Team:Tokyo-Nokogen/notebook

   







This chart suggests that we pick up part 1 and part 2 materials, ligate them, transform DH5a with them, inoculate that cell and do miniprep after cultivation. 【red light receptor】 Construct parts 【aggregation and lysis】 Order primers which designed to amplify antigen43. Construct parts. 

<p style="text-indent: 2em">★ 08/24/09 to 08/28/09 ---parts construction <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Construct parts <p style="text-indent: 3em">【red light receptor】 <p style="text-indent: 3em">Construct parts <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Extract genome contains antigen43 from E.coli K-12. <p style="text-indent: 3em">Amplify antigen43 by PCR using primers we made. <p style="text-indent: 3em">Construct parts

<p style="text-indent: 2em">★ 08/31/09 to 09/04/09 -parts construction <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Construct parts. <p style="text-indent: 3em">Make shortage plates and LB medium. <p style="text-indent: 3em">【red light receptor】 <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Check sequence of [a1] and [a2], but cannot read it. <p style="text-indent: 3em">Gel electrophoresis of antigen43 and expression vector pTrc99- Nde Ⅰ cut by Nde Ⅰ, <p style="text-indent: 3em">but cannot find appropriate bands. <td style="background-color:#666; color:#FFF" nowrap> <p style="text-indent: 2em">★ 09/07/09 to 09/11/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">【green light receptor】 <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Gel electrophoresis of antigen43 cut by Nde Ⅰand Spe Ⅰ. <p style="text-indent: 3em">Gel electrophoresis of expression vector pTrc99- Nde Ⅰ cut by Nde Ⅰand Xba Ⅰ <p style="text-indent: 3em">Do geneclean both of them, ligate them, transform DH5a with ligated sample and inoculate it. <p style="text-indent: 3em">Check sequence of [a1] and [a2] again, but cannot read it. <p style="text-indent: 2em">★ 09/14/09 to 09/18/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Sequence analysis. <p style="text-indent: 3em">【green light receptor】 <p style="text-indent: 3em">Sequence analysis <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em"> Pick up some single colonies from inoculated plate made last week and do direct colony PCR. After PCR, <p style="text-indent: 3em">we check presence of insert DNA in plasmid by electrophoresis but cannot find. <p style="text-indent: 3em">Order primers which break 6 Pst Ⅰsites not to change amino acid sequence. <p style="text-indent: 3em">Try reconstruction of [a1] to [a3]. <td style="background-color:#666; color:#FFF" nowrap> <p style="text-indent: 2em">★ 09/21/09 to 09/25/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">From results of sequence analysis, we find we couldn’t construct all of the above counter parts. <p style="text-indent: 3em">And start reconstruction of counter parts ([c1], [c2] [c4] and following parts.) <p style="text-indent: 3em">【green light receptor】 <p style="text-indent: 3em">Sequence analysis <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Gel electrophoresis of antigen43 cut by Nde Ⅰand Spe Ⅰ. <p style="text-indent: 3em">Gel electrophoresis of expression vector pTrc99- Nde Ⅰ cut by Nde Ⅰand Xba Ⅰ <p style="text-indent: 3em">Do geneclean both of them, ligate them, transform DH5a with ligated sample and inoculate it. <p style="text-indent: 3em">Try reconstruction of [a4] and [a5]. <p style="text-indent: 2em">★ 09/21/09 to 09/25/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Reconstruction of parts ([c3], [c6], [c12] and follwing part) <p style="text-indent: 3em">Check sequence of reconstruction parts. <p style="text-indent: 3em">【light receptor】 <p style="text-indent: 3em">Test red light receptor. <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Pick up some single colonies from inoculated plate made last week and do direct colony PCR. <p style="text-indent: 3em">After PCR, we check presence of insert DNA in plasmid by electrophoresis but cannot find. <p style="text-indent: 3em">Pick up all colonies and cultivate them and do induction in order to watch out occurrence of aggregation. <p style="text-indent: 3em">But we cannot see aggregation.

<td style="background-color:#666; color:#FFF" nowrap> <p style="text-indent: 2em">★ 09/28/09 to 10/02/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Reconstruction of parts ([c5] and following parts) <p style="text-indent: 3em">Sequence analysis <p style="text-indent: 3em">【light receptor】 <p style="text-indent: 3em">Test green light receptor. <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Try TA cloning (ligation of antigen43 with pGEMT vector). <p style="text-indent: 3em">Transform DH5a with ligated sample, inoculate it, cultivate it and do miniprep. <p style="text-indent: 3em">Try mutation 1st and 2nd PstⅠ sites. After mutagenesis, transform DH5a with ligated sample, inoculate it. <p style="text-indent: 3em">But there is no colony on plate.

<p style="text-indent: 2em">★ 10/5/09 to 10/09/09 <p style="text-indent: 3em">【counter】 <p style="text-indent: 3em">Sequence analysis <p style="text-indent: 3em">【aggregation and lysis】 <p style="text-indent: 3em">Check sequence of [a1] and [a2] again, but cannot read full length sequence. <p style="text-indent: 3em">→ Something wrong with Biobrick parts.

<td style="background-color:#666; color:#FFF" nowrap> <p style="text-indent: 2em">★ 10/12/09 to 10/16/09 <p style="text-indent: 3em">Start testing all parts. <p style="text-indent: 3em">Prepare for sending parts to MIT  <img src="http://2009.igem.org/wiki/images/9/90/TOP4.png" width="63" height="50" border="0"></a>

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