"

From 2010.igem.org

The Bacteria Game is a simple all-in-one kit to let homegrown bacteria compete against each other. March in lockstep with nature, breed your own creatures and become coach of your very own athletes. Simply use the included breeding kit to raise your own team and challenge your friends.

Included is everything you need to start instantly:

• 3 Petri dishes
• Bacteria starter set (bacteria culture, agar, toothpicks)
• 2 Game scenarios
• Different Game objects (barriers, forts)

Use the Agar to build different landscapes and as a nutrient for your creatures. Inculate your team onto the agar and watch the game begin. Use the included game objects to evolve your team.

Contents 1 Technical Description 2 Wetlab Notebook 2.1 30/09/2010 2.2 31/09/2010 2.3 01/10/2010

Technical Description

The concept of the game is based on the ability of some harmless wildtype bacteria to swim in soft media. Swimming enables the microbes to consume further nutrients if those in their vicinity are already consumed. All bacteria try to get away from the starting point as fast as possible to access fresh media. This mechanism can be employed for a game setup. Selection and culturing of best swimmers leads to propagation of ideal swimming characteristics. That is, why training may help gain a competitive edge. Those bacteria can easily be stored in the fridge with the supplied materials without any risk.

The showdown competition is run by synthetic bacteria. Predators and prey communicate and regulate each other's density. Via molecular signals, the predator cells kill the prey while living prey rescues predators. The diverse and colorful crowd surrounding the spectacle was genetically engineered to carry different pigments which has been appreciated at the iGEM competition 2009.

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 ([http://www.nature.com/nchembio/journal/v5/n12/pdf/nchembio.244.pdf Song et al., 2009]), ([http://www.nature.com/msb/journal/v4/n1/pdf/msb200824.pdf 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

30/09/2010

• transformation of TOP10 cells with plasmids (see table 1) out of the registry following [http://partsregistry.org/Help:Spring_2010_DNA_distribution#DNA_Kit_Plate_Instructions 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	[http://partsregistry.org/Part:BBa_K274110 BBa_K274110]	red	[http://partsregistry.org/Part:pSB1A2 pSB1A2]	2010 Kit Plate 3, 6J
pLA02	[http://partsregistry.org/Part:BBa_K274210 BBa_K274210]	orange	[http://partsregistry.org/Part:pSB1A2 pSB1A2]	2010 Kit Plate 3, 6N
pLA03	[http://partsregistry.org/Part:BBa_K274002 BBa_K274002]	purple	[http://partsregistry.org/Part:pSB1T3 pSB1T3]	2010 Kit Plate 3, 12B
pLA04	[http://partsregistry.org/Part:BBa_K274003 BBa_K274003]	dark green	[http://partsregistry.org/Part:pSB1K3 pSB1K3]	2010 Kit Plate 3, 20H
pLA05	[http://partsregistry.org/Part:BBa_K274004 BBa_K274004]	light green	[http://partsregistry.org/Part:pSB1K3 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

31/09/2010

• 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

01/10/2010

• preparation and conduction of microscopy experiments with predator-prey system following [http://www.nature.com/nchembio/journal/v5/n12/extref/nchembio.244-S1.pdf 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