Team:Heidelberg/Notebook/Killing II/6thweek

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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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6th week

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pSB1A3-Receiver-Colicin cloning

 * sequencing results of pSB1A3 - Receiver cloning -> cloning not sucessful
 * Primer sequences controlled

[back]

pSB1A3-Receiver-Colicin cloning
10  µl 5x Phusion HF Buffer 1.0 µl 10mM dNTPs 2.5 µl colE1/E9_prot_fw_BamHI 2.5 µl colE1_kil_prot_rv_SpeI/colE9_plasmid_rv_SpeI/colE9_lys_prot_rv_SpeI 2.0 µl Template DNA 0.5 µl Phusion DNA Polymerase 31.5 µl H2O --- 50.0 µl
 * Amplification of Colicins

10  µl 5x Phusion HF Buffer 1.0 µl 10mM dNTPs 2.5 µl T9002_lux_pR_fw_XbaI 2.5 µl T9002_lux_pR_rv_SpeI_BamHI_RBS 2.0 µl Template DNA (T9002 plasmid) 0.5 µl Phusion DNA Polymerase 31.5 µl H2O --- 50.0 µl
 * Amplification of Receiverpart (T9002) without GFP

program: 98 °C  30 sec 98 °C  10 sec 59 °C  30 sec 72 °C  45 sec 72 °C   5 min 4 °C  constant

HisTag cloning of Colicins for purification
10  µl 5x Phusion HF Buffer 1.0 µl 10mM dNTPs 2.5 µl colE1/E9_prot_fw_BamHI 2.5 µl colE1_prot_rv_HindIII/colE9_prot_rv_XmaI 2.0 µl Template DNA 0.5 µl Phusion DNA Polymerase 31.5 µl H2O --- 50.0 µl
 * Amplification of Colicins

program: 98 °C  30 sec 98 °C  10 sec 59 °C  30 sec 72 °C  45 sec 72 °C   5 min 4 °C  constant

[back]

pSB1A3-Receiver-Colicin cloning
20 µl DNA 6 µl H2O 4 µl NEBuffer 2 3 µl SpeI (NEB) 3 µl XbaI (NEB) 4 µl BSA 10x - 40 µl
 * Gelextraction Receiver PCR: 0.7% Agarose, 100 V, 60 min [[Image:080910_Rec_ohne_GFP_small.jpg |500 px | center]]
 * Gelextraction Receiver PCR: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O
 * Digestion of Receiver & pSB1A3 with SpeI and XbaI, 1 h 30 min 37 °C -> 10 min 65°C

10 µl Receiver DNA 2 µl Vector DNA pSB1A3 (sapped, from last week) 2 µl Buffer T4 DNA Ligase (Fermentas) 2 µl T4 DNA Ligase (Fermentas) 4 µl H2O Nuclease Free (Fermentas) - 20 µl - start thawing 50 µl competent E. coli MG1655 cells on crushed ice - add 5 µl ligation - incubate on ice for 20 minutes - heat shock at 42 °C for 45 sec - incubate 2 min on ice - add 200 µl preheated LB media - incubate at 37 °C for 1 h, 300 rpm - Plate 200 µl on preheated LB-Amp plate - incubate overnight
 * Gelextraction of pSB1A3 digestion: 0.7 % Agarose, 50 min, 135 V [[Image:080910_pSB1A3_gelex_small.jpg |500 px | center]]
 * Gelextraction of pSB1A3 digestion: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O, cutted pSB1A3 and T9002 -> not used
 * Ligation of pSB1A3 and Receiver: 1 h RT, 10 min 65 °C
 * Transformation of prior ligation: 5 µl per 50 µl E. coli MG1655
 * Controllgels of Colicin Amplification (Tuesday 080909): 1% Agarose, 37 min, 135V
 * Colicin E1 & E1 for HisTag [[Image:20080910_ColE1_His_ColE1_His_PCR080909_small.jpg |500 px | center]]
 * Colicin E9 lys & E9 for HisTag [[Image:20080910_ColE9_His_ColE9_lys_PCR080909_small.jpg |500 px | center]]
 * Colicin E9 whole plasmid [[Image:20080910_ColE9_PCR080909_small.jpg |500 px | center]]

HisTag cloning of Colicins for purification

 * Gelextraction of Colicin E1 and Colicin E9 PCR: 0,7%, 60 min, 100 V [[Image:080910_colE1His_colE9His_gelex.jpg |500 px | center]]
 * Gelextraction of Colicin E1 and Colicin E9 PCR: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O

Colicin activity test
-10 ml M9 media + 10 µl Kanamycin + E. coli Top10 with I20260 -10 ml M9 media + 10 µl Ampicilin + E. coli MG1655 with pKC67 (Colicin E9 plasmid) -10 ml M9 media + 10 µl Ampicilin + E. coli MG1655 with pKC67 (Colicin E9 plasmid) -10 ml M9 media + 200 µl 10% Glucose + E. coli JC411 (Colicin E1 plasmid) -10 ml M9 media + 200 µl 10% Glucose + E. coli JC411 (Colicin E1 plasmid) -10 ml LB media + 200 µl 10% Glucose + E. coli JC411 (Colicin E1 plasmid) -10 ml LB media + 200 µl 10% Glucose + E. coli JC411 (Colicin E1 plasmid)
 * inocculation of liquid cultures from glycerolstocks, incubation over night at 37 °C

[back]

pSB1A3-Receiver-Colicin cloning
10 µl Receiver DNA 2 µl Vector DNA pSB1A3 2 µl Buffer T4 DNA Ligase (Fermentas) 2 µl T4 DNA Ligase (Fermentas) 4 µl H2O Nuclease Free (Fermentas) - 20 µl
 * Digestion of pSB1A3 with XbaI and SpeI from cloning cultures of last week. We used these cultures because the bande where easier to distinguish. !!!
 * 2008-09-18 UPDATE: the different bands were probably from the religated pSB1A3 plasmid, which cannot be cutted with XbaI and SpeI. -> No cutted pSB1A3 backbone
 * Gelextraction of pSB1A3: 0.7 % Agarose, 1 h, 135 V [[Image:080911_colE9his_vec_and_pSB1A3_digestion_gelex_small.jpg |500 px | center]]
 * Gelextraction of pSB1A3: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O
 * Overnight ligation of pSB1A3(gelextraction, Thursday 2008-09-11) and T9002_without_GFP (gelextraction, Wednesday 2008-09-10)


 * Gelextraction colicin E1, colicin E9, colicin E9 lysis genes: 0.7 % Agarose, 1 h, 135 V [[Image:20080911_PCR_colicins_gelex_small.jpg |500 px | center]]
 * Gelextraction colicin E1, colicin E9, colicin E9 lysis genes: QIAquick Gel Extraction (Qiagen), eluted in 40 µl H2O -> stored ad -20 °C

HisTag cloning of Colicins for purification
20 µl DNA 6 µl H2O 4 µl NEBuffer 2/4 3 µl BamHI (NEB) 3 µl HindIII/XmaI (NEB) 4 µl BSA 10x - 40 µl
 * Miniprep of pQE-30 plasmids from ON-culture(QIAprep Spin Miniprep Kit (250), Qiagen)
 * Digestion of pQE-30 (E1 -> HindIII, E9 -> XmaI)

20 µl DNA 6 µl H2O 4 µl NEBuffer 2/4 3 µl BamHI (NEB) 3 µl HindIII/XmaI (NEB) 4 µl BSA 10x - 40 µl
 * Digestion of Colicin E1/E9 (E1 -> HindIII, E9 -> XmaI)

10 µl Colicin DNA 2 µl Vector DNA pQE-30 2 µl Buffer T4 DNA Ligase (Fermentas) 2 µl T4 DNA Ligase (Fermentas) 4 µl H2O Nuclease Free (Fermentas) - 20 µl
 * Gelextraction of pQE-30 and Colicin E1 digestion: 0.7 % Agarose, 1 h, 135 V [[Image:080911_colE1His_and_vector_digestion_gelex_small.jpg |500 px | center]]
 * Gelextraction of pQE-30 and Colicin E9 digestion: 0.7 % Agarose, 1 h, 135 V [[Image:080911_colE9his_vec_and_pSB1A3_digestion_gelex_small.jpg |500 px | center]]
 * Gelextraction of pQE-30 and Colicins digestion: QIAquick Gel Extraction (Qiagen), eluted in 30 µl H2O
 * Overnight ligation:

Colicin activity test
3:1 -> 3 ml M9 media + 1 ml supernatant + 200 µl reference promotor cells (I20260) 1:1 -> 2 ml M9 media + 2 ml supernatant + 200 µl reference promotor cells (I20260) 1:3 -> 1 ml M9 media + 3 ml supernatant + 200 µl reference promotor cells (I20260)
 * 9 am: One E9 - M9 culture, one E1 - M9 culture and one E1 - LB culture were stressed with 10 µl of 0.1 mg/ml Mytomycin C
 * 4 pm: The colicin E1 LB cultures were thrown away, because the grew well in M9. The MG1655 and colicin cultures were sonicated in a ultrasonic bath three time for 15 seconds. After sonication cell extract was observed under microscope -> lot of dead cells -> succesfull lysis.
 * Overnight-Activity-Assay:
 * lysed cells were centrifuged for 15 min at 400 rpm. The supernatants were transferd to new falcons and were used for a colicin activity test.
 * well plate scheme: [[Image:platescheme_small.jpg |500 px | center]]
 * ON-measurement for 14 h at 37 °C in Tecan well plate reader

[back]

pSB1A3-Receiver-Colicin cloning
10 µl Receiver DNA 6 µl Vector DNA pSB1A3 (sapped, from last week) 2 µl Buffer T4 DNA Ligase (Fermentas) 2 µl T4 DNA Ligase (Fermentas) - 20 µl - start thawing 50 µl competent E. coli TOP 10 cells on crushed ice - add 5 µl ligation - incubate on ice for 20 minutes - heat shock at 42 °C for 45 sec - incubate 2 min on ice - add 200 µl preheated LB media - incubate at 37 °C for 1 h, 300 rpm - Plate 200 µl on preheated LB-Amp plate - incubate overnight at 37 °C
 * New ligation because controll gel didn't show any bands for pSB1A3 -> probably a too low concentration: : 1 h 40 min RT, 15 min 65 °C
 * Transformation of ligation (Tuesday 2008-09-11) into E. coli TOP 10 cells: 5 µl per 50 µl E. coli BL21 cells
 * Controllgel of PCR: 1% Agarose, 30 min, 135 V [[Image:080912_controllgel_PCR_small.jpg |500 px | center]]

HisTag cloning of Colicins for purification
- start thawing 50 µl competent E. coli BL21 cells on crushed ice - add 5 µl ligation - incubate on ice for 20 minutes - heat shock at 42 °C for 45 sec - incubate 2 min on ice - add 200 µl preheated LB media - incubate at 37 °C for 1 h, 300 rpm - Plate 200 µl on preheated LB-Amp plate - incubate overnight at 37 °C
 * Transformation of ColE1 and ColE9 into BL21 cells: 5 µl per 50 µl E. coli BL21 cells

Colicin activity test

 * Colicin E1 and E9 kills other E. coli cells efficient

Other

 * Maxiprep of I15030 and T9002 for plasmid backbone. (Compact Prep Plasmid Maxi Kit (25), Qiagen)

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pSB1A3-Receiver-Colicin cloning

 * Transformation results: we picked and plated 8 colonies of each transformation on LB-Ampicilin plates and in addition we inocculated liquid cultures.

HisTag cloning of Colicins for purification

 * Transformation results: We plated our transformation on LB-Kanamycin because BL21 cells contain a helper plasmid which has an Kanamycin resistance. But pQE-30 plasmids have an Ampicilin resistance. Because of that we transderd colonies from an bacteria lawn to Ampicilin/Kanamycin plates.

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pSB1A3-Receiver-Colicin cloning
10 µl DNA 3  µl H2O 2  µl NEBuffer 2 1.5 µl XbaI (NEB) 1.5 µl SpeI (NEB) 2  µl BSA 10x -- 20 µl
 * Miniprep of pSB1A3-Receiver-Cloning from ON-cultures(QIAprep Spin Miniprep Kit (250), Qiagen)
 * Digestion of pSB1A3-Rec XbaI and SpeI, 1h 30 min -> 37 °C, 15 min -> 65 °C

34 µl pSB1A3 (digested) 4 µl SAP Buffer 1 µl SAP Enzyme - 39 µl 6 µl Receiver DNA 4 µl Vector DNA pSB1A3 2 µl Buffer T4 DNA Ligase (NEB) 2 µl T4 DNA Ligase (NEB) 6 µl H2O - 20 µl
 * Gel for selection of positive clones: Estimated fragments ~2150 bp pSB1A3 backbone and ~1088 bp T9002 without GFP. No bands at 1088 bps. Colony-PCR-Screen on Mondayday.
 * Since selection of positive clones failed, we started a new ligation
 * Sapping: 30 min 37°C, 10 min 65°C
 * Ligation of pSB1A3 and T9002 without GFP: 18 h 16 °C, 20 min 65 °C, 4 °C const

HisTag cloning of Colicins for purification
9 µl ColE1/E9 His 3 µl Vector DNA pQE-30 2 µl Buffer T4 DNA Ligase (NEB) 2 µl T4 DNA Ligase (NEB) 4 µl H2O - 20 µl
 * Plated colonies from Saturdayurday (2008-09-13) were not grown.
 * Retransformation of ligation:
 * ColE1His: 2.5 µl ligation on 50 µl BL-21 cells, 10 µl ligation on 100 µl TOP 10 cells
 * ColE9His: 2.5 µl ligation on 50 µl BL-21 cells, 10 µl ligation on 100 µl TOP 10 cells
 * New Ligation with other insert/plasmid ration
 * Ligation of pQE-30 and Colicins: 18 h 16 °C, 20 min 65 °C, 4 °C const

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