IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-7-5

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Contents

Plasmid Characteristics

We decided our plasmid should contain the following:

  1. LuxABC/DE(?)
  2. KaiABC
  3. Constitutive / controlled promoter
  4. Amp/Kan/Kat

Control

Transform PCC 7942 with AM 2195.

Experiment Possibilities

  1. One plasmid
    • Knockout the Kai.
    • Mutate 2195 for BB comp.
    • Insert BioBrick’d KaiABC to Am2185 -> HU0000.
    • Transform PCC 7942 with HU0000.
    • PCC 7942.
      • +AM2195 [NS 2.1]
      • +(AM2195 + KaiABC) [NS 2.1]
    • Pro:
      • Can be performed in parallel
    • Cons:
      • Size
  2. Two plasmids
    • Mutate some other plasmid (with different resistance from AM2195) for BB comp. -> HU0001.
    • Insert KaiABC BB into HU0001.
    • Transform control strain with HU0001.
    • PCC 7942.
      • +AM2195 [NS 2.1]
      • +KaiABC [NS 2.1]
    • Pro:
      • Modularity
      • Size
    • Cons:
      • Time

Plasmid Selections

  • pAM1570
  • pAM2195
  • pAM2314
  • NS 2.1 (Cm/Ap)
  • NS 1.0 (Sp/Sm)
  • NS 2.2?
  • NS 2.0?
  • 1579 NS

Incubator status

click here
click here

Two of our PCC 6803 flasks from 2006-6-22 have now turned deep green. A third flask is pale green. Unfortunately our PCC 6803 cultures from 2006-6-26 still aren't growing. Maybe the thiosulfate in the medium is a problem?

The closed-but-not-sealed clean agar plate is now dried up. We took it out of the incubator, as well as the dried PCC 7942 plate. Now we know that unsealed plates will dry up in a week or less. We should always seal our plates with parafilm in the future.

We added two plates: one streaked with 6803 culture (single colony 2006-6-22), and the other with frozen 6803 stock. The agar media contains thiosulfate, so if these plates don't grow, we should probably try making new plates without thiosulfate.

  • Added
    • Plate (BG-11 medium with thiosulfate) streaked with frozen PCC 6803
    • Plate (BG-11 medium with thiosulfate) streaked with PCC 6803 (single colony 2006-6-22)
  • Removed
    • Dried PCC 7942 plate
    • Dried clean plate (closed but not sealed)
  • Switched
    • PCC 6803 single colony (2006-6-22) from upper shelf with PCC 7942 single colony (2006-6-22) on lower shelf
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