Team:Heidelberg/Notebook/Killing II/12thweek

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12th week

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Contents

Monday 10/20/2008

pSB1A3-Receiver-Colicin cloning

  • Minipreps of colE1/E9/E9lys-Receiver with QiaCube, Qiagen
  • Send probes to GATC for sequencing


Sender cloning: constitutive promotor-sender

  • Minipreps of J23107-Receiver with QiaCube, Qiagen
  • Send probe to GATC for sequencing


Tuesday 10/21/2008

Sequencing results of ready cloned BioBrick parts

EcoRI-site mutation PstI-site 1 mutation PstI-site 2 mutation PstI-site 3 mutation Prefix Suffix complete sequence
colE1_BB_57-1 + + + + + + +
colE1_BB_57-2 + + + + + + +
colE1_BB_57+2 ? + + ? + ? missing sequence
colE1_BB_67+1 + + + + + ? sequencing failure
colE9_BB_(2) + XXX XXX XXX + + +
colE9lys_BB XXX XXX XXX XXX + + +
sender_BB XXX XXX XXX XXX + + +

--> colE1_BB_57-1, colE9_BB_(2), colE9lys_BB and sender_BB gave positive sequencing results in all criteria and will be sent to the registry

Controldigestion of parts with EcoRI and PstI

  • colE1_BB_57-1, colE9_BB_(2), colE9lys_BB and sender_BB was digested with EcoRI and PstI: 1h 30 min -> 37 °C
 0.5 µl EcoRI (NEB)
 0.5 µl PstI (NEB)
 3.0 µl DNA
 2.0 µl EcoRI Buffer (NEB)
 2.0 µl BSA 10x
12.0 µl H2O
-------
20.0 µl
  • Gelresults: The digestion pattern looks like expected. 1% Agarose, 135 V, 30 min
081021-controldigestion.jpg

Control of antibiotics resistance

  • LB-media containing tetracycline, chloramphenicol, ampicilin or kanamycin was inoculated with the different parts to the theri antibiotics resistance.

Wednesday 10/22/2008

  • Antibiotics test: Each part only grew in ampicilin media as expected

Thursday 10/23/2008

pSB1A2-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Colicin activity test

Friday 10/24/2008

pSB1A3-Receiver-Colicin cloning

HisTag cloning of Colicins for purification

Sender Cloning: pBAD - sender

Sender Cloning: constitutive promotor - sender

Saturday 10/25/2008

Killer-prey system test and lysis test of killer cells

Theoretical work for documentation

Sunday 10/26/2008

Theoretical work for documentation.

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