Team:Heidelberg/Notebook/Killing II/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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4th week

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pSB1A3-Receiver-Colicin-Cloning
25.0 µl Phusion MasterMix 2.5 µl Primer fw(ColE1_prot_fw_BamHI/ColE9_plasmid_rv_SpeI) 2.5 µl Primer rv(ColE1_kil_prot_rv_SpeI/ColE9_plasmid_rv_SpeI) 17.0 µl H2O 3.0 µl pColE1/pColE9-J --- 50.0 µl
 * PCR of colicin E1 and E9 operon with specific primers:

program: 98 °C  30 sec 98 °C  10 sec   | 57 °C  20 sec   | 25 cycles 72 °C  45 sec   | 72 °C   8 min 4 °C  constant

Activity Test

 * Inoculation of 5 ml liquid ONC with...
 * pColE1 in JC411 -> 0.1% Glucose Medium
 * pColE9 in MG1655
 * TOP 10
 * MG 1655

pBAD-Sender Cloning
10 µl DNA (320-330 ng/µl) 3 µl Tango Buffer 10x (Fermentas) 2 µl DraI (Fermentas) 15 µl H2O - 30 µl
 * Controldigestion of BBa_F1610 with DraI: 1.5 - 2 h -> 37 °C
 * Gel of digestion: Results are not perfect but expected bands can be estimated. [[Image:080825_Gel_Verdau_F1610.jpg |500 px | center]]

Activity Test

 * Inoculation of pBAD-BBa_F1610 in 8 ml M9 Kana + 0.1% Arabinose. (8 colonies)

General

 * Inoculation of liquid ONC of BBa_J23107, BBa_J23102, BBa_R0011, BBa_R0040 in 5 ml LB Amp. (For Minipreps and Glycerolstocks)

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pSB1A3-Receiver-Colicin-Cloning
20.0 µl DNA (PCR product, purified) 13.0 µl H2O 4.0 µl NEBuffer 3 (NEB) 3.0 µl BamHI (NEB) 4.0 µl BSA 10x (NEB) --- 44.0 µl
 * PCR Purification of T9002 without GFP (08/22/08) and colicin E1/E9 operon: Qiagen PCR Purification Kit.
 * Digestion of purified products with BamHI: 2h -> 37 °C
 * Gelextraction of digestion: Cutted the bands and freezed for purification tomorrow.

Activity Test

 * 2nd step of colicin test based on ONC from Tuesdaysday:
 * pColE1 (JC411):
 * inoculation of 12 ml LB ONC + 0.1% Glucose -> unstressed
 * inoculation of 12 ml LB ONC + 0.1% Glucose + 1µg/ml Mytomycin C
 * EDIT 09/02/08: Too high Mytomycin C concentrations
 * 100 µl ONC + 300 µl LB + 200 µl 10% glucose plated on LB-Agar
 * pColE9 (MG1655):
 * inoculation of 12 ml LB ONC-> unstressed
 * inoculation of 12 ml LB ONC + 1µg/ml Mytomycin C
 * EDIT 09/02/08: Too high Mytomycin C concentrations
 * 100 µl ONC + 500 µl LB plated on LB-Agar
 * MG1655 + TOP10:
 * inoculation of 12 ml LB ONC-> unstressed
 * 100 µl ONC + 500 µl LB plated on LB-Agar

pBAD-Sender Cloning

 * Control of Cloning -> see activity test

Activity Test

 * To check if we have one positive clone we perform the sender activity test again (see Fridayday 08/08/22 for plate scheme and details). After the first measurements an increase in GFP expression by BBa_T9002 was observed for colonies 9, 10, 11. Because of that we inoculated liquid ONC with LB-Kana for miniprepes and glycerolstocks

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pSB1A3-Receiver-Colicin-Cloning
10 µl DNA I 10 µl DNA II 4 µl 10x T4 DNA Ligase Buffer 2 µl T4 Ligase 14 µl H2O - 40 µl 20 µl DNA 6 µl H2O 4 µl NEBuffer 2 3 µl SpeI 3 µl XbaI 4 µl BSA 10x - 40 µl
 * Gelextraction of BamHI digestion: Qiagen Gelextraction kit
 * Ligation of receiver with colicins ratio 1:1: 1h -> RT, 10 min -> 65 °C
 * Gelextaction of Ligation: ligated construct (~3000 bp) was extracted and purified. Qiagen Gel Extraction Kit
 * Digestion of ligated product with XbaI and SpeI: 1.5 h -> 37 °C and 65 °C -> 20 min
 * Gel of E1 and E9 digestion: no fragments were visible
 * Gel of BBa_T9002 digestion: Expected bands cannot be differentiated because fragment sizes are too similar ~1950 bp and ~2150 bp.
 * Send probes of pColE9 (colE9_prot_fw_XbaI), pColE1(colE1_prot_fw_XbaI/colE1_prot_rv_SpeI/colE1_imm_fw_SpeI) and BBa_T9002 (pSB_ins_fw/pSB_ins_rv) to GATC for sequencing.

Activity Test

 * Creation of Supernatant of stressed and unstressed colicin cultures.
 * Plate the 4 different supernatants and the 4 different cultures on the prepared ColE1, ColE9, MG1655 and TOP10 plates.

pBAD-Sender Cloning

 * Miniprep and Glycerolstocks of pBAD18-BBa_F1610 colony 9, 10, 11
 * Send miniprep of pBAD18-BBa_F161 cloning colony 9 (VIC121/VIC122 align on pBAD18)to GATC for sequencing.

Activity Test

 * 24h measurement of Colicin test

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pSB1A3-Receiver-Colicin-Cloning
25.0 µl Phusion MasterMix 2.5 µl Primer fw 2.5 µl Primer rv  2.0 µl DNA Template 18.0 µl H2O --- 50.0 µl 34.0 µl pSB1A3 DNA (XbaI/SpeI cutted) 4.0 µl SAP Buffer (Fermentas) 1.0 µl SAP enzymes (Fermentas) 6.0 µl Receiver DNA 2.0 µl Vector DNA 2.0 µl Buffer T4 DNA Ligase 8.0 µl H2O --- 20.0 µl
 * Sequencing results: ColE1_prot correct. Others were not sequenced.
 * New attempt of second digestion: no bands are visible.
 * New cloning strategy:
 * Cloning of AHL-receiver without GFP into pSB1A3 (Amplification from BBa_T9002 with T9002_XbaI_fw and T9002_SpeI_BamHI_RBS_rv primers)
 * Cloning of colicin operon behind LuxPr promoter.
 * In addtion:
 * PCR of BBa_T9002 with new primers standardprotocol Phusion MasterMix (Finnzymes, NEB)
 * PCR Purification and Analytical gel -> right fragment size
 * Digestion of PCR product and vector with XbaI and SpeI
 * Gelextraction: expected bands (~1061 bp LuxpR-receiver and ~2157 bp pSB1A3-backbone) could be separated and were eluted in 34 µl H2O.
 * Sapping of vector to avoid self-ligation: 30 min 37 °C -> 15 min 65 °C
 * Ligation: 16 °C -> Overnight

Activity Test

 * Results: No effects were obtained
 * Preparation of probes for inocculation over night to measure colicin E1 activity:
 * 1 x TOP 10
 * 1 x TOP 10 + 2 ml ColE1 supernatant unstressed
 * 1 x TOP 10 + 2 ml ColE1 supernatant stressed
 * 1 x TOP 10 + 2 ml ColE9 supernatant unstressed
 * 1 x TOP 10 + 2 ml ColE9 supernatant stressed
 * 1 x TOP 10 + 2 ml MG1655

pBAD-Sender Cloning

 * Sequencing results: pBAD18-BBa_F1610 is the right construct.

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pSB1A3-Receiver-Colicin-Cloning

 * Transformation of ON-Ligation

Activity Test
Results:
 * OD Measurement of colicin treated ONCs. An effect in cultures of Mytomycin C stressed cells were observed. But this could also be the toxic effect of Mytomycin C. In addition we obtained that we used a too high concentration of Mytomycin C (1 µg/ml instead of 0.1 µg/ml).
 * Measured cell growth curve over night: Logistic growth could be observed but in general growth curve does not look that good. [[Image:080829-growth_curve_small.jpg |500 px | center]]

General

 * Seminar on Synthetic Biology: Chris
 * Breakfast
 * Project Presentation for Frankfurter Allgemeine Zeitung
 * Campus TV
 * Team Meeting

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