|
|
| (127 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:
| |
| - | <br>- BBa_B0010 (terminator), resistance: A, well kit plate 1: 13D
| |
| - | <br>- BBa_R0010 (promoter LacI), resistance: A, well kit plate 1: 1D
| |
| - | <br>- BBa_B0030 (RBS), resistance: A, well kit plate 1: 1H
| |
| - | <br>- BBa_E0240 (RBS-GFP-TER), resistance: A, well kit plate 1: 12M
| |
| - | <br>- BBa_I13507(RBS-mRFP-TER), resistance: A, well kit plate 1: 22O
| |
| - | <br>- BBa_J13002(pTetR-RBS), resistance: A, well kit plate 1: 13B
| |
| - | <br>- BBa_I6007 (inverter TetR), resistance: A, well kit plate 2: 1C
| |
| - |
| |
| - | {| class="wikitable" width="80%" align="center"
| |
| - | |+ Parts&Characteristics
| |
| - | |-
| |
| - | ! scope=col | Part
| |
| - | ! scope=col | Resistance
| |
| - | ! scope=col | Well (Kit Plate)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | BBa_B0010 (terminator)
| |
| - | | width="34%" |
| |
| - | A
| |
| - | | width="33%" rowspan="2" |
| |
| - | 13D (1)
| |
| - | |-
| |
| - | | width="33%" |
| |
| - | BBa_R0010 (promoter LacI)
| |
| - | | width="34%" |
| |
| - | A
| |
| - | |-
| |
| - | | align="center" colspan="3" |
| |
| - | Ligne 1
| |
| - | |}
| |
| - |
| |
| - | ===09.07.09===
| |
| - |
| |
| - | ===10.07.09===
| |
| - |
| |
| - | ==August==
| |
| - |
| |
| - | </div><div CLASS="epfl09bouchon"></div>
| |
"
