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Team:Heidelberg/Notebook/Killing II/12thweek
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12th week
Contents |
Monday 10/20/2008
pSB1A3-Receiver-Colicin cloning
- Minipreps of colE1/E9/E9lys-Receiver with QiaCube, Qiagen
- Send probes to GATC for sequencing
Sender cloning: constitutive promotor-sender
- Minipreps of J23107-Receiver with QiaCube, Qiagen
- Send probe to GATC for sequencing
Tuesday 10/21/2008
Sequencing results of ready cloned BioBrick parts
| EcoRI-site mutation | PstI-site 1 mutation | PstI-site 2 mutation | PstI-site 3 mutation | Prefix | Suffix | complete sequence | |
|---|---|---|---|---|---|---|---|
| colE1_BB_57-1 | + | + | + | + | + | + | + |
| colE1_BB_57-2 | + | + | + | + | + | + | + |
| colE1_BB_57+2 | ? | + | + | ? | + | ? | missing sequence |
| colE1_BB_67+1 | + | + | + | + | + | ? | sequencing failure |
| colE9_BB_(2) | + | XXX | XXX | XXX | + | + | + |
| colE9lys_BB | XXX | XXX | XXX | XXX | + | + | + |
| sender_BB | XXX | XXX | XXX | XXX | + | + | + |
--> colE1_BB_57-1, colE9_BB_(2), colE9lys_BB and sender_BB gave positive sequencing results in all criteria and will be sent to the registry
Controldigestion of parts with EcoRI and PstI
- colE1_BB_57-1, colE9_BB_(2), colE9lys_BB and sender_BB was digested with EcoRI and PstI: 1h 30 min -> 37 °C
0.5 µl EcoRI (NEB) 0.5 µl PstI (NEB) 3.0 µl DNA 2.0 µl EcoRI Buffer (NEB) 2.0 µl BSA 10x 12.0 µl H2O ------- 20.0 µl
- Gelresults: The digestion pattern looks like expected. 1% Agarose, 135 V, 30 min
Control of antibiotics resistance
- LB-media containing tetracycline, chloramphenicol, ampicilin or kanamycin was inoculated with the different parts to the theri antibiotics resistance.
Wednesday 10/22/2008
- Antibiotics test: Each part only grew in ampicilin media as expected
Thursday 10/23/2008
pSB1A2-Receiver-Colicin cloning
HisTag cloning of Colicins for purification
Colicin activity test
Friday 10/24/2008
pSB1A3-Receiver-Colicin cloning
HisTag cloning of Colicins for purification
Sender Cloning: pBAD - sender
Sender Cloning: constitutive promotor - sender
Saturday 10/25/2008
Characterization: ColicinE1-Receiver Activitytest: Killer-prey system test and lysis test of killer cells
- afternoon: Inoculation of the following cultures:
- constitutive Sender with GFP(J23107-F1610 + I20260)(TB-Kana_Amp)
- ColE1Rec pBAD-mCherry (TB-Kana-Amp-Arab)
- T9002 without GFP pBAD-mCherry (TB-Kana-Amp-Arab)
- ColE1Rec + I20260 (TB-Kana-Amp)
- T9002 without GFP + I20260 (TB-Kana-Amp)
- 8.30 pm: Preparing mixtures for the plate:
- plate scheme:
Theoretical work for documentation
Sunday 10/26/2008
Theoretical work for documentation.
