class='s6'>only pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) grown<br/>colony PCR can't examine pcI(SP)+RBS+crtZ+terminator(XP)(anti-A)<br/>because pcI is too small(49bp)<br/>incubate pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) in LB-medium<td class='s7'><td ></tr><tr><td class='s4'>6/28/2011<td class='s7'><td class='s6'>extract plasmid pcI(SP)+RBS+crtZ+terminator(XP)(anti-A)<br/>digest RBS+crtZ+terminator(XP)(zip)(anti-A)again<br/>digest pcI(ES&SP)(anti-A)again<br/>digest PSB1K3(EP)(anti-K)again<br/>digest PSB1C3(EP)(anti-C)again<br/>ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)again<br/> 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)again<br/> 3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)again<br/> 4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)again<br/> 5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A)again<td class='s7'><td ></tr><tr><td class='s4'>6/29/2011<td class='s7'><td class='s6'>transform 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)again<br/> 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)again<br/> 3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)again<br/> 4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)again<br/> 5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A)again<td class='s7'><td ></tr><tr><td class='s4'>6/30/2011<td class='s5'>ligation RBS+ilvC and RBS+ilvD and RBS+malss<td class='s6'>only 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) grown<br/> 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) grown<br/> 5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A) grown<br/>colony PCR above-mentioned<br/>electrophoresis check<br/>sequencing above-mentioned<td class='s5'>incubate 37RBS_CrtY_Terminator<td ></tr><tr><td class='s4'>7/1/2011<td class='s5'>transform RBS+malss ; digest PSB1A3 ; ligation and transform B0034+kivd<td class='s9'><td class='s5'>extract 37RBS_CrtY_Terminator digest 37RBS_CrtY_Terminator(X,P)<td ></tr><tr><td class='s4'>7/4/2011<td class='s5'>RBS+malss colony PCR<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/5/2011<td class='s5'>transform RBS+ilvC and RBS+ilvD ; ligation Ptet+vioABCE<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/6/2011<td class='s5'>RBS+ilvC and RBS+ilvD colony PCR<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/7/2011<td class='s5'>Electrophoresis of RBS+ilvC and RBS+ilvD and RBS+malss<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/9/2011<td class='s7'><td class='s6'>sequencing result → fail<br/>digest RBS+crtZ+terminator(XP)(zip)(anti-A)<br/>digest RBS+crtZ+terminator(XP)(purify)(anti-A)<br/>digest pcI(ES&SP)(anti-A)<br/>digest PSB1K3(EP)(anti-K)<br/>digest PSB1C3(EP)(anti-C)<br/>ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)<br/> 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)<br/> 3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)<br/> 4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)<br/> 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A)<td class='s7'><td ></tr><tr><td class='s4'>7/11/2011<td class='s5'>Ptet+vioABCE colony PCR<td class='s6'>transform 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)<br/> 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)<br/> 3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)<br/> 4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)<br/> 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A)<td class='s7'><td ></tr><tr><td class='s4'>7/12/2011<td class='s5'>Electrophoresis of Ptet+vioABCE <td class='s6'>only 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) grown<br/> 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) grown<br/> 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) grown<td class='s7'><td ></tr><tr><td class='s4'>7/13/2011<td class='s5'>Ptet(SP) and vioABCE(XP) gel purified ; ligation Ptet+vioABCE<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/14/2011<td class='s5'>transform Ptet+vioABCE<td class='s6'>Flow cytometry: K098988, K098987 (in 25,37,42 temperature degree ) colony PCR 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) <br/> 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) <br/> 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) <br/>electrophoresis check<br/>sequencing 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) <br/> 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) <br/> 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) <td class='s7'><td ></tr><tr><td class='s4'>7/16/2011<td class='s7'><td class='s9'><td class='s5'>digest CrtEBI(E,S)<td ></tr><tr><td class='s4'>7/17/2011<td class='s7'><td class='s6'>sequencing result 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) → successs<br/>incubate cI+pcI-K098995 in LB-medium<td class='s7'><td ></tr><tr><td class='s4'>7/18/2011<td class='s5'>ligation and transform B0034+kivd ; Ptet+vioABCE colony PCR<td class='s6'>extract plasmid cI+pCI<td class='s5'>ligase CrtEBI+37RBS_CrtY_Terminator<td ></tr><tr><td class='s4'>7/19/2011<td class='s5'>Electrophoresis of Ptet+vioABCE <td class='s6'>digest cI+pcI(ES)(anti-A)<br/>digest RBS+ctrZ+terminator(XP)(anti-A)<br/>digest PSB1K3(EP)(anti-K)<br/>ligate cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)<td class='s5'>Transform CrtEBI+37RBS_CrtY_Terminator<td ></tr><tr><td class='s4'>7/20/2011<td class='s5'>ligation RBS+Alss ; prepare plasmid of Ptet+vioABCE,kivd,ilvC,ilvD,Alss ; B0034+kivd colony PCR ; incubate LB of PSB1A3 and PSB1K3 <td class='s6'>transform cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)<td class='s7'><td ></tr><tr><td class='s4'>7/21/2011<td class='s5'>prepare plasmid of PSB1K3 ; Electrophoresis of B0034+kivd ; transform RBS+Alss<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/25/2011<td class='s5'>ligation RBS+Alss ; RBS+Alss colony PCR<td class='s6'>colony PCR cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)<br/>electrophoresis check<br/>sequencing cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)<td class='s5'>ligase CrtEBI+37RBS_CrtY_Terminator<td ></tr><tr><td class='s4'>7/26/2011<td class='s5'>digest B0034+ilvC and B0034+ilvD ; transform RBS+Alss <td class='s9'><td class='s5'>Transform CrtEBI+37RBS_CrtY_Terminator<td ></tr><tr><td class='s4'>7/27/2011<td class='s5'>ligation K098995+B0034 ; RBS+Alss colony PCR ; ligation (B0034+ilvC)+(B0034+ilvD)<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/28/2011<td class='s5'> RBS+Alss colony PCR again ; transform K098995+B0034 and (B0034)ilvC+ilvD<td class='s9'><td class='s5'>colony PCR CrtEBI+37RBS_CrtY_Terminator→success<td ></tr><tr><td class='s4'>7/29/2011<td class='s5'> (B0034)ilvC+ilvD colony PCR ; ligation (B0034)ilvC+ilvD and K098995+B0034<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/30/2011<td class='s5'>transform (B0034)ilvC+ilvD and K098995+B0034 ; digest RBS+ilvD(ES)<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>7/31/2011<td class='s5'>K098995+B0034 and (B0034)ilvC+ilvD colony PCR<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>8/1/2011<td class='s5'>colony PCR of ilvD+terminator and ligation RBS+ilvC+ilvD(PSA1C3)and then transform<td class='s6'>sequencing result cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) → successs<br/><td class='s7'><td ></tr><tr><td class='s4'>8/2/2011<td class='s5'>抽plasmid ptet+vioABCE+terminator and colony PCR RBS+ilvC+RBS+ilvD and incubate37C RBS+tetR+terminator<td class='s6'>Flow cytometry: K098988 in 42 temperature degree<td class='s7'><td ></tr><tr><td class='s4'>8/3/2011<td class='s5'>ligase (B0030,B0032)ilvC+ilvD and digest B0032+COO12,BOO32+Alss and 抽plasmid Ptet+vioABCE+terminator then digest it and transform 37C RBS+tetR+terminator and (B0030,B0032)ilvC+ilvD<td class='s9'><td class='s5'>ligase CrtEBI+Plac CrtEBI+Ptet<td ></tr><tr><td class='s4'>8/4/2011<td class='s5'>1.ligaes 2.check digestion product 3.抽plasmid 37C RBS+tetR+terminator 4.transform ligastion pruduct Plac+Alss,RBS+ilvD+37C RBS+tetR+terminator 5.colony PCR ilvC+ilvD<td class='s6'>1.digest B0015 (XP) and check by electrophoresis 2.ligate B0030 or B0034 +kivd and B0015<td class='s5'>transform CrtEBI+Plac CrtEBI+Ptet ligase J23103_CrtEBI+37RBS_CrtY_Terminator<td ></tr><tr><td class='s4'>8/5/2011<td class='s5'>1.colony PCR RBS+ilvD+37C RBS+tetR+terminator 2.colony PCR Plac+Alss 3.<td class='s6'>1. transform 8/4 ligation products 2.purify Boo15, J61048, Ptet and check by electrophoresis 3. ligate Ptet(purify) and kivd, J61048(purify) and kivd, B0015(purify) and kivd 4.Flow cytometry <td class='s5'>Transform J23103_CrtEBI+37RBS_CrtY_Terminator<td ></tr><tr><td class='s4'>8/6/2011<td class='s5'>1.ligase RBS+ilvD+37C RBS+tetR+terminator <td class='s6'>1.PCR kivd+Boo15 2. Check PCR products by electrophoresis. Four samples reach expected bands. 3. transform 8/5 ligation products 4. inbate 988 DH5-alpha, 988 EPI300 5. four zone culture kivd+B0015<td class='s5'>colony PCR<td ></tr><tr><td class='s4'>8/7/2011<td class='s7'><td class='s6'>1.extract plasmid:EPI300 K098988, DH5-alpha K098988 and check py electrophoresis 2.sequencing kivd+J61048, Ptet+kivd<td class='s5'>purify CrtEBIY<td ></tr><tr><td class='s4'>8/8/2011<td class='s5'>1.transform RBS+ilvD+37C RBS+tetR+terminator <td class='s6'>1.transform 988 to EPI300 2.sequencing kivd+B0015<td class='s7'><td ></tr><tr><td class='s4'>8/9/2011<td class='s5'>1.ligase PCI +B0034 and (B0032)ilvC+ilvD+37C RBS+tetR+terminator and B0032+C0012+R+LuxR+terminator 2.transform (B0032)ilvC+ilvD+37C RBS+tetR+terminator 3.digest B0034 and (B0032)ilvC+ilvD and Plac+B0032+Alss<td class='s6'>1.digest kivd+J61048, Ptet+kivd<td class='s5'>ligase Plac+CrtEBIY(purify) digest J23103CrtEBI(E,S).(S,P)<td ></tr><tr><td class='s4'>8/10/2011<td class='s5'>1.ligate Plac+Alss+ilvC+ilvD 2.digest(B0032)ilvC+IlvD and B0030,32,34+ilvC 3.check digestion product 4.colony PCR RBS+ilvD+37C RBS+tetR+terminator 5.transform (B0030,34)ilvC+ilvD and Plac+Alss+ilvC+ilvD and (B0032)ilvC+IlvD and pCI+B0034<td class='s6'>1.ligate Ptet+kivd+terminator with different methods 2.Flow cytometry: incubate K098988(EPI300) at 32, 37, 42 temperature degree<td class='s5'>Transform J23103CrtEBI+37RBS_CrtY_Terminator<td ></tr><tr><td class='s4'>8/11/2011<td class='s5'>1.ligate Plac+Alss+RBS+ilvC and ilvC+ilvD+37C RBS+tetR+terminator 2.colony PCR Plac+Alss+ilvC+iulvD and pCI+B0034 and ilvC+ilvD+37C RBS+tetR+terminator 3.transform ligastion products 5.ligate Plac+Alss+ilvC+ilvD<td class='s9'><td class='s5'>ligase J23103CrtEBI+37RBS_CrtY_Terminator colony PCR →success<td ></tr><tr><td class='s4'>8/12/2011<td class='s5'>1.ligate Plac+Alss+ilvC and iulvC+ilvD+37C RBS+tetR+terminator 2.check digestion products(8/10,11) 3.colony PCR Plac+Alss+ilvC and (B0034)ilvC+ilvD and ilvC+ilvD+37C RBS+tetR+terminator 4.transform Plac+Alss+ilvC+ilvD and ilvC+ilvD+37C RBS+tetR+terminator <td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>8/13/2011<td class='s5'>1.colony PCR 8/12transformation products 2.liagte B0030,34+ilvC+ilvD and Plac+Alss+RBS+ilvC and then transform<td class='s6'>1.digest plasmids of kivd EcoR1 and Pst1 2.ligate kivd with psb1c3<td class='s7'><td ></tr><tr><td class='s4'>8/14/2011<td class='s5'>1.digest ilvC+ilvD+37C RBS+tetR+terminator 2.check digestion products 3.ligate Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminator(PSB1K3) 4.digest Plac+Alss<td class='s6'>transform 8/13 ligation products<td class='s7'><td ></tr><tr><td class='s4'>8/15/2011<td class='s5'>1.ligate Plac+Alss+B0030,32,34+ilvC and (B0030,34)ilvC+ilvD 2.抽plasmid Ptet+Alss+ilvC+ilvD 3.incubate LB of transformation products 4.transform Plac+Alss+B0030,32,34+ilvC and Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminator and B0030,34+ilvC+ilvD<td class='s6'>1.PCR 8/14 sample and check by electrophoresis->fail 2.digest plasmids of crtZ EcoR1 and Pst1 3.ligate crtZ and psb1c3<td class='s7'><td ></tr><tr><td class='s4'>8/16/2011<td class='s5'>1.抽plasmid of LB products(8/15) 2.digest Plac+Alss+ilvC+ilvD<td class='s9'><td class='s5'>incubate psb3T5<td ></tr><tr><td class='s4'>8/17/2011<td class='s5'>1.check dugestion products 2.colony PCR Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminator<td class='s6'>transform 8/15 ligation products<td class='s5'>extract plasmid psb3T5<td ></tr><tr><td class='s4'>8/18/2011<td class='s5'>1.ligate ilvC+ilvD and Plac+Alss+RBS+ilvC and pCI+B0034 2.colony PCR B0034+C0012+37C RBS+tetR+terminator 3.transform ligation products<td class='s6'>PCR kivd and check by electrophoresis->success<td class='s5'>digest k098995_CrtZ_Terminator(X,P) psb3T5(E,P) ligase 1.J23103CrtEBIY+K098995_CrtZ_Terminator+psb1c3/psb3T5 2.J23103CrtEBIY+psb1c3/psb3T5<td ></tr><tr><td class='s4'>8/19/2011<td class='s5'>1.colony PCR 8/18 transformation products 2.transform Plac+Alss+RBS+ilvC and pCI+B0034<td class='s6'>PCR crtZ and check by electrophoresis->success<td class='s5'>transform 1.J23103CrtEBIY+K098995_CrtZ_Terminator+psb1c3/psb3T5 2.J23103CrtEBIY+psb1c3/psb3T5<td ></tr><tr><td class='s4'>8/20/2011<td class='s7'><td class='s6'>incubate kivd in LB<td class='s5'>colony PCR <td ></tr><tr><td class='s4'>8/21/2011<td class='s7'><td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>8/22/2011<td class='s5'>1.colony PCR 8/19 transformation products <td class='s6'>1.ligate Ptet+kivd+terminator+psb1c3 2.transform 3.incubate crtZ in LB 4.extract plasmid of kivd and check by electrophoresis<td class='s5'>extract plasmid J23103_CrtEBIY digest J23103_CrtEBIY(E,P) and check by electrophoresis ligase J23103_CrtEBIY+K098995_CrtZ_Terminator <br/><td ></tr><tr><td class='s4'>8/23/2011<td class='s5'>1.digest Plac+Alss+ilvC+ilvD 2.ligase(Plac+Alss+ilvC+ilvD)and(B0032+ilvC+B0032+ilvD) + (37C RBS+tetR+terminator) 3.colony PCR 8/18 pCI+B0034 4.transform ligation products <td class='s6'>1.extract plasmid of crtZ and check by electrophoresis<td class='s5'>digest 1.psb3T5(E,P) 2.psb1c3(E,P) 3.J23103_CrtEBIY(E,S) 4.K098995_CrtZ_Terminator(E,X) 5.CrtY(E,P) 6.CrtY_Terminator(E,P) purify 1.psb3T5(E,P) 2.psb1c3(E,P) 3.J23103_CrtEBIY(E,S) 4.K098995_CrtZ_Terminator(E,X) ligase 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3 transform J23103_CrtEBIY+K098995_CrtZ_Terminator<br/><td ></tr><tr><td class='s4'>8/24/2011<td class='s5'>1.colony PCR Plac+Alss+ilvC 2.ligase and transform Plaac+Alss+ilvC+ilvD+37C RBS +tetR+terminator ,(B0034)ilvC+ilvD 3.incubate LB of B0034+C0012+37C RBS+tetR+terminator <td class='s6'>1.ligate PcI+rbs+crtZ+T and psb1c3 2.PCR Ptet+kivd+T<td class='s5'>colony PCR J23103_CrtEBIY+K098995_CrtZ_Terminator transform 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3<td ></tr><tr><td class='s4'>8/25/2011<td class='s5'>1.colony PCR 8/24 transformation products 2.ligase Plac+Alss+B0032,34+ilvC ,(B0032)ilvC+ilvD+37C RBS+tetR+terminator 3.抽plasmid of 8/24 LB<td class='s6'>transform PcI+crtZ+terminator<td class='s5'>colony PCR 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3 →success<td ></tr><tr><td class='s4'>8/26/2011<td class='s5'>1.transform 8/25 ligation products 2.digest K098995,37C RBS+tetR+terminator,(B0030)ilvC+ilvD 3.ligase K098995+B0034<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>8/27/2011<td class='s5'>1.colony PCR BOO32+ilvC+ilvD+37C RBS+tetR+terminator ,(B0034)ilvC+ilvD 2. transform 8/26 ligation products <td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>8/28/2011<td class='s5'>1. colony PCR 8/27 transform products and K098995+B0034<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>8/29/2011<td class='s7'><td class='s6'>digest psb4A5<td class='s7'><td ></tr><tr><td class='s4'>8/30/2011<td class='s7'><td class='s6'>1.digest Ptet+kivd+T EcoR1 and Pst1 2. transform Ptet+kivd+T+psb4A5<td class='s7'><td ></tr><tr><td class='s4'>8/31/2011<td class='s5'>1.digest PSB1C3 2.ligase Plac+Alss+ilvC+ilvD ,(B0034)ilvC+ilvD ,37C RBS+tetR+terminator ,B0034+ilvC ,Plac+Alss+B0030+ilvC ,B0030+ilvD+37C RBS+tetR+terminator 3.transform Plac+Alss+B0032,34+ilvC<td class='s6'>1.digest 37RBS+tetR+T,Ptet+kivd+T 2. transform Ptet+kivd+T+psb4A5 3.digest Plac+Alss+ilvc+ilvd (SP) and 37RBS+tetR+T(XP)<td class='s7'><td ></tr><tr><td class='s4'>9/1/2011<td class='s5'>in morning :1.ligation for change parts to 1c3 backbone 2.PCR pLac+Alss+Boo32+ilvC and check by electrophoresis //in afternoon: 3.transform ligation product in the morning for change parts to 1C3 backbone parts are a. b0030+ilvc b.b0034+ilvc c. b0030+ilvD d. b0032+ilvd e. Plac +Alss+ilvc+ilvD f.b0034+lacI +37℃RBS+tetR+T g. B circuit 6-1 7-1 4. PCR 8/31 product and check by electrophoresis // 5.select&culture PSB1A3 and Dcircuit inLB <td class='s6'>1.ligation 37RBS+tetR+T with Ptet+kivd+T and transform they 2.PCR 8/30 sample3.purify and ligate Plac+Alss+ilvc+ilvd (SP) and 37RBS+tetR+T(XP) 4.transform Plac+Alss+ilvc+ilvd + 37RBS+tetR+T <td class='s5'>digest 1.J23103_CrtEBIY(E,S) 2.K098995_CrtZ_Terminator(E,P) 3. psb4A5(E,P)<br/><td ></tr><tr><td class='s4'>9/2/2011<td class='s5'>in morning : 1. extract plasmid of LB cultured last night //in afternoon: 2. PCR 9/1 change backbone product and check by electrophoresis 3. transform 9/1 product totally 16 tubes<td class='s9'><td class='s5'>ligase K098995_CrtZ_Terminator+psb4A5<td ></tr><tr><td class='s4'>9/3/2011<td class='s5'>1.PCR 9/2 product & check by electrophoresis => success parts:pLac+alss+B0030+ilvC //B0034+ilvC+ilvD 2.culture right parts<td class='s6'>PCR 37RBS+tetR+T+Ptet+kivd+T and sequence one of the samples<td class='s5'>transform K098995_CrtZ_Terminator+psb4A5<td ></tr><tr><td class='s4'>9/4/2011<td class='s5'>1.ligation D circuit to PSB1C3&PSB3K3 for change backbone 2.ligation B circuit & B0032+ilvD &(B0030)pLac+alss+ilvc+ilvd to PSB1C3 for change backbone 3. ligation a. (pLac +alss) with (B0032/34+ilvc) b.(B0034+ilvc) with (B0034+ilvD) <td class='s9'><td class='s5'>colony PCR K098995_CrtZ_Terminator+psb4A5 →success<br/><td ></tr><tr><td class='s4'>9/5/2011<td class='s5'>1. PCR 9/3 product & check by electrophoresis<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>9/6/2011<td class='s5'>in morning :1.transform 9/4 ligation product //in afternoon:1. select bacteria and delivery sequencing<td class='s9'><td class='s5'>extract plasmid K098995_CrtZ_Terminator+psb4A5<td ></tr><tr><td class='s4'>9/7/2011<td class='s7'><td class='s9'><td class='s5'>cotransform J23103_CrtEBIY_psb1k3+K098995_CrtZ_Terminator_psb4A5<td ></tr><tr><td class='s4'>9/8/2011<td class='s5'>1.PCR 9/6 transformation product <td class='s9'><td class='s5'>incubate cotransform product in different temperature<td ></tr><tr><td class='s4'>9/9/2011<td class='s5'>1.check 9/8 PCR product by electrophoresis<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>9/10/2011<td class='s5'>digest (K098995+b0034) &vioC<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>9/14/2011<td class='s11'>1. prepare for spreading the incubated LB on plates &incubated in 37 ℃ 2. ligation ptet +vioABE +PSB1c3 3. digest (K098995+B0034) E.P&E.S and PSB1c3 4.ligation parts for change backbone to 1C3 :a. B circuit 7-1 7-2 b.B0030+ilvD c. B0032+ilvD d. B0030+ilvC e. B0034+ilvC f. D circuit -2&-3<td class='s9'><td class='s5'>cotransform J23103_CrtEBIY_psb3T5+K098995_CrtZ_Terminator_psb4A5<td ></tr><tr><td class='s4'>9/15/2011<td class='s12'>in morning :1. digest PSB1A3 2. transform PSB1A3 &(vioABE + PSB1C3) 3. incubated colony to show different color in different temperature 4. inactive vioC+T digest product //in afternoon : 1. ligation(K098995 +B0034) with (vioC) 2. transform product for change backbone //at night: incbated D circuit in LB<td class='s9'><td class='s5'>incubate cotransform product<br/><td ></tr><tr><td class='s4'>9/16/2011<td class='s12'>1. PCR 9/15 transform product &check by electrophoresis 2. PCR vioABE+1C3// night: 3.transform PSB1A3 &(K098995+B0034+vioC+T) 4. ligation the other failed parts for change backbone to 1C3 5. extract plasmid of D circuit and check by electrophoresis 6. incubated (B0034+ilvC) (B0030+ilvD) (B circuit 7-1) (K098995 +B0034) (vioABE )=>1C3 inLB in37℃<td class='s9'><td class='s5'>spread the incubated LB on plates and incubate in different temperature <td ></tr><tr><td class='s4'>9/17/2011<td class='s12'>1. transform 9/16 product 2. religation PSB1A3 &(K098995+B0034+vioC+T) <td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>9/18/2011<td class='s12'>1.transform 9/17 product 2.PCR (B0032+alss) (B0030+ilvD) (B0030+ilvC) &check by electrophoresis <td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>9/19/2011<td class='s12'>1. incubated parts in LB 2. digest 9/16 plasmid to check 3. ligation C circuit <td class='s6'>Flow cytometry: 2010 IGEM <td class='s7'><td ></tr><tr><td class='s4'>9/20/2011<td class='s12'>1. transform C circuit 2. check (D circuit -1/-2)(B0034+ilvc-1/-2)(B0030+ilvd-1/-2)(K098995+B0034-1/-2)(B circuit-1/-2)(PSB1A3 E.P) by electrophoresis 3. extract plasmid of (B0030+ilvD)(B0032+ALss) and digest it<td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>9/21/2011<td class='s12'>1.digest K098995+B0034 2. check 9/20 digest product by electrophoresis <td class='s9'><td class='s7'><td ></tr><tr><td class='s4'>9/22/2011<td class='s13'><td class='s6'>digest Plac+alss+ilvc+ilvd (EP) and ligate is with psb3K3<td class='s7'><td ></tr><tr><td class='s4'>9/23/2011<td class='s13'><td class='s6'>1.transform Plac+alss+ilvc+ilvd in the morning 2.incubate Plac+alss+ilvc+ilvd in LB at night<td class='s7'><td ></tr><tr><td class='s4'>9/24/2011<td class='s13'><td class='s6'>1.extract plasmid of Plac+alss+ilvc+ilvd 2.cotransform Plac+alss+ilvc+ilvd(3K3) and Ptet+kivd+T(4A5)<td class='s7'><td ></tr><tr><td class='s4'>9/27/2011<td class='s13'><td class='s9'><td class='s5'>incubate cotransform product in different temperature <br/><td ></tr></table> | ||
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Latest revision as of 13:40, 5 October 2011
| Date | G1 | G2 | G2-2 | G2-1 | |
| 4/26/2011 | colon PCR vioABCE+terminator | digest Ptet ES site, Ligate CrtEBI+Ptet +1K3 overnight | |||
| 4/27/2011 | extract Plac plasmid, ligation Plac+37RBS | Ptet+crt EBI transform | |||
| 4/28/2011 | Electrophoresis of vioABCE+terminator & incubate LB ; transform Plac+37RBS | ligate Ptet+crtEBI+1c3(zip) and transform | |||
| 4/29/2011 | prepare plasmid of vioABCE+terminator | Yesterday transform failed.ligate ptet+crtEBI+1c3 again.(non-purify 1C3) | |||
| 4/30/2011 | ligate ptet+crtEBI+1c3(purify) overnight. | ||||
| 5/1/2011 | transform Plac+37RBS | extract Ptet plasmid. Transform Ptet+crtEBI+1c3 | |||
| 5/2/2011 | transform Ptet+vioABCE+terminator | ||||
| 5/3/2011 | Digest crtEBI XP and Ptet SP | ||||
| 5/4/2011 | Ptet digest ES ; vioABCE+terminator digest XP ;ligation Ptet+vioABCE+terminator | Ligate ptet(purify) Amp (digest SP)+crtEBI(purify)(digestXP). Ligate ptet(ES)+crtEBI(XP)+1c3(EP) | |||
| 5/5/2011 | transform Ptet+vioABCE+terminator | Transform 5/4 two ligations.Ligate ptet(purify)Amp (SP)+crtEBI(purify)(XP). | |||
| 5/6/2011 | Transform ptet(purify)Amp(SP)+crtEBI(purify)(XP). | ||||
| 5/7/2011 | incubate Ptet,CrtEBI | ||||
| 5/8/2011 | extract Ptet CrtEBI plasmid | ||||
| 5/10/2011 | Plac+37RBS colony PCR ; prepare plasmid of Plac+37RBS | Ligate Ptet+CrtEBI+1c3(original method) and Ligate Ptet+CrtEBI+1c3(purify) | |||
| 5/11/2011 | Ptet+vioABCE+terminator colony PCR ;Plac+37RBS extract plasmid | Transform Ptet+CrtEBI+1c3(original method) ,Plac+CctEBIY,Plac+CrtEBI | |||
| 5/12/2011 | ligation LuxR+terminator | PCR Ptet+CrtEBI+1c3 | CrtY point mutation PCR | ||
| 5/13/2011 | transform LuxR+terminator | Electrophoresis.PCR Ptet+CrtEBI+1c3 again | CrtY self-ligation | ||
| 5/14/2011 | ligation LuxR+terminator | Electrophoresis. | CrtY transform | ||
| 5/15/2011 | transform LuxR+terminator | ||||
| 5/16/2011 | LuxR+terminator colony PCR | ||||
| 5/17/2011 | LuxR+terminator incubate LB | Ptet digest sp ,crtEBI digestXP (purify both),Ligation. | CrtY colonyPCR | Digest C0012 ES & XP; B0034 ES | |
| 5/18/2011 | prepare plasmid of LuxR+terminator ; ligation J24679+LuxR+terminator; prepare plasmid of Ptet & digest EP SP; incubateLB of k274003 | Transform Ptet SP+CrtEBI XP | Ligase C0012+37RBS +tetR+terminator & B0034+C0012 Digest B0034+GFP+terminator | ||
| 5/19/2011 | ligation Ptet+vioABCE+terminator ; prepare plasmid of k274003 ; transform J24679+LuxR+terminator | transform C0012+37RBS +tetR+terminator & B0034+C0012 | |||
| 5/20/2011 | J24679+LuxR+terminator colony PCR ; incubate LB of Ptet | CrtY colonyPCR | PCR C0012+37RBS +tetR+terminator | ||
| 5/21/2011 | prepare plasmid of Ptet ; ligation and transform J24679+LuxR+terminator ; ligation and transform Ptet+vioABCE+terminator | digest CrtY(S) →success | |||
| 5/22/2011 | Ptet+vioABCE+terminator colony PCR | dilute plasmid RBS+crtZ from 2011plate19B-I742158 | |||
| 5/24/2011 | digest CrtY(E,S) Terminator(E,X) | ||||
| 5/25/2011 | ligation and transform J24679+LuxR+terminator | ligase CrtY+Terminator | |||
| 5/26/2011 | think of new ligation and transform protocol of J24679+LuxR+terminator | transform CrtY+Terminator | |||
| 5/27/2011 | follow new protocol to ligation and transform protocol of J24679+LuxR+terminator (add 1mM ATP when ligation) | ||||
| 5/28/2011 | J24679+LuxR+terminator colony PCR & incubate LB | ||||
| 5/29/2011 | prepare plasmid of J24679+LuxR+terminator X2 | ||||
| 5/30/2011 | prepare plasmid of J24679+LuxR+terminator X5 | ||||
| 5/31/2011 | check J24679+LuxR+terminator plasmid | ||||
| 6/1/2011 | transform RBS+crtZ | colony PCR CrtY+Terminator | |||
| 6/2/2011 | colony PCR RBS+crtZ electrophoresis check sequencing RBS+crtZ | ||||
| 6/3/2011 | ligase CrtY+Terminator | ||||
| 6/4/2011 | transform CrtY+Terminator | ||||
| 6/7/2011 | sequencing result RBS+crtZ → successs incubate RBS+crtZ in LB-medium | colony PCR CrtY+Terminator →success | |||
| 6/8/2011 | extract plasmid RBS+crtZ | ||||
| 6/10/2011 | digest RBS+crtZ(ES)(anti-A) digest terminator-J61048(XP&EX)(anti-A) digest PSB1K3(EP)(anti-K) ligate 1.RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K) 2.RBS+crtZ(ES)+terminator(EX)(anti-A) | ||||
| 6/11/2011 | transform 1.RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K) 2.RBS+crtZ(ES)+terminator(EX)(anti-A) | incubate 1.37RBS 2.CrtY_Terminator 3.psb1k3 4.psb1c3 | |||
| 6/12/2011 | only RBS+crtZ(ES)+terminator(EX)(anti-A) grown colony PCR above-mentioned electrophoresis check sequencing RBS+crtZ(ES)+terminator(EX)(anti-A) digest RBS+crtZ(ES)(anti-A)again digest terminator-J61048(XP)(anti-A)again digest PSB1K3(EP)(anti-K)again ligate RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)again | extract plasmid digest 1.37RBS(E,S).(S,P) 2.CrtY_Terminator(X,P) 3.psb1c3(E,P) ligase 37RBS+CrtY_Terminator | |||
| 6/13/2011 | transform RBS+crtZ(ES)+terminator(XP)+PSB1K3(EP)(anti-K)again | transform 37RBS+CrtY_Terminator | |||
| 6/17/2011 | sequencing result RBS+crtZ(ES)+terminator(EX)(anti-A) → successs | ||||
| 6/23/2011 | digest 37RBS and kivd ; incubate LB of B0030,B0032,B0034 | ||||
| 6/24/2011 | prepare plasmid of B0030,B0032,B0034 & check ; ligation 37RBS+kivd | incubate RBS+crtZ+terminator(anti-A) in LB-medium | |||
| 6/25/2011 | digest ilvC,ilvD,malss,B0030,B0032,B0034 | extract plasmid RBS+crtZ+terminator(anti-A) digest RBS+crtZ+terminator(XP)(anti-A) digest pcI(ES&SP)(anti-A) digest PSB1K3(EP)(anti-K) ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) 2.pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) | colony PCR 37RBS+CrtY_Terminator→success | ||
| 6/26/2011 | check digest product | transform 1.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) 2.pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) | |||
| 6/27/2011 | only pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) grown colony PCR can't examine pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) because pcI is too small(49bp) incubate pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) in LB-medium | ||||
| 6/28/2011 | extract plasmid pcI(SP)+RBS+crtZ+terminator(XP)(anti-A) digest RBS+crtZ+terminator(XP)(zip)(anti-A)again digest pcI(ES&SP)(anti-A)again digest PSB1K3(EP)(anti-K)again digest PSB1C3(EP)(anti-C)again ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)again 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)again 3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)again 4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)again 5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A)again | ||||
| 6/29/2011 | transform 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K)again 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K)again 3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C)again 4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C)again 5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A)again | ||||
| 6/30/2011 | ligation RBS+ilvC and RBS+ilvD and RBS+malss | only 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) grown 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) grown 5.pcI(SP)+RBS+crtZ+terminator(XP)(zip)(anti-A) grown colony PCR above-mentioned electrophoresis check sequencing above-mentioned | incubate 37RBS_CrtY_Terminator | ||
| 7/1/2011 | transform RBS+malss ; digest PSB1A3 ; ligation and transform B0034+kivd | extract 37RBS_CrtY_Terminator digest 37RBS_CrtY_Terminator(X,P) | |||
| 7/4/2011 | RBS+malss colony PCR | ||||
| 7/5/2011 | transform RBS+ilvC and RBS+ilvD ; ligation Ptet+vioABCE | ||||
| 7/6/2011 | RBS+ilvC and RBS+ilvD colony PCR | ||||
| 7/7/2011 | Electrophoresis of RBS+ilvC and RBS+ilvD and RBS+malss | ||||
| 7/9/2011 | sequencing result → fail digest RBS+crtZ+terminator(XP)(zip)(anti-A) digest RBS+crtZ+terminator(XP)(purify)(anti-A) digest pcI(ES&SP)(anti-A) digest PSB1K3(EP)(anti-K) digest PSB1C3(EP)(anti-C) ligate 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) 3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C) 4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C) 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) | ||||
| 7/11/2011 | Ptet+vioABCE colony PCR | transform 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) 3.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1C3(EP)(anti-C) 4.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1C3(EP)(anti-C) 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) | |||
| 7/12/2011 | Electrophoresis of Ptet+vioABCE | only 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) grown 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) grown 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) grown | |||
| 7/13/2011 | Ptet(SP) and vioABCE(XP) gel purified ; ligation Ptet+vioABCE | ||||
| 7/14/2011 | transform Ptet+vioABCE | Flow cytometry: K098988, K098987 (in 25,37,42 temperature degree ) colony PCR 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) electrophoresis check sequencing 1.pcI(ES)+RBS+crtZ+terminator(XP)(zip)+PSB1K3(EP)(anti-K) 2.pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) | |||
| 7/16/2011 | digest CrtEBI(E,S) | ||||
| 7/17/2011 | sequencing result 5.pcI(SP)+RBS+crtZ+terminator(XP)(purify)(anti-A) → successs incubate cI+pcI-K098995 in LB-medium | ||||
| 7/18/2011 | ligation and transform B0034+kivd ; Ptet+vioABCE colony PCR | extract plasmid cI+pCI | ligase CrtEBI+37RBS_CrtY_Terminator | ||
| 7/19/2011 | Electrophoresis of Ptet+vioABCE | digest cI+pcI(ES)(anti-A) digest RBS+ctrZ+terminator(XP)(anti-A) digest PSB1K3(EP)(anti-K) ligate cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) | Transform CrtEBI+37RBS_CrtY_Terminator | ||
| 7/20/2011 | ligation RBS+Alss ; prepare plasmid of Ptet+vioABCE,kivd,ilvC,ilvD,Alss ; B0034+kivd colony PCR ; incubate LB of PSB1A3 and PSB1K3 | transform cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) | |||
| 7/21/2011 | prepare plasmid of PSB1K3 ; Electrophoresis of B0034+kivd ; transform RBS+Alss | ||||
| 7/25/2011 | ligation RBS+Alss ; RBS+Alss colony PCR | colony PCR cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) electrophoresis check sequencing cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) | ligase CrtEBI+37RBS_CrtY_Terminator | ||
| 7/26/2011 | digest B0034+ilvC and B0034+ilvD ; transform RBS+Alss | Transform CrtEBI+37RBS_CrtY_Terminator | |||
| 7/27/2011 | ligation K098995+B0034 ; RBS+Alss colony PCR ; ligation (B0034+ilvC)+(B0034+ilvD) | ||||
| 7/28/2011 | RBS+Alss colony PCR again ; transform K098995+B0034 and (B0034)ilvC+ilvD | colony PCR CrtEBI+37RBS_CrtY_Terminator→success | |||
| 7/29/2011 | (B0034)ilvC+ilvD colony PCR ; ligation (B0034)ilvC+ilvD and K098995+B0034 | ||||
| 7/30/2011 | transform (B0034)ilvC+ilvD and K098995+B0034 ; digest RBS+ilvD(ES) | ||||
| 7/31/2011 | K098995+B0034 and (B0034)ilvC+ilvD colony PCR | ||||
| 8/1/2011 | colony PCR of ilvD+terminator and ligation RBS+ilvC+ilvD(PSA1C3)and then transform | sequencing result cI+pcI(ES)+RBS+crtZ+terminator(XP)+PSB1K3(EP)(anti-K) → successs | |||
| 8/2/2011 | 抽plasmid ptet+vioABCE+terminator and colony PCR RBS+ilvC+RBS+ilvD and incubate37C RBS+tetR+terminator | Flow cytometry: K098988 in 42 temperature degree | |||
| 8/3/2011 | ligase (B0030,B0032)ilvC+ilvD and digest B0032+COO12,BOO32+Alss and 抽plasmid Ptet+vioABCE+terminator then digest it and transform 37C RBS+tetR+terminator and (B0030,B0032)ilvC+ilvD | ligase CrtEBI+Plac CrtEBI+Ptet | |||
| 8/4/2011 | 1.ligaes 2.check digestion product 3.抽plasmid 37C RBS+tetR+terminator 4.transform ligastion pruduct Plac+Alss,RBS+ilvD+37C RBS+tetR+terminator 5.colony PCR ilvC+ilvD | 1.digest B0015 (XP) and check by electrophoresis 2.ligate B0030 or B0034 +kivd and B0015 | transform CrtEBI+Plac CrtEBI+Ptet ligase J23103_CrtEBI+37RBS_CrtY_Terminator | ||
| 8/5/2011 | 1.colony PCR RBS+ilvD+37C RBS+tetR+terminator 2.colony PCR Plac+Alss 3. | 1. transform 8/4 ligation products 2.purify Boo15, J61048, Ptet and check by electrophoresis 3. ligate Ptet(purify) and kivd, J61048(purify) and kivd, B0015(purify) and kivd 4.Flow cytometry | Transform J23103_CrtEBI+37RBS_CrtY_Terminator | ||
| 8/6/2011 | 1.ligase RBS+ilvD+37C RBS+tetR+terminator | 1.PCR kivd+Boo15 2. Check PCR products by electrophoresis. Four samples reach expected bands. 3. transform 8/5 ligation products 4. inbate 988 DH5-alpha, 988 EPI300 5. four zone culture kivd+B0015 | colony PCR | ||
| 8/7/2011 | 1.extract plasmid:EPI300 K098988, DH5-alpha K098988 and check py electrophoresis 2.sequencing kivd+J61048, Ptet+kivd | purify CrtEBIY | |||
| 8/8/2011 | 1.transform RBS+ilvD+37C RBS+tetR+terminator | 1.transform 988 to EPI300 2.sequencing kivd+B0015 | |||
| 8/9/2011 | 1.ligase PCI +B0034 and (B0032)ilvC+ilvD+37C RBS+tetR+terminator and B0032+C0012+R+LuxR+terminator 2.transform (B0032)ilvC+ilvD+37C RBS+tetR+terminator 3.digest B0034 and (B0032)ilvC+ilvD and Plac+B0032+Alss | 1.digest kivd+J61048, Ptet+kivd | ligase Plac+CrtEBIY(purify) digest J23103CrtEBI(E,S).(S,P) | ||
| 8/10/2011 | 1.ligate Plac+Alss+ilvC+ilvD 2.digest(B0032)ilvC+IlvD and B0030,32,34+ilvC 3.check digestion product 4.colony PCR RBS+ilvD+37C RBS+tetR+terminator 5.transform (B0030,34)ilvC+ilvD and Plac+Alss+ilvC+ilvD and (B0032)ilvC+IlvD and pCI+B0034 | 1.ligate Ptet+kivd+terminator with different methods 2.Flow cytometry: incubate K098988(EPI300) at 32, 37, 42 temperature degree | Transform J23103CrtEBI+37RBS_CrtY_Terminator | ||
| 8/11/2011 | 1.ligate Plac+Alss+RBS+ilvC and ilvC+ilvD+37C RBS+tetR+terminator 2.colony PCR Plac+Alss+ilvC+iulvD and pCI+B0034 and ilvC+ilvD+37C RBS+tetR+terminator 3.transform ligastion products 5.ligate Plac+Alss+ilvC+ilvD | ligase J23103CrtEBI+37RBS_CrtY_Terminator colony PCR →success | |||
| 8/12/2011 | 1.ligate Plac+Alss+ilvC and iulvC+ilvD+37C RBS+tetR+terminator 2.check digestion products(8/10,11) 3.colony PCR Plac+Alss+ilvC and (B0034)ilvC+ilvD and ilvC+ilvD+37C RBS+tetR+terminator 4.transform Plac+Alss+ilvC+ilvD and ilvC+ilvD+37C RBS+tetR+terminator | ||||
| 8/13/2011 | 1.colony PCR 8/12transformation products 2.liagte B0030,34+ilvC+ilvD and Plac+Alss+RBS+ilvC and then transform | 1.digest plasmids of kivd EcoR1 and Pst1 2.ligate kivd with psb1c3 | |||
| 8/14/2011 | 1.digest ilvC+ilvD+37C RBS+tetR+terminator 2.check digestion products 3.ligate Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminator(PSB1K3) 4.digest Plac+Alss | transform 8/13 ligation products | |||
| 8/15/2011 | 1.ligate Plac+Alss+B0030,32,34+ilvC and (B0030,34)ilvC+ilvD 2.抽plasmid Ptet+Alss+ilvC+ilvD 3.incubate LB of transformation products 4.transform Plac+Alss+B0030,32,34+ilvC and Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminator and B0030,34+ilvC+ilvD | 1.PCR 8/14 sample and check by electrophoresis->fail 2.digest plasmids of crtZ EcoR1 and Pst1 3.ligate crtZ and psb1c3 | |||
| 8/16/2011 | 1.抽plasmid of LB products(8/15) 2.digest Plac+Alss+ilvC+ilvD | incubate psb3T5 | |||
| 8/17/2011 | 1.check dugestion products 2.colony PCR Plac+Alss+ilvC+ilvD+37C RBS+tetR+terminator | transform 8/15 ligation products | extract plasmid psb3T5 | ||
| 8/18/2011 | 1.ligate ilvC+ilvD and Plac+Alss+RBS+ilvC and pCI+B0034 2.colony PCR B0034+C0012+37C RBS+tetR+terminator 3.transform ligation products | PCR kivd and check by electrophoresis->success | digest k098995_CrtZ_Terminator(X,P) psb3T5(E,P) ligase 1.J23103CrtEBIY+K098995_CrtZ_Terminator+psb1c3/psb3T5 2.J23103CrtEBIY+psb1c3/psb3T5 | ||
| 8/19/2011 | 1.colony PCR 8/18 transformation products 2.transform Plac+Alss+RBS+ilvC and pCI+B0034 | PCR crtZ and check by electrophoresis->success | transform 1.J23103CrtEBIY+K098995_CrtZ_Terminator+psb1c3/psb3T5 2.J23103CrtEBIY+psb1c3/psb3T5 | ||
| 8/20/2011 | incubate kivd in LB | colony PCR | |||
| 8/21/2011 | |||||
| 8/22/2011 | 1.colony PCR 8/19 transformation products | 1.ligate Ptet+kivd+terminator+psb1c3 2.transform 3.incubate crtZ in LB 4.extract plasmid of kivd and check by electrophoresis | extract plasmid J23103_CrtEBIY digest J23103_CrtEBIY(E,P) and check by electrophoresis ligase J23103_CrtEBIY+K098995_CrtZ_Terminator | ||
| 8/23/2011 | 1.digest Plac+Alss+ilvC+ilvD 2.ligase(Plac+Alss+ilvC+ilvD)and(B0032+ilvC+B0032+ilvD) + (37C RBS+tetR+terminator) 3.colony PCR 8/18 pCI+B0034 4.transform ligation products | 1.extract plasmid of crtZ and check by electrophoresis | digest 1.psb3T5(E,P) 2.psb1c3(E,P) 3.J23103_CrtEBIY(E,S) 4.K098995_CrtZ_Terminator(E,X) 5.CrtY(E,P) 6.CrtY_Terminator(E,P) purify 1.psb3T5(E,P) 2.psb1c3(E,P) 3.J23103_CrtEBIY(E,S) 4.K098995_CrtZ_Terminator(E,X) ligase 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3 transform J23103_CrtEBIY+K098995_CrtZ_Terminator | ||
| 8/24/2011 | 1.colony PCR Plac+Alss+ilvC 2.ligase and transform Plaac+Alss+ilvC+ilvD+37C RBS +tetR+terminator ,(B0034)ilvC+ilvD 3.incubate LB of B0034+C0012+37C RBS+tetR+terminator | 1.ligate PcI+rbs+crtZ+T and psb1c3 2.PCR Ptet+kivd+T | colony PCR J23103_CrtEBIY+K098995_CrtZ_Terminator transform 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3 | ||
| 8/25/2011 | 1.colony PCR 8/24 transformation products 2.ligase Plac+Alss+B0032,34+ilvC ,(B0032)ilvC+ilvD+37C RBS+tetR+terminator 3.抽plasmid of 8/24 LB | transform PcI+crtZ+terminator | colony PCR 1.CrtY+psb1c3 2.CrtY_Terminator+psb1c3 →success | ||
| 8/26/2011 | 1.transform 8/25 ligation products 2.digest K098995,37C RBS+tetR+terminator,(B0030)ilvC+ilvD 3.ligase K098995+B0034 | ||||
| 8/27/2011 | 1.colony PCR BOO32+ilvC+ilvD+37C RBS+tetR+terminator ,(B0034)ilvC+ilvD 2. transform 8/26 ligation products | ||||
| 8/28/2011 | 1. colony PCR 8/27 transform products and K098995+B0034 | ||||
| 8/29/2011 | digest psb4A5 | ||||
| 8/30/2011 | 1.digest Ptet+kivd+T EcoR1 and Pst1 2. transform Ptet+kivd+T+psb4A5 | ||||
| 8/31/2011 | 1.digest PSB1C3 2.ligase Plac+Alss+ilvC+ilvD ,(B0034)ilvC+ilvD ,37C RBS+tetR+terminator ,B0034+ilvC ,Plac+Alss+B0030+ilvC ,B0030+ilvD+37C RBS+tetR+terminator 3.transform Plac+Alss+B0032,34+ilvC | 1.digest 37RBS+tetR+T,Ptet+kivd+T 2. transform Ptet+kivd+T+psb4A5 3.digest Plac+Alss+ilvc+ilvd (SP) and 37RBS+tetR+T(XP) | |||
| 9/1/2011 | in morning :1.ligation for change parts to 1c3 backbone 2.PCR pLac+Alss+Boo32+ilvC and check by electrophoresis //in afternoon: 3.transform ligation product in the morning for change parts to 1C3 backbone parts are a. b0030+ilvc b.b0034+ilvc c. b0030+ilvD d. b0032+ilvd e. Plac +Alss+ilvc+ilvD f.b0034+lacI +37℃RBS+tetR+T g. B circuit 6-1 7-1 4. PCR 8/31 product and check by electrophoresis // 5.select&culture PSB1A3 and Dcircuit inLB | 1.ligation 37RBS+tetR+T with Ptet+kivd+T and transform they 2.PCR 8/30 sample3.purify and ligate Plac+Alss+ilvc+ilvd (SP) and 37RBS+tetR+T(XP) 4.transform Plac+Alss+ilvc+ilvd + 37RBS+tetR+T | digest 1.J23103_CrtEBIY(E,S) 2.K098995_CrtZ_Terminator(E,P) 3. psb4A5(E,P) | ||
| 9/2/2011 | in morning : 1. extract plasmid of LB cultured last night //in afternoon: 2. PCR 9/1 change backbone product and check by electrophoresis 3. transform 9/1 product totally 16 tubes | ligase K098995_CrtZ_Terminator+psb4A5 | |||
| 9/3/2011 | 1.PCR 9/2 product & check by electrophoresis => success parts:pLac+alss+B0030+ilvC //B0034+ilvC+ilvD 2.culture right parts | PCR 37RBS+tetR+T+Ptet+kivd+T and sequence one of the samples | transform K098995_CrtZ_Terminator+psb4A5 | ||
| 9/4/2011 | 1.ligation D circuit to PSB1C3&PSB3K3 for change backbone 2.ligation B circuit & B0032+ilvD &(B0030)pLac+alss+ilvc+ilvd to PSB1C3 for change backbone 3. ligation a. (pLac +alss) with (B0032/34+ilvc) b.(B0034+ilvc) with (B0034+ilvD) | colony PCR K098995_CrtZ_Terminator+psb4A5 →success | |||
| 9/5/2011 | 1. PCR 9/3 product & check by electrophoresis | ||||
| 9/6/2011 | in morning :1.transform 9/4 ligation product //in afternoon:1. select bacteria and delivery sequencing | extract plasmid K098995_CrtZ_Terminator+psb4A5 | |||
| 9/7/2011 | cotransform J23103_CrtEBIY_psb1k3+K098995_CrtZ_Terminator_psb4A5 | ||||
| 9/8/2011 | 1.PCR 9/6 transformation product | incubate cotransform product in different temperature | |||
| 9/9/2011 | 1.check 9/8 PCR product by electrophoresis | ||||
| 9/10/2011 | digest (K098995+b0034) &vioC | ||||
| 9/14/2011 | 1. prepare for spreading the incubated LB on plates &incubated in 37 ℃ 2. ligation ptet +vioABE +PSB1c3 3. digest (K098995+B0034) E.P&E.S and PSB1c3 4.ligation parts for change backbone to 1C3 :a. B circuit 7-1 7-2 b.B0030+ilvD c. B0032+ilvD d. B0030+ilvC e. B0034+ilvC f. D circuit -2&-3 | cotransform J23103_CrtEBIY_psb3T5+K098995_CrtZ_Terminator_psb4A5 | |||
| 9/15/2011 | in morning :1. digest PSB1A3 2. transform PSB1A3 &(vioABE + PSB1C3) 3. incubated colony to show different color in different temperature 4. inactive vioC+T digest product //in afternoon : 1. ligation(K098995 +B0034) with (vioC) 2. transform product for change backbone //at night: incbated D circuit in LB | incubate cotransform product | |||
| 9/16/2011 | 1. PCR 9/15 transform product &check by electrophoresis 2. PCR vioABE+1C3// night: 3.transform PSB1A3 &(K098995+B0034+vioC+T) 4. ligation the other failed parts for change backbone to 1C3 5. extract plasmid of D circuit and check by electrophoresis 6. incubated (B0034+ilvC) (B0030+ilvD) (B circuit 7-1) (K098995 +B0034) (vioABE )=>1C3 inLB in37℃ | spread the incubated LB on plates and incubate in different temperature | |||
| 9/17/2011 | 1. transform 9/16 product 2. religation PSB1A3 &(K098995+B0034+vioC+T) | ||||
| 9/18/2011 | 1.transform 9/17 product 2.PCR (B0032+alss) (B0030+ilvD) (B0030+ilvC) &check by electrophoresis | ||||
| 9/19/2011 | 1. incubated parts in LB 2. digest 9/16 plasmid to check 3. ligation C circuit | Flow cytometry: 2010 IGEM | |||
| 9/20/2011 | 1. transform C circuit 2. check (D circuit -1/-2)(B0034+ilvc-1/-2)(B0030+ilvd-1/-2)(K098995+B0034-1/-2)(B circuit-1/-2)(PSB1A3 E.P) by electrophoresis 3. extract plasmid of (B0030+ilvD)(B0032+ALss) and digest it | ||||
| 9/21/2011 | 1.digest K098995+B0034 2. check 9/20 digest product by electrophoresis | ||||
| 9/22/2011 | digest Plac+alss+ilvc+ilvd (EP) and ligate is with psb3K3 | ||||
| 9/23/2011 | 1.transform Plac+alss+ilvc+ilvd in the morning 2.incubate Plac+alss+ilvc+ilvd in LB at night | ||||
| 9/24/2011 | 1.extract plasmid of Plac+alss+ilvc+ilvd 2.cotransform Plac+alss+ilvc+ilvd(3K3) and Ptet+kivd+T(4A5) | ||||
| 9/27/2011 | incubate cotransform product in different temperature |
