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| - | {{EPF-Lausanne09}}
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| - | <div CLASS="epfltrick">__TOC__
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| - | </div><div CLASS="epfl09">
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| | | | |
| - | =Wet Lab=
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| - | ==July==
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| - | ===06.07.09===
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| - | LOVTAP plasmid AND TrpR plasmid were transformed in competent E. Coli following received protocol, and grown overnight (see Lab book for more details).
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| - | <br>One problem: we actually don't know TrpR plasmid resistance, so we tried with three resistances available in the lab: Amp., Kana. and Chl.
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| - | LOVTAP is in a plasmid called pCal-n (see picture below):
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| - |
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| - | [[Image: pCAL-n.jpg|500px|thumb|center|pCal-n plasmid]]
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| - |
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| - | <br>Some comments on the plasmid:
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| - | <br>-CBP is a small peptide with which we could purify LOVTAP protein
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| - | <br>-Thrombin target is a nucleotidic sequence that can be recognized by thrombin a peptidase. This peptidase will cut CBP once LOVTAP is purified
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| - |
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| - | ===07.07.09===
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| - | We have to grow the 3 strains generously sent by [mailto:j.beatty@ubc.ca Tom Beatty]
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| - |
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| - | The three strains are :
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| - | :*''R.Palustris'' CEA001 (wild type) ; should be grown on LB medium only
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| - | :*''R.Palustris'' BPHP1+ ; should be grown on LB with gentamycin (100 micrograms/ml)
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| - | :*''E.Coli'' DH10B (pBPH/hmu0) ; should be grown on LB with gentamycin (20 micorgrams/ml)
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| - |
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| - | The transformed LOVTAP and TrpR worked well (N.B. the plasmid of TrpR is pUC19 so the antibiotic resistance is Amp -> see below)
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| - |
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| - |
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| - |
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| - | [[Image: RTEmagicC_puc19_2.gif.gif|500px|thumb|center|pUC19 plasmid]]
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| - | We did the glycerol stock, located in the -80 fridge, first floor of the iGEM compartement.
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| - |
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| - | Then, a miniprep was done with both cultures.
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| - | A LOVTAP plasmid aliquot was done, a TrpR plasmid aliquot was done, located in the -20 fridge, 2nd floor.
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| - |
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| - | ===08.07.09===
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| - | 1. R. Palustris culture grew. A glycerol stock has been done.
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| - | A pellet is on the fridge level 2, waiting for a miniprep.
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| - |
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| - | 2. iGEM parts have been transformed:
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| - |
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| - | {| class="wikitable" width="70%"
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| - | |+ <big> '''Parts&Characteristics''' </big>
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| - | |-
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| - | ! scope=col | Part
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| - | ! scope=col | Characteristic
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| - | ! scope=col | Resistance
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| - | ! scope=col | Well (Kit Plate)
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| - | |-
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| - | | width="33%" |
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| - | [http://partsregistry.org/Part:BBa_B0010 BBa_B0010]
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| - | | width="33%" align="center"|
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| - | Terminator
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| - | | width="33%" align="center" |
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| - | A
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| - | | width="50%" align="center" |
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| - | 13D (1)
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| - | |-
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| - | | width="33%" |
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| - | [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]
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| - | | width="33%" align="center"|
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| - | Promoter LacI
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| - | | width="34%" align="center" |
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| - | A
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| - | | width="50%" align="center" |
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| - | 1D (1)
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| - | |-
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| - | | width="33%" |
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| - | [http://partsregistry.org/Part:BBa_B0030 BBa_B0030]
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| - | | width="33%" align="center"|
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| - | RBS
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| - | | width="34%" align="center" |
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| - | A
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| - | | width="50%" align="center" |
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| - | 1H (1)
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| - | |-
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| - | | width="33%" |
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| - | [http://partsregistry.org/Part:BBa_E0240 BBa_E0240]
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| - | | width="33%" align="center"|
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| - | RBS-GFP-TER
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| - | | width="34%" align="center" |
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| - | A
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| - | | width="50%" align="center" |
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| - | 12M (1)
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| - | |-
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| - | | width="33%" |
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| - | [http://partsregistry.org/Part:BBa_I13507 BBa_I13507]
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| - | | width="33%" align="center"|
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| - | RBS-mRFP-TER
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| - | | width="34%" align="center" |
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| - | A
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| - | | width="50%" align="center" |
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| - | 22O (1)
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| - | |-
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| - | | width="33%" |
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| - | [http://partsregistry.org/Part:BBa_J13002 BBa_J13002]
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| - | | width="33%" align="center"|
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| - | pTetR-RBS
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| - | | width="34%" align="center" |
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| - | A
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| - | | width="50%" align="center" |
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| - | 13B (1)
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| - | |-
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| - | |}
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| - |
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| - | ===09.07.09===
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| - | 1. Miniprep and isolations of the yesterday transformed plasmids. (cf. 08.09.09 subpart)
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| - | Concentrations of the plasmids: cf. lab notebook pp. 8-9
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| - |
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| - | 2. PCR of pCal-n to PCR up LOVTAP with the different primers designed the 06.09.09, the products are:
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| - | <br>- Prom_T7-RBS-CBP-LOVTAP
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| - | <br>- RBS-CBP-LOVTAP
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| - | <br>- CBP-LOVTAP
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| - | <br>- LOVTAP
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| - |
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| - |
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| - | 3. An agarose gel was runned to check PCR products
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| - |
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| - | 4. PCR products were digested with EcorI and SpeI and BBa_B0010(plasmid chosen containing the terminator) was digested with EcorI and XbaI, the digestion products were treated with phosphatase. Then, PCR products were purified.
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| - |
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| - | Finally, LOVTAP (PCR products) were ligated on BBa_B0010 (plasmid chosen containing the terminator).
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| - |
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| - |
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| - | 5. Two more iGEM parts have been transformed:
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| - | {| class="wikitable" width="80%" align="center"
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| - | |+ <big> '''Parts&Characteristics''' </big>
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| - | |-
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| - | ! scope=col | Part
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| - | ! scope=col | Resistance
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| - | ! scope=col | Well (Kit Plate)
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| - | |-
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| - | | width="33%" |
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| - | BBa_I6007 (inverter TetR)
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| - | | width="34%" align="center" |
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| - | A
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| - | | width="34%" align="center" |
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| - | 1C (2)
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| - | |-
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| - | | width="33%" |
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| - | BBa_P1010 (death gene)
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| - | | width="34%" align="center" |
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| - | C
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| - | | width="34%" align="center" |
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| - | 5E (1)
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| - | |-
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| - | |}
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| - | Remarks: BBa_P1010, the death gene has to be transformed in ccdB (death gene) resistant cells: One Shot ccdB survival 2T1 E. Coli
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| - |
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| - | ===10.07.09===
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| - |
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| - | ==August==
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| - |
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| - | </div><div CLASS="epfl09bouchon"></div>
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