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Team:Weimar-Heidelberg Arts/Project/Bacteria Game/Wetlab
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Wetlab Notebook
- usage of E. chromi principle (pigmented E. coli cells)
- as a colorful crowd
- plasmids available in iGEM Spring 2010 DNA Distribution (see table 1)
- as a colorful crowd
- testing of a synthetic E.coli predator-prey system (Song et al., 2009), (Ballagaddé et al., 2008)
- for final assault on the swarming plate and under the microscope
- kindly provided by R. Smith (see table 2)
- for final assault on the swarming plate and under the microscope
- wildtype E. coli
- for the game-kit
- offered by A. Kern
- for the game-kit
30/09/2010
- transformation of TOP10 cells with plasmids (see table 1) out of the registry following standard recommendations
- over-night growth on selective LB agar plates (10 g Tryptone, 5 g Yeast extract, 10 g NaCl and 15 g Agar ad 1 L ddH2O + required antibiotics)
| plasmid | Part | pigment color | backbone | registry location |
|---|---|---|---|---|
| pLA01 | BBa_K274110 | red | pSB1A2 | 2010 Kit Plate 3, 6J |
| pLA02 | BBa_K274210 | orange | pSB1A2 | 2010 Kit Plate 3, 6N |
| pLA03 | BBa_K274002 | purple | pSB1T3 | 2010 Kit Plate 3, 12B |
| pLA04 | BBa_K274003 | dark green | pSB1K3 | 2010 Kit Plate 3, 20H |
| pLA05 | BBa_K274004 | light green | pSB1K3 | 2010 Kit Plate 3, 20J |
- glycerol stocks (prepared directly after arrived) from predator and prey cells (see table 2) were plated on selective LB agar plates (s. a.)
| sample | name/function | cell strain | plasmid | marker |
|---|---|---|---|---|
| 1a | predator | MG1655 | ptetLuxRLasI-luxCcdA(SC101), placCcdBs-tetGFPuv(LVA) | Cm, Kan |
| 3a | prey | MG1655 | pLasRLuxI-luxCcdBs, ptet-mCherry(ColE1) | Cm, Kan |
01/10/2010
- inoculation of day cultures with colonies from the previous day
- in 5 mL LB + required antibiotic
- preparation of TB soft agar plates (10 g Tryptone, 5 g NaCl and 3 g Agar ad 1 L ddH2O + required antibiotics swarm plates) for swarming
- inoculation by carefully pipetting 3 μL of liquid culture into the solidified agar
- incubation of swarm plates at room temperature for 20 h
- sufficient humidity was ensured by petri dishes filled with water
- a photo was taken (by Canon EOS 5D Mark II) every 30 sec. for 6 h
- inoculation of over-night cultures from the day cultures
02/10/2010
- preparation and conduction of microscopy experiments with predator-prey system following liquid-phase protocols
- snapshots were taken every second for one minute at beginning and at the very end of the experiments
- time-lapse took 3 h with a picture each 20 seconds using given filters for GFPuv and mCherry
