Team:Heidelberg/Notebook/Killing II/5thweek

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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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5th week

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go back to the overview

pSB1A3-Receiver-Colicin-Cloning

 * Transformationresults of ligation pSB1A3-RecluxpR no colonies
 * Retransformation of ligation into TOP 10 -> standard protocoll

Activity Test

 * Inoculation of TOP10 ONC for colicin-test next day:
 * +2 ml MG1655
 * +0.002 µg/ml Mytomycin C
 * 2 ml ColE1 unstressed
 * 2 ml ColE1 stressed
 * 2 ml ColE9 unstressed
 * 2 ml ColE9 stressed
 * Inoculation for growthcurve measurement over the day (tomorrow):
 * ColE1 unstressed
 * ColE1 stressed
 * ColE9 unstressed
 * ColE9 stressed
 * DH5alpha
 * TOP10
 * MG1655

pBAD-Sender Cloning

 * Double transformation of pBAD-F1610-9 and AI-2-Sender int TOP 10 and DH5 alphha
 * Inoculation of ONC with pBAD-F1610 colony 9 (from glycerolstock)

General

 * Preparation of M9 media

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pSB1A3-Receiver-Colicin-Cloning

 * Inoculation of liquid cultures from Transformation of ligation: pSB1A3-RecluxpR.

pBAD-Sender Cloning

 * Miniprep of pBAD-F1610,9: 179,6 ng/ul; Qiagen Miniprepkit
 * Inoculation of liquid cultures from Transformation of double-transformation.

Activity Test

 * Results of Colicintest: You can see that stressed Colicin E1 cells are able to kill TOP 10. But this could also be caused by Mytomycin C. [[Image:080902-colicin-activity_ON_measurement_small.jpg |500 px | center]]
 * Growthcurve:
 * Inoculation of TOP10s with 1ml/2ml supernatant of:
 * MG1655
 * ColicinE1 unstressed
 * ColicinE1 stressed
 * ColicinE9 unstressed
 * ColicinE9 stressed
 * OD was measured hourly in tecan plate reader
 * New inoculation of same cultures in LB media.

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pSB1A3-Receiver-Colicin-Cloning
concentrations: R1: 152,3 ng/ul R2: 150,4 ng/ul R3: 76,7 ng/ul R4: 132,8 ng/ul R5: 142,1 ng/ul R6: 138,4 ng/ul R7: 136,8 ng/ul R8: 187,7 ng/ul 10.0 µl DNA 2.0 µl NEBuffer 2 2.0 µl BamHI 2.0 µl XbaI 2.0 µl BSA 10x 2.0 µl H2o --- 20.0 µl
 * Minipreps of the ligation ONCsvfrom 02.09.:
 * Controldigestions of ligation with XbaI and SpeI
 * Gel of digestion: [[Image:080903-controldigestion_luxpR_cloning_small.jpg |500 px | center]]
 * Gelresults: Expected fragments 2150 bp and 1088 bp. The 2150 bp fragments are there but there is a 1500 bp fragment instead of the 1088 bp fragment. Despite this we made some new glycerolstocks.

Activity Test

 * Results of growth curve test from yesterday: The results were very diffusing so that we perform this test again. [[Image:080903-growthcurve_colicin_test_small.jpg |500 px | center]]
 * New hourly growth curve measurement (see Tuesdaysday for details). No significant effect of colicins can be seen. Only Mytomycin C kills the TOP 10 cells very effectively [[Image:080903-growthcurve_colicin_test_2_small.jpg |500 px | center]]

pBAD-Sender Cloning
concentrations prey_Top10,1: 123,6 ng/ul prey_Top10,2: 157,4 ng/ul prey_DH5-a,1: 202,0 ng/ul prey_DH5-a,2: 174,2 ng/ul
 * Minipreps of doubletransformations:

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pSB1A3-Receiver-Colicin-Cloning
25.0 µl Phusion MasterMix 2.5 µl colE1prot/colE9prot_fw_BamHI 2.5 µl colE1prot/colE9prot_rv_SpeI 18.0 µl H2O 2.0 µl DNA templates --- 50.0 µl
 * Despite of the strange digestion pattern we try the next step of the cloning.
 * PCR of colicins:

program: 98 °C 30 sec 98 °C 10 sec 59 °C 20 sec 72 °C 45 sec 72 °C  8 min 4 °C constant Vector: 6.0 µl DNA 2.0 µl NEBuffer 2 2.0 µl SpeI 2.0 µl BamHI 2.0 µl BSA10x 6.0 µl H2O --- 20.0 µl
 * PCR purification and analytic gel: Expected fragments are there [[Image:080904-colicin_PCR_purification.jpg |500 px | center]]
 * Digestion of purified PCR products with BamHI and SpeI: 1h -> 37 °C

Insert: 10.0 µl DNA 2.0 µl NEBuffer 2 2.0 µl SpeI 2.0 µl BamHI 2.0 µl BSA10x 2.0 µl H2O --- 20.0 µl
 * Gel & Gelextraction: The expected bands were visible. After cutting we purified them using the Qiagen Gel Extraction Kit.
 * Sequencing: Send R1 (LuxR-Receiver) to GATC.
 * Inoculation of 4 receiver colonies

Activity Test

 * New plans for characterization:
 * Cloning of Colicin proteins into pQE-30 vector for His-Tag purification of colicins. Therefore we designed reverse primers with HindIII (ColE1) and XmaI(ColE9) sites.

pBAD-Sender Cloning

 * Inoculation of 4 cotransformations for activity tests

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pSB1A3-Receiver-Colicin-Cloning

 * Ligation and Transformation of pRecluxpR with ColE1/ColE9: no results

Activity Test

 * Activity test over the day: no results

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go to 6th week