All entries for Wednesday 25 January 2006
January 25, 2006
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.