Team:Heidelberg/Notebook/Killing II/11thweek

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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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11th week

go back to 10th week

go back to the overview

mCherry

 * Transformationresults: Cultures were grown, also on plates with wrong plasmid - insert ratio
 * Inoculation of liquid cultures from transformation with wrong ratio in the morning.
 * Miniprep of liquid cultures in the evening: eluted in 35 µl H2O, (Qiagen Miniprepkit)
 * Inoculation of liquid cultures from transformation with right ratio.

Mutagenesis of pSB1A2-ColicinE1-Receiver

 * MiniPreps of colE1 after the 2nd mutagenesis (colonies 1.14, 1.16, 5.1, 5.7) and of colE9 (colonies 2 and 4, still with the pre-/suffix) each on pSB1A2 after receiver --> sent for sequencing
 * Colonies picked from the transformations of the mutated colE9 parts (with the correct pre-/suffix) --> inocculated and MiniPreped (for digestion and sequencing)

Characterization: ColicinE1-Receiver Activitytest

 * 4 pm: Inoculation of:
 * 30 ml TB-Kana with BBa_I20260
 * 30 ml TB-Amp with T9002-Receiver without GFP
 * 30 ml TB-Amp with pSB1A2-ColicinE1-Receiver
 * 8.30 pm: Preparing mixtures for the plate:
 * plate scheme: [[Image:081013_colicin_activity_test_plate_scheme.png |500 px | center]]
 * Killer:Prey 1:0.25 -> 200 µl Referencepromotercells + 800 µl Receivercells
 * Killer:Prey 1:1   -> 200 µl Referencepromotercells + 200 µl Receivercells + 600 µl TB media
 * Killer:Prey 1:5   -> 200 µl Referencepromotercells +  40 µl Receivercells + 760 µl TB media
 * Killer:Prey 1:10  -> 200 µl Referencepromotercells +  20 µl Receivercells + 780 µl TB media
 * 9.30 pm: Starting measurement

Characterization: Sender Activity Test

 * Characterization: Sender-Test measured every hour over the day. See Saturdayurday for more details.
 * Layout of the 96 well plate: [[Image: 081011-plate_scheme_sender_amplifier_test.jpg | 800 px | center]]
 * Results:
 * AI-1 GFP/OD: [[Image:081013-AI-1_GFP-OD_small.jpg |500 px | center]]
 * AI-1 OD: [[Image:081013-AI-1_OD_small.jpg |500 px | center]]
 * Sender GFP/OD: [[Image:081013-sender_GFP-OD_small.jpg |500 px | center]]
 * Sender OD: [[Image:081013-sender_OD_small.jpg |500 px | center]]
 * Amplifier GFP/OD: [[Image:081013-amplifier_GFP-OD_small.jpg |500 px | center]]
 * Amplifier OD: [[Image:081013-amplifier_od_small.jpg |500 px | center]]
 * In most cases the later supernatants have a higher GFP/OD rate: But there in some cases earlier concentrations are much higher. A problem was that the plate stand at 4 °C for 2 days so this could cause these outliers. In the next days we repeat this experiment to characterize these parts properly.

[back]

mCherry
10.0 µl pBAD-mCherry DNA (3rd mutation) 4.0 µl BSA 10x (NEB) 4.0 µl NEBuffer 4 (NEB) 0.5 µl SacI (NEB) 0.7 µl XbaI (NEB) 20.8 µl H2O --- 40.0 µl
 * Controldigestion of Cloning: 2 h 20 min -> 37 °C
 * Gel of digestion: 1% Agarose, 135 V, 30 min [[Image:081014-pBAD-mcherry_control_digest_small.jpg |500 px | center]]
 * Results of Digestion:
 * Expected fragments for correct mutation: ~1100 bp & ~4600 bp
 * Coolonies 1, 2, 4 & 5 shows the right bands. -> Sucessful cloning.

Mutagenesis of pSB1A2-Receiver-ColicinE1
10.0 µl ColE1-Receiver DNA (3rd mutation) 4.0 µl BSA 10x (NEB) 4.0 µl NEBuffer 3 (NEB) 0.5 µl PstI (NEB) 21.5 µl H2O --- 40.0 µl 5.0 µl pfu buffer 1.0 µl forward primer (10 µM Col E1_mut_Pst_2_fw) 1.0 µl reverse primer (10 µM Col E1_mut_Pst_2_rv) 1.0 µl dNTPs (12.5 mM, 50x) 0.5 µl template DNA - 5 to 50 ng plasmid DNA 40.5 µl milliQ water 1.0 µl turbo pfu polymerase (Stratagene) 50.0 µl
 * Miniprep of ON liquid cultures 3rd colicin E1 mutagenesis: eluted in 35 µl H2O, Qiagen Miniprepkit
 * Controldigestion of 3rd Mutagenesis: 2 h 20 min -> 37 °C
 * Gel of digestion: 1% Agarose, 135 V, 30 min [[Image:081014-controldigestion_3rd_mutagenesis_colE1_rec_small.jpg |500 px | center]]
 * Results of Digestion:
 * Expected fragments for correct mutation: ~695 bp & ~4600 bp
 * The gel shows the right bands. This indicates that our three mutations were succesful.
 * Mutagenesis PCR of Miniprep: Mutagenesis of 3rd PstI site

95 °C  30 sec 95 °C  30 sec    | 55 °C  60 sec    | 16 cycles 68 °C  11 min    | 4 °C  constant - start thawing 100 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 400 µl preheated LB media - incubate at 37 °C for 1 h, 500 rpm - Plate 500 µl on preheated LB-Amp plate - incubate overnight
 * Digestion with DpnI (NEB) of the third mutagenesis to cut parental plasmid: Added 1 µl of DpnI to the finished Mutagenesis PCR. DpnI cuts at methylated GATC sites, so that only parental plasmids without the mutation are cutted. 2 h 35 min -> 37 °C
 * Controlgel of third Mutagenesis (PstI_3): 1 % Agarose, 135 V, 30 min; DNA only visible in products from the colonie 1 (1.14 and 1.16) -> probably the mutagenesis worked on this plasmid.
 * PCR Purification Kit (Qiagen) to purify the plasmids. Eluted in 35 µl H2O
 * Transformation of prior mutagenesis: 5 µl per 100 µl E. coli TOP 10 competent cells

Characterization: ColicinE1-Receiver Activitytest

 * Results:
 * Killer:Prey ratio 1:0.25 GFP: [[Image:081014-colicin_activity_test_1-0.25_GFP_small.jpg |500 px | center]]
 * Killer:Prey ratio 1:0.25 OD: [[Image:081014-colicin_activity_test_1-0.25_OD_small.jpg |500 px | center]]
 * Killer:Prey ratio 1:1 GFP: [[Image:081014-colicin_activity_test_1-1_GFP_small.jpg |500 px | center]]
 * Killer:Prey ratio 1:1 OD: [[Image:081014-colicin_activity_test_1-1_OD_small.jpg |500 px | center]]
 * Killer:Prey ratio 1:5 GFP: [[Image:081014-colicin_activity_test_1-5_GFP_small.jpg |500 px | center]]
 * Killer:Prey ratio 1:5 OD: [[Image:081014-colicin_activity_test_1-5_OD_small.jpg |500 px | center]]
 * Killer:Prey ratio 1:10 GFP: [[Image:081014-colicin_activity_test_1-10_GFP_small.jpg |500 px | center]]
 * Killer:Prey ratio 1:10 OD: [[Image:081014-colicin_activity_test_1-10_OD_small.jpg |500 px | center]]

Sender cloning: constitutive promotor-sender
[back]

mCherry

 * Sequencing results: Colonies 1, 2, 4 & 5 are positive clones. -> double transformation

Standardization

 * Inoculation of 4th mutagenesis transformation cultures
 * Miniprep of ColE9-Receiver BioBrickstandard: 5 colonies of ColE9-Receiver(2) and 5 colonies of ColE9-Receiver(4) (Qiagen, Miniprepkit) --> all 10 sent for sequencing

Activity Test

 * Results

Sender

 * Sendertest

Visualization
I20260 + constitutive sender in TOP 10 I20260 in TOP 10 I20260 + ColicinE1-Receiver in TOP 10 I20260 + T9002_without_GFP-Receiver in TOP 10 pBAD-mCherry + ColicinE1-Receiver in TOP 10 pBAD-mCherry + T9002_without_GFP-Receiver in TOP 10 pBAD-mCherry + ColicinE1-Receiver in HCB33 pBAD-mCherry + T9002_without_GFP-Receiver in HCB33 pBAD-mCherry + ColicinE1-Receiver in MG1655 pBAD-mCherry + T9002_without_GFP-Receiver in MG1655
 * Doubletransformation of:

[back]

pSB1A2-Receiver-Colicin cloning
Sequencing results of ColE9-Receiver cloning


 * Miniprep of ONC

2.0 µl DNA Template 2.5 µl Primer fw 2.5 µl Primer rv 18.0 µl H2O 25.0 µl Phusion MasterMix --- 50.0 µl program: 98 °C   2 min 98 °C  10 sec   | 53 °C  30 sec   | 25 cycles 72 °C  90 sec   | 72 °C   5 min 4 °C  constant 2.0 µl T4 DNA Ligase (Fermentas) 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.6 µl pSB1A2 (6,5 ng/µl) 13.4 µl ColE1Receiver PCR Product (5,6 ng/µl) - start thawing 100 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 400 µl preheated LB media - incubate at 37 °C for 1 h, 500 rpm - Plate 500 µl on preheated LB-Amp plate - incubate overnight
 * Controldigestion of Minipreps:
 * Gel of Digestion:
 * PCR of Minipreps:
 * Gel of PCR
 * Gelextraktion
 * Ligation ColicinE1-Receiver (BioBrick Standard) with pSB1A2 (3:1): 1 h -> 22 °C
 * Transformation of prior ligation: 5 µl per 100 µl E. coli TOP 10 competent cells

Colicin activity test
After adjustment of the OD of the different cultures (killer, reference killer, prey), mixtures of different rations were done and transfered to a 96 well plate for ON measurement after following schemes





Sender

 * Results of sender test

[back]

Sender Cloning: constitutive promotor - sender
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ColE1 Cloning for standardization
25.0 µl Taq Master Mix (Fermentas) 2.5 µl T9002_LuxpR_Not_Eco_Xba_G_fw (Tm=80,96°C) 2.5 µl ColE1_kil_prot_rv_A_SpeI (Tm=66,94°C) 20.0 µl H2O 1   colony --- 50.0 µl program 1: 98 °C   10 sec 98 °C   30 sec         | 57 °C   30 sec         | 25 cycles 72 °C   1 min 45 sec   | 72 °C   10 min 4 °C  constant program 2: 98 °C   10 sec 98 °C   30 sec         | 67 °C   30 sec         | 25 cycles 72 °C   1 min 45 sec   | 72 °C   10 min 4 °C  constant 38.5 µl DNA (of Gelextraction) 0.5 µl EcoRI (20 000 U/ml, NEB) 1.0 µl SpeI (10 000 U/ml, NEB) 5.0 µl BSA 10x 5.0 µl EcoRI Buffer(NEB) --- 50.0 µl 1.Ligation attempt (+): 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.0 µl T4 DNA Ligase (Fermentas) 1.8 µl pSB1A2 14.2 µl receiver-colE1-insert (From PCR-Purification) --- 20.0 µl 2.Ligation attempt (-): 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.0 µl T4 DNA Ligase (Fermentas) 1.1 µl pSB1A2 14.9 µl receiver-colE1-insert (From PCR-Purification) --- 20.0 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 250 µl preheated LB media - incubate at 37 °C for 20 min, 500 rpm - Plate 300 µl on preheated LB-Amp plate - incubate overnight at 37°C
 * PCR-Amplification of Receiver-Col E1-Insert with the right Prefix and Suffix
 * Gel of Receiver-colE1 PCR-amplification: 0,7% Agarose, 135 V, 30 min
 * Gel-Results of colE1 PCR-amplification: Bands of PCR-A
 * Gelextraction with Qiagen Kit: eluted in 40 µl H2O
 * Digestion of receiver-colicinE1-insert for 2 h at 37°C
 * PCR-Purification
 * Ligation of receiver-colE1-insert into pSB1A2 (1):
 * Transformation of receiver-colE1-pSB1A2 BioBrick: 5 µl DNA (Ligation) per 50 µl E. coli TOP 10 competent cells

Sender and Amplifier Activity Test - 24h
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ColE1 Cloning for standardization
XbaI digestion: 10.0 µl DNA 2.0 µl BSA 10x (NEB) 2.0 µl NEBuffer 2 0.5 µl XbaI (NEB) 5.5 µl H2O --- 20.0 µl SpeI digestion: 10.0 µl DNA 2.0 µl BSA 10x (NEB) 2.0 µl NEBuffer 2 1.0 µl SpeI (NEB) 5.0 µl H2O --- 20.0 µl SpeI digestion: 10.0 µl DNA 2.0 µl BSA 10x (NEB) 2.0 µl NEBuffer 2 0,5 µl XbaI 1.0 µl SpeI (NEB) 4.5 µl H2O --- 20.0 µl
 * Inoculation of colonies from transformation:
 * 5 colonies from each plate in 10 ml LB-amp medium respectively:
 * 57°C(+)-1
 * 57°C(+)-2
 * 57°C(-)-1
 * 57°C(-)-2
 * 67°C(+)-1
 * 67°C(+)-3
 * 67°C(-)-1
 * 67°C(-)-3
 * Minipreps:
 * 57°C(+)-1
 * 57°C(+)-2
 * 57°C(-)-1
 * 57°C(-)-2
 * 67°C(+)-1
 * 67°C(+)-3
 * 67°C(-)-1
 * 67°C(-)-3
 * Controldigestion with XbaI, SpeI and XbaI+SpeI of BioBrick Cloning: Receiver-ColicinE1-pSB1A3
 * Controlgel: 0,7% Agarose, 135 V, 30 min
 * Gelresults:
 * Expected Fragments:
 * XbaI digestion (X): ~5200 bp (linear plasmid)
 * SpeI digestion (S): ~5200 bp (linear plasmid)
 * XbaI+SprI digestion (X+S): ~3200 bp (Insert) + ~2000 bp (Vector)
 * Undigested Plasmid (P): ~3200 bp


 * The following clones are positive ColE1 BioBricks:
 * 57°C(+)-1
 * 57°C(+)-2
 * 57°C(-)-1
 * 57°C(-)-2
 * 67°C(+)-1
 * 67°C(-)-1

Sender Cloning: constitutive promotor - sender
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