Addition of heat-killed bacteria to Dictyostelium transformamts

Contributed by Etienne Joly, Jane Kirk, Keith Jermyn, and Jeffrey Williams in the Dicty Newsletter.
Published in Lloyd, Ceccarelli and Williams (1990).

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Heat-killed bacteria preparation:

1. E. coli (or Klebsiella aerogenes) are grown overnight in LB, centrifuged at 6000 rpm for 5 minutes and then washed three times in the growth volume of KK₂ buffer (6.5 mM KH₂PO₄, 3.8 mM K₂HPO₄, pH 6.2)
2. 
   
3. The last pellet is resuspended in 1/10^th of the initial volume of KK₂ and an aliquot is diluted to measure the OD₆₅₀.
4. 
   
5. The volume of the bacterial suspension is then adjusted to a density of 10¹² cells/ml (assuming that an OD₆₅₀ of 0.1 corresponds to 10⁸ cells/ml).
6. 
   
7. Cells are heat-killed at 70°C for 10 minutes and then stored in aliquots at -20°C.

For use with Dictyostelium transformations:

1. One aliquot of heat-killed bacteria is thawed and this suspension is added at 1% vol/vol to the initial selective medium, i.e., at the time the G418 is first added (normally after overnight recovery ).
2. 
   
3. Tetracycline may be included at 15µg/ml in the medium, which is changed every two to three days but which is replaced with medium unsupplemented by bacteria after the first addition.
4. 
   
5. After two medium changes (6 to 8 days), very few bacteria remain visible in the medium and transformants have formed visible colonies.
6. 
   

Note: No further increase in efficiency is found if the suspension of bacteria is also added in all media changes. This latter finding suggests that the addition of bacteria is only helpful for the newly transformed amoebae at the critical, early stage when they are isolated and surrounded by dying cells.

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