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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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| - |
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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="80%"
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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. [https://2009.igem.org/Team:EPF-Lausanne/Notebook/Wet_Lab#08.07.09 08.09.09 subpart])
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| - |
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| - | Concentrations of the plasmids: cf. lab notebook pp. 8-9
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| - |
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| - |
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| - | 2. PCR of pCal-n to PCR up LOVTAP with the different primers designed the [https://2009.igem.org/Team:EPF-Lausanne/Notebook/Cloning_Strategy#06.07.09 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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| - | Result: Prom_T7-RBS-CBP-LOVTAP didn't worked, the other were ok.
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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 [http://partsregistry.org/Part:BBa_B0010 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 [http://partsregistry.org/Part:BBa_B0010 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%"
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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_I6007 BBa_I6007]
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| - | | width="33%" align="center"|
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| - | Double repressor: called Inverter TetR
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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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| - | 1C (2)
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| - | |-
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| - | | width="33%" |
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| - | [http://partsregistry.org/Part:BBa_P1010 BBa_P1010]
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| - | | width="33%" align="center"|
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| - | Death Cassette
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| - | | width="34%" align="center" |
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| - | C
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| - | | width="50%" align="center" |
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| - | 5E (1)
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| - | |-
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| - | |}
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| - |
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| - |
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| - | Remarks: [http://partsregistry.org/Part:BBa_P1010 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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| - | Miniprep of the yesterday transformations were done, glycerol stock of [http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)] and [http://partsregistry.org/Part:BBa_P1010 Death Cassette (BBa_P1010)] were done and finally they were put at -80°C.
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| - |
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| - | As Prom_T7-RBS-CBP-LOVTAP PCR product migration on the gel didn't worked (in fact there wasn't enough products) July the 9th, a second PCR was done with more cycles (40) to check wether the the primers were accurately designed.
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| - |
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| - | Results: The primers are correct -> Prom_T7-RBS-CBP-LOVTAP was correctly amplified.
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| - |
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| - | A digestion-ligation according iGEM special protocol [[Media: BioBrick_Assembly_Manual.pdf|Biobrick_Assembly_Manual]] was done with LacI and RBS. And Term was digested (with E and X) to linearize the plasmid.
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| - |
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| - | As [http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)] and [http://partsregistry.org/Part:BBa_P1010 Death Gene (BBa_P1010)] were transformed with a contaminated SOC, we did a PCR with the isolated plasmids to check if the plasmids were correct.
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| - |
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| - | Results: [http://partsregistry.org/Part:BBa_P1010 Death Cassette (BBa_P1010)] sample contain the correct plasmid, [http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)] doesn't.
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| - |
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| - |
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| - | Finally, a gel extraction was done to purify the digested [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)] (linearized plasmid)
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| - |
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| - | ===13.07.09===
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| - | [http://partsregistry.org/Part:BBa_I6007 Inverter TetR (BBa_I6007)] was transformed again. And the new plasmid (created on July the 10th, [https://2009.igem.org/Team:EPF-Lausanne/Notebook/Wet_Lab#10.07.09 10.07.09]) LacI-RBS was transformed on DH5-alpha competent cells.
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| - |
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| - | We failed in purifing our [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)], do it again tomorrow, beginning with the digestion, etc.
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| - |
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| - | ===14.07.09===
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| - | The digestion of the [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)] was done, in order to purify it once more, using gel extraction.
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| - |
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| - | Miniprep of LacI-RBS and Inverter TetR, and PCR of the two of them.
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| - |
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| - | The linearized [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)] was runned on an agarose gel and purified with a gel extraction kit.
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| - |
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| - | Even though linearized [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)] concentration was very low, we tried to ligate the previously amplified LOVTAP [https://2009.igem.org/Team:EPF-Lausanne/Notebook/Wet_Lab#09.07.09 (08.07.09)] in linearized [http://partsregistry.org/Part:BBa_B0010 Terminator (BBa_B0010)]
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| - |
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| - | ===15.07.09===
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| - | Miniprep of LacI-RBS (again, because we had a very low concentration yesterday). We obtained a better concentration.
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| - |
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| - | Transformation of LovTAP-Term (LB + plates).
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| - | LB-Agar plates without antibiotic and with chlorophenicol & Ampicilin have been made.
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| - |
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| - | ===16.07.09===
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| - | Our plates and tubes from yesterday transformation are the same for the negative control and the LovTAP-Term. So either the ligation or the transformation didn't work. Actually we think that it is the transformation, because we put the wrong antibiotics : Chl instead of Amp. As we don't have any ligation products any more, we did the digestion, gel extraction and ligation again.
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| - | We also did a PCR of LacI-RBS product of the second transformation (of 15.07.09), as the concentration for the transformation of 14.07.09 was too low. We then ran the gel.
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| - | Gel extraction of Term.
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| - |
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| - | Preparation of LB plates : AMP plates and Chl plates.
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| - |
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| - | Then ligation of LovTAP and Term.
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| - | Finally digestion of the ligation product.
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| - |
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| - | ===17.07.09===
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| - |
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| - | ===20.07.09===
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| - |
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| - | ==August==
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| - | </div><div CLASS="epfl09bouchon"></div>
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