Team:Heidelberg/Notebook/Killing II/10thweek

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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>

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

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10th week

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pSB1A2-Receiver-Colicin cloning

 * Gel of E9 lys PCR-Screen: 1% Agarose, 135 V, 35 min [[Image:081006-PCR_Screen_ColE9lys_standardization_small.jpg |500 px | center]]
 * Gel-Results of E9-PCR-Screen:
 * Estimated Bands: ~2150bp
 * Estimated Bands visible in many tubes -> Screening of single colonies.
 * PCR-Screen of single colonies of pSB1A2-Receiver-Col E9 lys

25.0 µl Taq Master Mix (Fermentas) 2.5 µl T9002_LuxpR_Not_Eco_Xba_G_fw (Tm=80,96°C) 2.5 µl ColE9_lyProt_rv_A_SpeI (Tm=64,29°C) 20.0 µl H2O 1   colony --- 50.0 µl

program: 95 °C   3 min 95 °C   1 min          | 64 °C   1 min          | 30 cycles 72 °C   1 min 30 sec   | 72 °C   10 min 4 °C  constant


 * Gel of E9 lys PCR-Screen: 1% Agarose, 135 V, 35 min [[Image:081006-PCR_single_screen_colE9lys_Standardization_small.jpg |500 px | center]]
 * Gel-Results of E9-PCR-Screen: Colonies 1, 2, 3, 4, 7 & 8 positive; Inoculation of 5 ml LB-Amp media with this colonies for miniprep, 37 °C -> ON

Colicin Activity Test

 * Plan for activity tests of Colicin-Receivers:
 * Overnight measurement (Monday - Tuesday) in Tecan plate reader with reference promotor cells (BBa_I20260) in combination with pSB1A2-Rec-ColE9 lys lysed supernatant.
 * Plate scheme (click for detailed image): [[Image: 08-10-05_-_Colicintest_Col_E9_lys.jpg | 800 px | center]]

[back]

pSB1A2-Receiver-Colicin cloning
colonies 1 & 8: 5.0 µl DNA (~65-80 ng/µl) 25.5 µl H2O 4.0 µl BSA 10x (NEB) 4.0 µl NEBuffer 2 (NEB) 0.5 µl SpeI (NEB) 1.0 µl XbaI (NEB) --- 40.0 µl
 * Colicin-Activity-Test: Cultures were not grown -> new inoculation
 * Miniprep of pSB1A2-Receiver-ColE9lys (BioBrick Standard): colonies 1, 2, 3, 4, 7 & 8, eluted in 35 µl H2O
 * Digestion of pSB1A2-Receiver-ColE9lys (BioBrick Standard): colonies 1, 2, 3, 4, 7 & 8

colonies 2, 3 & 4: 2.0 µl DNA (~245-275 ng/µl) 28.5 µl H2O 4.0 µl BSA 10x (NEB) 4.0 µl NEBuffer 2 (NEB) 0.5 µl SpeI (NEB) 1.0 µl XbaI (NEB) --- 40.0 µl

colony 7: 2.0 µl DNA (~125 ng/µl) 28.5 µl H2O 4.0 µl BSA 10x (NEB) 4.0 µl NEBuffer 2 (NEB) 0.5 µl SpeI (NEB) 1.0 µl XbaI (NEB) --- 40.0 µl
 * Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:081007-digestion_BB_Rec_colE9lys_small.jpg |500 px | center]]
 * Digestion results:
 * Expected fragments:
 * 2 x ~2000 (insert & vector), if XbaI site is recovered
 * 1 x ~4000, if XbaI site is not recovered
 * In each colony there is only one fragment. Probably we have no positive clones with the BioBrick standard. To check the sequence we send the probes for sequencing.
 * Send probes 1, 2, 3, 4, 7, & 8 to GATC for sequencing: Primers VF2 & VR
 * Growth test of each Rec-Colicin glycerol stock: Inoculation in 2 ml LB-Amp and 2 ml TB-Amp. 37 °C -> ON

Sender-activity-test

 * Inoculation of 15 ml TB-Amp media with sender (J23107 + F1610) from glycerol stock
 * Inoculation of 15 ml TB-Amp media with GFP-receiver (BBa_T9002)
 * Inoculation of 15 ml LB-Amp media with sender (J23107 + F1610) from glycerol stock
 * Inoculation of 15 ml LB-Amp media with GFP-receiver (BBa_T9002)


 * Plan for Wednesdaynesday:
 * Inoculation of 7 ml TB media with 150 µl from sender ONC
 * Inoculation of 7 ml TB media with 7 ml from GFP-receiver ONC
 * Count amount of cells from ONC dilution t = 0 h (counting chamber)
 * Measurement of optical density (OD) of ONC dilution t = 0 h
 * Measurement of OD of one probe half-hourly for 8 - 10 h
 * Creation of supernatant the measured probe by centrifugation and filtration half-hourly for 8 - 10 h
 * Storing supernatant at 4 °C
 * Count amount of cells from last probe (counting chamber)
 * Start overnight measurement in Tecan plate reader
 * plate scheme (click for detailed image): [[Image: 081007-pipetting_scheme_sender_test.jpg | 800 px | center ]]

[back]

Standardization of pSB1A2-colE9lys-Receiver
'XbaI/SpeI:' 5.0 µl DNA (~240 ng/µl) 5.0 µl NEBuffer 2 (NEB) 5.0 µl BSA 10x (NEB) 0.5 µl XbaI (20 000 Units/ml, NEB) 1.0 µl SpeI (10 000 Units/ml, NEB) 33.5 µl H2O --- 50.0 µl 'XbaI:' 5.0 µl DNA (~240 ng/µl) 5.0 µl NEBuffer 2 (NEB) 5.0 µl BSA 10x (NEB) 0.5 µl XbaI (20 000 Units/ml, NEB) 34.5 µl H2O --- 50.0 µl
 * Sequencing results: Colonies 1, 2, 3, 4 & 8 have the right sequence. Our first colicin Receiver fullfills BioBrick standard and is ready for activity tests. Colony 7 has mistakes inside the receiver and is thrown away.
 * Digestion of pSB1A2-Receiver-ColE9lys colony 2 with XbaI/SpeI, XbaI, SpeI, EcoRI/PstI:

'SpeI:' 5.0 µl DNA (~240 ng/µl) 5.0 µl NEBuffer 2 (NEB) 5.0 µl BSA 10x (NEB) 1.0 µl SpeI (10 000 Units/ml, NEB) 34.0 µl H2O --- 50.0 µl

'EcoRI/PstI:' 5.0 µl DNA (~240 ng/µl) 5.0 µl NEBuffer 2 (NEB) 5.0 µl BSA 10x (NEB) 0.5 µl EcoRI (20 000 Units/ml, NEB) 0.5 µl PstI (20 000 Units/ml, NEB) 34.0 µl H2O --- 50.0 µl

Colicin Acitivity test

 * Inoculation of ONC for colicin activity test on Thursday, 2008-10-09:
 * 10 ml TB-Kana with reference promoter cells from glycerolstocks (BBa_I20260)
 * 10 ml TB-Amp with pSB1A2-Receiver, pSB1A2-Receiver-ColE9lys, pSB1A2-Receiver-ColE1 from Glycerolstocks

Sender-activity-test

 * Inoculation of 7 ml TB media with 150 µl from sender ONC
 * Inoculation of 7 ml TB media with 7 ml from GFP-receiver ONC
 * Measurement of optical density (OD) of ONC dilution t = 0 h
 * Measurement of OD of one probe half-hourly for 7 h
 * Creation of supernatant the measured probe by centrifugation and filtration half-hourly for 8 - 10 h
 * Storing supernatant at 4 °C
 * Start overnight measurement in Tecan plate reader
 * plate scheme (click for detailed image): [[Image: 081007-pipetting_scheme_sender_test.jpg | 800 px | center ]

[back]

Mutagenesis of pSB1A2-Receiver-Colicin E1/E9
5.0 µl pfu buffer 1.0 µl forward primer (10 µM) 1.0 µl reverse primer (10 µM) 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 cloned pfu polymerase (Stratagene)
 * Mutagenesis PCR:

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 200 µl on preheated LB-Amp plate - incubate overnight
 * Digestion with DpnI (NEB) 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 -> 37 °C
 * PCR Purification Kit (Qiagen) to purify the plasmids.
 * Transformation of prior mutagenesis: 5 µl per 100 µl E. coli TOP 10 competent cells
 * Controlgel of Mutagenesis: 1 % Agarose, 135 V, 30 min; DNA is visible -> probably the mutagenesis worked. [[Image:081009-mutagenese_controll_gel_small.jpg |500 px | center]]

Colicin Activity tests

 * 10 am: Inocullation of TB media from ONC
 * 50 ml TB-Kana with 1.4 ml ONC of reference promoter cells (BBa_I20260)
 * 3 x 50 ml TB-Amp with 1.4 ml ONC of pSB1A2-Receiver, pSB1A2-Receiver-ColE9lys, pSB1A2-Receiver-ColE1
 * 12.30 am: OD -> 0.320. Separation of the 3 Receiver cultures into 8 tubes with 3 ml. Activation with 8 different Autoinducer(detailed information, Sigma) concentrations: 0 nM, 1 nM, 5 nM, 10 nM, 25 nM, 50 nM, 75 nM & 100 nM.
 * 8.00 pm: Preparing the test
 * plate scheme: [[Image:081009-plate_scheme_colicin_activity_test.jpg |500 px | center]]
 * Preparation of probes: [[Image:081009-plate_scheme_colicin_activity_test_mix.jpg |500 px | center]]
 * 10 pm start of measurement

Sender-activity-test

 * Results of ON measurements: The graph shows the GFP intensities over time for the different supernatants and one AI-1 concentration (c = 1 nM). The GFP induction is much lower for the supernatants in comparison to the AI-1. Other tests with lower AI-1 concentration have to be performed to determine the AI-1 production rate. [[Image: 081008-sender_Activity_test_GFP_sup_besch_small.jpg | 700 px | center ]]

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Mutagenesis of pSB1A2-Receiver-Colicin E1/E9
5.0 µl pfu buffer 1.0 µl forward primer (10 µM) 1.0 µl reverse primer (10 µM) 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)
 * Inoculation of liquid cultures from transformation of the first mutagenesis.
 * Miniprep of liquidcultures: eluted in 35 µl H2O (Qiagen Miniprepkit)
 * 2nd mutagenesis PCR of Colicin E1 - Receiver (mutation of first PstI site):
 * Mutagenesis PCR:

95 °C  30 sec 95 °C  30 sec    | 55 °C  60 sec    | 16 cycles 68 °C  11 min    | 4 °C  constant

Sender Cloning: constitutive promotor - sender
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mCherry
20.0 µl DNA 0.5 µl SacI (20 000 U/ml, 100 % Activity in NEBuffer 4, NEB) 0.7 µl XbaI (20 000 U/ml, 75 % Activity in NEBuffer 4, NEB) 5.0 µl BSA 10x 5.0 µl NEBuffer 4 (NEB) 18.8 µl H2O --- 50.0 µl upper band 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.0 µl T4 DNA Ligase (Fermentas) 0.5 µl pBAD (11.4 ng/µl) 15.5 µl CheY-mCherry (8.3 ng/µl) --- 20.0 µl
 * For the visualization of our killer cells we need a RFP or mCherry reporter plasmid with kanamycin resistance. We decided to clone a cheY-mCherry fusion protein into pBAD18 vector.
 * Digestion of pBAD18 plasmid & pES16-CheY-mCherry with SacI/XbaI: 2h 37 °C, 20 min 65 °C
 * Gel of Digestion: 0.7% Agarose, 135 V, 30 min [[Image:081011-cheY-mCherry_pBAD-digestion_small.jpg |500 px | center]]
 * Gelresults:
 * Expected Fragments:
 * pBAD -> ~4600 bp
 * CheY-mCherry -> ~1100 bp
 * We were able to cut both fragments.
 * Gelextraction with Qiagen Kit: eluted in 35 µl H2O
 * Ligation of pBAD & mCherry: 11 h -> 16 °C, 20 min -> 65 °C
 * EDIT 10/12/202008: calculated DNA ratio wrong

RFP
20.0 µl DNA 1.0 µl EcoRI (20 000 U/ml, NEB) 1.0 µl PstI (10 000 U/ml, NEB) 5.0 µl BSA 10x 5.0 µl EcoRI Buffer(NEB) 18.5 µl H2O --- 50.0 µl upper band 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.0 µl T4 DNA Ligase (Fermentas) 8.5 µl pSB3K3 (upper Band) 7.5 µl BBa_J23102 --- 20.0 µl
 * In addition we try to clone RFP gene cassette from J23102 into pSB3K3.
 * Digestion of BBa_J23102 & BBa_I15030 with EcoRI/PstI: 2 h -> 37 °C, 20 min -> 65 °C
 * Gel of Digestion: 0.7% Agarose, 135 V, 30 min [[Image:081011-J23102___pSB3K3_small.jpg |500 px | center]]
 * Gelresults:
 * Expected Fragments:
 * J23102 -> ~945 bp
 * I15030 -> ~1909 bp
 * pSB3K3 -> ~2750 bp
 * J23102 was cutted out of the gel. For pSB3K3 we were not sure which was the right fragment so we go on with both fragments.
 * Gelextraction with Qiagen Kit: eluted in 35 µl H2O
 * Ligation of J23102 and pSB3K3 (5:1):

lower band 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.0 µl T4 DNA Ligase (Fermentas) 11.6 µl pSB3K3 (lower band) 4.4 µl BBa_J23102 --- 20.0 µl
 * EDIT 10/12/202008: calculated DNA ratio wrong

Mutagenesis of pSB1A2-Receiver-Colicin E1/E9
- 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 200 µl on preheated LB-Amp plate - incubate overnight 10.0 µl DNA 2.0 µl BSA 10x (NEB) 0.5 µl EcoRI (NEB) 2.0 µl Buffer EcoRI 5.5 µl H2O --- 20.0 µl
 * Digestion of overnight PCR probes with DpnI for 2 h -> 37 °C.
 * PCR purification of probes: eluted in 35 µl H2O (Qiagen Kit)
 * Transformation of prior mutagenesis: 5 µl per 100 µl E. coli TOP 10 competent cells
 * Controlgel of Mutagenesis: 1 % Agarose, 135 V, 30 min; DNA is visible -> probably the mutagenesis worked. [[Image:081011-controlgel_2nd_mutagenesis.jpg |500 px | center]]
 * Controlldigestion of first mutagenesis of Colicin E1 & E9 - Receiver with EcoRI: 2h -> 37 °C
 * Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:081011-controlgel_digestion_mutagenesis_small.jpg |500 px | center]]
 * Gelresults: Mutated Colicin E1 and E9 Receivers were cutted only once by EcoRI. -> Mutagenesis sucessful.

Colicin E9 operon - parts: Colicin E9
20.0 µl DNA 1.0 µl EcoRI (20 000 U/ml, NEB) 1.0 µl PstI (10 000 U/ml, NEB) 5.0 µl BSA 10x 5.0 µl EcoRI Buffer(NEB) 18.5 µl H2O --- 50.0 µl upper band 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.0 µl T4 DNA Ligase (Fermentas) 6.1 µl pSB3K3 (upper Band) 9.9 µl Colicin E9 --- 20.0 µl
 * Digestion of Colicin E9 PCR product & pSB3K3 (from BBa_I15030)
 * Gel of Digestion: 0.7% Agarose, 135 V, 30 min [[Image:081011-colicinE9-digestion_small.jpg |500 px | center]]
 * Gelresults:
 * Expected Fragments:
 * Colicin E9 -> ~620 bp
 * Colicin E9 was cutted out of the gel.
 * Gelextraction with Qiagen Kit: eluted in 35 µl H2O
 * Ligation of colicin E9 and pSB3K3 (5:1):

lower band 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.0 µl T4 DNA Ligase (Fermentas) 9.5 µl pSB3K3 (lower band) 8.5 µl Colicin E9 --- 20.0 µl EDIT 10/12/202008: calculated DNA ratio wrong

Sender activity test

 * Constitutive sender and amplifier (BBa_I15030) activity test
 * 7x Inoculation of 7 ml TB media with 140 µl from sender ONC
 * 7x Inoculation of 7 ml TB media with 140 µl from amplifier ONC
 * Inoculation of 7 ml TB media with 7 ml from GFP-receiver ONC
 * Measurement of optical density (OD) of ONC dilutions t = 0 h
 * Measurement of OD of one probe hourly for 6 h
 * Creation of supernatant of the measured probe by centrifugation and filtration hourly for 6 h
 * Storing supernatant at 4 °C
 * Count amount of cells from last probe (counting chamber)
 * plate scheme: [[Image: 081011-plate_scheme_sender_amplifier_test.jpg | 800 px | center]]
 * Unfortunately we were not able to measure ON because the Tecan plate reader was not recognized by the PC.

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mCherry
- 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 at 37°C 2.0 µl T4 DNA Ligase Buffer (Fermentas) 2.0 µl T4 DNA Ligase (Fermentas) 6.05 µl pBAD (11.4 ng/µl) 9.95 µl CheY-mCherry (8.3 ng/µl) --- 20.0 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 at 37°C
 * Transformation of pBAD-mCherry Ligation of saturday, 10/11/2008 (wrong vector-insert ratio): 5 µl per 100 µl E. coli TOP 10 competent cells
 * Ligation of pBAD and mCherry (1:5): 1 h -> 22 °C, 20 min -> 65 °C
 * Reason: Transformation on Saturdayurday was carried out with wrong ratio of vector and insert (calculation error).
 * Transformation of pBAD-mCherry Ligation: 5 µl per 100 µl E. coli TOP 10 competent cells

RFP
- 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 at 37°C
 * Transformation of J23102 and pSB3K3 Ligation of saturday, 10/11/2008 (wrong vector-insert ratio): 5 µl per 100 µl E. coli TOP 10 competent cells

Mutagenesis of pSB1A2-Receiver-Colicin E1
10.0 µl DNA (Miniprep) 2.0 µl BSA 10x (NEB) 0.5 µl PstI(NEB) 2.0 µl Buffer EcoRI 5.5 µl H2O --- 20.0 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
 * Inoculation of Col E1 second mutagenesis step in 10 ml LB-amp; 5 h -> 37 °C
 * Colonies 1-8 of Col E1 (5)
 * Colonies 10-17 of Col E1 (1)
 * Miniprep of liquidcultures (Col E1 (1.14;1.16;5.1;5.7): eluted in 35 µl H2O (Qiagen Miniprepkit)
 * Controlldigestion of second mutagenesis of Colicin E1 - Receiver with PstI: 2 h -> 37 °C
 * Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:081012-control_digestion_PstI_colE1_Rec_2nd_mut.jpg |500 px | center]]
 * Gelresults: It can not be concluded weather the mutation worked. The fragments that are expected with and without the mutated PstI-Site are very similar.
 * Third Mutagenesis PCR:

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_2): 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. [[Image:081012-3rd_mutagenesis_controll_gel_small.jpg |500 px | center]]
 * PCR Purification Kit (Qiagen) to purify the plasmids. Eluated in 35 µl H2O
 * Transformation of prior mutagenesis: 5 µl per 100 µl E. coli TOP 10 competent cells

Standardization of pSB1A2-colE9plasmid-Receiver
2.5 µl Primer fw T9002 (10 µM) 2.5 µl Primer rv (10 µM) 18.0 µl H2O 25.0 µl Phusion MasterMix (Finnzymes, NEB) 2.0 µl Template (ColE9-Receiver mutated) --- 50.0 µl
 * PCR to add right BioBrick prefix and suffix to mutated ColicinE9-Receiver (without EcoRI site, colonies 2 & 4)

98 °C   30 sec 98 °C   10 sec          | 56 °C   30 sec          | 25 cycles 72 °C   1 min 30 sec    | 72 °C   10 min 4 °C   constant 6.5 µl H2O 5.0 µl BSA 10x (NEB) 5.0 µl EcoRI Buffer (NEB) 0.5 µl EcoRI (NEB) 1.0 µl SpeI (NEB) 32.0 µl DNA --- 50.0 µl ColicinE9-Receiver (4) 1.1 µl pSB1A2 14.9 µl ColicinE9-Receiver 2.0 µl T4 DNA Ligase 2.0 µl T4 DNA Ligase Buffer --- 20.0 µl
 * Gel to purify PCR products: 0.7%, 135 V, 30 min [[Image:081012-standardization_colE9_rec_gelex_PCR_small.jpg |500 px | center]]
 * Gelextraction: eluted in 35 µl H2O (Qiagen, Gelextraction Kit)
 * Digestion of ColE9-Receiver PCR product with EcoRI and SpeI: 2h -> 37 °C
 * Ligation of ColicinE9-Receiver (BioBrick) with pSB1A2 (3:1): 1h -> 22 °C, 20 min 65 °C

ColicinE9-Receiver (2) 1.8 µl pSB1A2 14.2 µl ColicinE9-Receiver 2.0 µl T4 DNA Ligase 2.0 µl T4 DNA Ligase Buffer --- 20.0 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
 * Transformation of prior ligation: 5 µl per 100 µl E. coli TOP 10 competent cells

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