|
|
| (98 intermediate revisions not shown) |
| Line 1: |
Line 1: |
| - | {{EPF-Lausanne09}}
| |
| - | <div CLASS="epfltrick">__TOC__
| |
| - | </div><div CLASS="epfl09">
| |
| | | | |
| - | =Wet Lab=
| |
| - | ==July==
| |
| - | ===06.07.09===
| |
| - | LOVTAP plasmid AND TrpR plasmid were transformed in competent E. Coli following received protocol, and grown overnight (see Lab book for more details).
| |
| - | <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.
| |
| - | LOVTAP is in a plasmid called pCal-n (see picture below):
| |
| - |
| |
| - | [[Image: pCAL-n.jpg|500px|thumb|center|pCal-n plasmid]]
| |
| - |
| |
| - | <br>Some comments on the plasmid:
| |
| - | <br>-CBP is a small peptide with which we could purify LOVTAP protein
| |
| - | <br>-Thrombin target is a nucleotidic sequence that can be recognized by thrombin a peptidase. This peptidase will cut CBP once LOVTAP is purified
| |
| - |
| |
| - | ===07.07.09===
| |
| - | We have to grow the 3 strains generously sent by [mailto:j.beatty@ubc.ca Tom Beatty]
| |
| - |
| |
| - | The three strains are :
| |
| - | :*''R.Palustris'' CEA001 (wild type) ; should be grown on LB medium only
| |
| - | :*''R.Palustris'' BPHP1+ ; should be grown on LB with gentamycin (100 micrograms/ml)
| |
| - | :*''E.Coli'' DH10B (pBPH/hmu0) ; should be grown on LB with gentamycin (20 micorgrams/ml)
| |
| - |
| |
| - |
| |
| - | The transformed LOVTAP and TrpR worked well (N.B. the plasmid of TrpR is pUC19 so the antibiotic resistance is Amp -> see below)
| |
| - |
| |
| - |
| |
| - |
| |
| - | [[Image: RTEmagicC_puc19_2.gif.gif|500px|thumb|center|pUC19 plasmid]]
| |
| - |
| |
| - |
| |
| - | We did the glycerol stock, located in the -80 fridge, first floor of the iGEM compartement.
| |
| - |
| |
| - | Then, a miniprep was done with both cultures.
| |
| - | A LOVTAP plasmid aliquot was done, a TrpR plasmid aliquot was done, located in the -20 fridge, 2nd floor.
| |
| - |
| |
| - | ===08.07.09===
| |
| - | 1. R. Palustris culture grew. A glycerol stock has been done.
| |
| - | A pellet is on the fridge level 2, waiting for a miniprep.
| |
| - |
| |
| - | 2. iGEM parts have been transformed:
| |
| - |
| |
| - | {| class="wikitable" width="100%"
| |
| - | |+ <big> '''Parts&Characteristics''' </big>
| |
| - | |-
| |
| - | ! scope=col | Part
| |
| - | ! scope=col | Characteristic
| |
| - | ! scope=col | Resistance
| |
| - | ! scope=col | Well (Kit Plate)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | [http://partsregistry.org/Part:BBa_B0010 BBa_B0010]
| |
| - | | width="33%" align="center"|
| |
| - | Terminator
| |
| - | | width="33%" align="center" |
| |
| - | A
| |
| - | | width="100%" align="center" |
| |
| - | 13D (1)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | [http://partsregistry.org/Part:BBa_R0010 BBa_R0010(promoter LacI)]
| |
| - | | width="34%" align="center" |
| |
| - | A
| |
| - | | width="34%" align="center" |
| |
| - | 1D (1)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | [http://partsregistry.org/Part:BBa_B0030 BBa_B0030(RBS)]
| |
| - | | width="34%" align="center" |
| |
| - | A
| |
| - | | width="34%" align="center" |
| |
| - | 1H (1)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | [http://partsregistry.org/Part:BBa_E0240 BBa_E0240(RBS-GFP-TER)]
| |
| - | | width="34%" align="center" |
| |
| - | A
| |
| - | | width="34%" align="center" |
| |
| - | 12M (1)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | [http://partsregistry.org/Part:BBa_I13507 BBa_I13507(RBS-mRFP-TER)]
| |
| - | | width="34%" align="center" |
| |
| - | A
| |
| - | | width="34%" align="center" |
| |
| - | 22O (1)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | [http://partsregistry.org/Part:BBa_J13002 BBa_J13002(pTetR-RBS)]
| |
| - | | width="34%" align="center" |
| |
| - | A
| |
| - | | width="34%" align="center" |
| |
| - | 13B (1)
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | ===09.07.09===
| |
| - | 1. Miniprep and isolations of the yesterday transformed plasmids. (cf. 08.09.09 subpart)
| |
| - |
| |
| - | Concentrations of the plasmids: cf. lab notebook pp. 8-9
| |
| - |
| |
| - |
| |
| - | 2. PCR of pCal-n to PCR up LOVTAP with the different primers designed the 06.09.09, the products are:
| |
| - | <br>- Prom_T7-RBS-CBP-LOVTAP
| |
| - | <br>- RBS-CBP-LOVTAP
| |
| - | <br>- CBP-LOVTAP
| |
| - | <br>- LOVTAP
| |
| - |
| |
| - |
| |
| - | 3. An agarose gel was runned to check PCR products
| |
| - |
| |
| - |
| |
| - | 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.
| |
| - |
| |
| - | Finally, LOVTAP (PCR products) were ligated on BBa_B0010 (plasmid chosen containing the terminator).
| |
| - |
| |
| - |
| |
| - | 5. Two more iGEM parts have been transformed:
| |
| - | {| class="wikitable" width="80%" align="center"
| |
| - | |+ <big> '''Parts&Characteristics''' </big>
| |
| - | |-
| |
| - | ! scope=col | Part
| |
| - | ! scope=col | Resistance
| |
| - | ! scope=col | Well (Kit Plate)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | BBa_I6007 (inverter TetR)
| |
| - | | width="34%" align="center" |
| |
| - | A
| |
| - | | width="34%" align="center" |
| |
| - | 1C (2)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | BBa_P1010 (death gene)
| |
| - | | width="34%" align="center" |
| |
| - | C
| |
| - | | width="34%" align="center" |
| |
| - | 5E (1)
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | Remarks: BBa_P1010, the death gene has to be transformed in ccdB (death gene) resistant cells: One Shot ccdB survival 2T1 E. Coli
| |
| - |
| |
| - | ===10.07.09===
| |
| - |
| |
| - | ==August==
| |
| - |
| |
| - | </div><div CLASS="epfl09bouchon"></div>
| |
"
