GMU talk:Synthetic Biology/Bacteria Game/iGEM Wiki

iGem Wiki preparation page

Introduction

Bacteria Game

Small introductory text.

Description

A in depth description of the non-technical concept.

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 (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

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)

table 1: overview of used iGEM constructs

plasmid	Part	pigment color	backbone	registry location
pLA01	Template:Part	red	Template:Part	2010 Kit Plate 3, 6J
pLA02	Template:Part	orange	Template:Part	2010 Kit Plate 3, 6N
pLA03	Template:Part	purple	Template:Part	2010 Kit Plate 3, 12B
pLA04	Template:Part	dark green	Template:Part	2010 Kit Plate 3, 20H
pLA05	Template:Part	light green	Template:Part	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 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