January 25, 2006

Update on the labwork

Meeting with Isabelle. Labwork will start mid-february. We will fuse a luciferace reporter Lux A/B (I believe) with the glnAP2 promoter to track transcription dynamics of glnG. Steps are:

  • Amplify glnAP2 region including the lac1 operator sites with PCR.
  • Add resctriction enzyme sites to the sequence to create the sticky ends.
  • Insert the promoter region in front of the Lux A/B gene (restriction sites are in front of the gene sequence) which comes on a plasmid.
  • Single out plasmid carrying cells by the ampillicin resistance gene Amp which is also on the plasmid.

There were other issues to think about..

  • When cultures sit on a plate, they create a local environment by secretion so that they no longer need the ampicillin resistance. The chemostat circumvented that problem by constantly refreshing the medium.
  • In liquid culture the clock could be startet by first adding and then removing a certain chemical. On plates its not that easy to manipulate the medium. What to do? Maybe back to liquid culture – there was something about plates with many small cylinders of which luminescence could be measured.

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Ulrich Janus

January 2006

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