IGEM:IMPERIAL/2009/Assays Protocols/M3 Assays

Assays for Cell Death Strategy

Considerations:

• Test each RE separately and together.
  
  • Together we can use the cell death assays, seperately we can perform an experimental setup to determine DNA cleavage.
• 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.
  
• However, this method relies on damaged cell membranes to assess whether cells are 'dead' or 'alive.' As we use restriction enzymes to kill our cells, this would leave the cell membranes untouched. Question - will the cell membranes degrade naturally once the cell genetics have been destroyed? Can be used to test this theory.

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.

Measuring Cell Death by counting Colony Forming Units (CFUs)
CFU Method: By knowing the density of cells present in a media using OD measurements, and depositing a set amount of this media (and therefore a set no. of cells) onto an agar plate, we can know the proportion of viable cells still present in our colony after restriction enzyme production. This gives a quantifiable method for the calculation of the efficiency of our killing strategy.

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)

We must ensure we do many repetitions to get reliable data

GFP, CFU & PoPS

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