Team:Weimar-Heidelberg Arts/Project/Bacteria Game/Wetlab

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)
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)
wildtype E. coli
- for the game-kit
  - offered by A. Kern

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)

**table 1:** used iGEM constructs
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.)

*table 2:* used predator-prey system, gift from R. Smith
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

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

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