Team:Heidelberg/Notebook/Sensing Group/Notebook/4thweek

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   Home    Team   Overview    Advisors  </a></li>  Undergraduates  </a></li>  University  </a></li>  DKFZ  </a></li>  BioQuant  </a></li>  BioRegion Rhein-Neckar  </a></li> </ul> </li>  Project</a> <ul class="DropDownMenu" id="MB1-DDM1">  Overall Project  </a></li>  Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Sensing"> Sensing  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_I"> Killing I  - Phages  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Killing_II"> Killing II - Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Project/Visualization"> Visualization  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Parts" style="color: white">Parts</a> <ul class="DropDownMenu" id="MB1-DDM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts"> Submitted Parts  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Parts/Characterization"> Characterization  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Modeling" style="color: white">Modeling</a> <ul class="DropDownMenu" id="MB1-DDM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling"> Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Chemotaxis"> Chemotaxis-Colicin  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Modeling/Phage"> Phage Dynamics model  </a></li> </ul> </li> <li> <a href="http://2008.igem.org/Team:Heidelberg/Notebook/Overview" style="color: white">Notebook</a> <ul class="DropDownMenu" id="MB1-DDM5"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Sensing >  </a> <ul class="SideMenu" id="MB1-DDM2-SM1"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Sensing_Group/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Killing I - Phages >  </a> <ul class="SideMenu" id="MB1-DDM2-SM2"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_I/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Killing II - Colicin >  </a> <ul class="SideMenu" id="MB1-DDM2-SM3"> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Cloning"> Cloning strategy </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/Notebook"> Notebook </a></li> </ul> </li> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/visualization"> Visualization  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/material"> Material & Methods  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/team_meetings"> Team Meetings  </a> <li><a href="http://2008.igem.org/Team:Heidelberg/Notebook/seminar"> Seminar on Synthetic Biology  </a> </ul> </li> <li style="width: 160px"> <a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview" style="color: white">Human Practice</a> <ul class="DropDownMenu" id="MB1-DDM4"> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Project_Overview"> Project Overview  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Phips_the_Phage"> Phips the Phage  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Essay"> Essay  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Surveys"> Surveys  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Open_Day"> Open Day  </a></li> <li><a href="http://2008.igem.org/Team:Heidelberg/Human_Practice/Nobel_Prize"> Nobel Prize  </a></li> </ul> </li>

<li> <a href="http://2008.igem.org/Team:Heidelberg/Sponsors" style="color: white">Sponsors</a> </li> </ul>

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Back to the overview

Monday, 08/25/2008
--> not successful
 * sequentiell and simultaneous digestion of pDk48 NcoI/NdeI and NcoI/KpnI
 * preparation of O/N culture for pDK48 cells

Cloning

 * test digestion of pDK48 with XbaI (NEBuffer 2 + BSA) and PstI (NeBuffer 3 + BSA) to check wehther the vector is the right one
 * digestion of Fusion-1 (from 08/21/2008) with NcoI/NdeI, with subsequent gel extraction
 * Ligation of Fusion-1 fragment and pDk48 plasmid and transformation into DH5α competent cells

LuxS Test
Material: 500 mM IPTG-stock (A), 10 mM IPTG-stock (B)
 * 1) negative control: just 1 ml cell suspension
 * 2) 10 µM IPTG induced (1 µl stock B + 999 µl cell suspension)
 * 3) 20 µM IPTG induced (2 µl stock B + 998 µl cell suspension)
 * 4) 50 µM IPTG induced (5 µl stock B + 995 µl cell suspension)
 * 5) 100 µM IPTG induced (10 µl stock B + 990 µl cell suspension)
 * 6) 500 µM IPTG induced (1 µl stock A + 999 µl cell suspension)
 * 7) 1 mM IPTG induced (2 µl stock A + 998 µl celll suspension)

Measurement was done like described in the Materials & Methods section <br style="clear: both;"/>

Wednesday, 08/27/2008

 * PCR of LuxQ from V. harveyi with Phusion Mastermix
 * PCR cycle: 5min @ 98°C || 10s @ 98°C | 30s @ 50°C | 1min @ 72°C || 5min @ 72°C | 4°C hold (30 cycles)
 * NO PCR product
 * preparation of pTrc99α and pDK48 O/N cultures for Maxipreps

Thursday, 08/28/2008
--> No result
 * Maxiprep of pDK48 and pTrc99α (product eluted in TB buffer, not water!!)
 * Digestion of LuxQ-Tar fusion (A,B) and LuxQ 1-10 with the follwing enzymes:
 * Q(1-10):
 * XbaI (NEBuffer 2 + BSA) --> if insert: 736 + 1204 bp band; if no insert: 4176 bp band
 * EcoRV (NEBuffer 3 + BSA) --> if insert: 5074 bp + 866 band; if no insert: 4176 bp
 * F1:
 * BamHI (NEBuffer 3 + BSA) --> if insert: 6101 bp band; if no insert: 5768 + 833 bp band
 * PstI (NEBuffer 3 + BSA --> if insert: 6101 bp; if no insert: 5768 + 833 bp band

Friday, 08/29/2008
Go to 5th week