IGEM:IMPERIAL/2009/Assays Protocols/M3 Assays

Assays for Cell Death Strategy

Considerations:

• Test each RE separately and together.
  
• Test with Dam +ve and -ve strains (put under control of tight, non-leaky promoter pBAD).
  
• Test thermoinduction system separately.
  
• Qualitative - GFP under pBAD promoter.
  
• Quantitative - colony-forming units (CFU).
  

FACS (Fluorescent Automated Cell Sorter)

Media:Cell Death Assay.pdf   Qualitative and quantitative

• This is an in vitro assay.
  
• Can quantitate the number of dead cells in the population.
  

Using GFP marker

Basic genetic circuit for the restriction enzyme testing construct:

Here, there are 3 possibilities for the expression of the GFP, and these are shown below:

i) Non-functional restriction enzyme co-expressed with the GFP.
ii) Basal levels of GFP expression suggesting that the promoter is not functioning properly.
iii) Initial increase in fluorescence, followed by a plateauing of the fluorescence, as the restriction enzymes destroy the genetic material within the cell.

By Colony Forming Units

• A measure of viable cell numbers in CFU/ml (colony-forming units per millilitre).

1) Plating dilution series on TSB Agar Plates
2) Counting of colonies after overnight incubation
3) Determining the number of colony forming units (number of cells still alive)

GFP, CFU & PoPS

There is the potential to relate both measurement to get obtain quantifiable data in terms of PoPS (see here.