Team:EPF-Lausanne/Notebook/Wet Lab

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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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[[Image: pCAL-n.jpg|500px|thumb|center|pCal-n plasmid]]
 
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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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===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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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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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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[[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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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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===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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2. 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_B0010 (terminator)
 
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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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13D (1)
 
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|-
 
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| width="33%" |
 
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BBa_R0010 (promoter LacI)
 
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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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1D (1)
 
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|-
 
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| width="33%" |
 
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BBa_B0030 (RBS)
 
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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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1H (1)
 
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|-
 
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| width="33%" |
 
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BBa_E0240 (RBS-GFP-TER)
 
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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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12M (1)
 
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|-
 
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| width="33%" |
 
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BBa_I13507(RBS-mRFP-TER)
 
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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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22O (1)
 
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|-
 
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| width="33%" |
 
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BBa_J13002(pTetR-RBS)
 
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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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13B (1)
 
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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 yesterdey 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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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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3. An agarose gel was runned to check PCR products
 
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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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Finally, LOVTAP (PCR products) were ligated on BBa_B0010 (plasmid chosen containing the terminator).
 
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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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===10.07.09===
 
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==August==
 
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</div><div CLASS="epfl09bouchon"></div>
 

Latest revision as of 08:05, 28 July 2009