IGEM:IMPERIAL/2009/Assays Protocols/M3 Assays

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m (Using GFP marker)
m (Using GFP marker)
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ii) Basal levels of GFP expression suggesting that the promoter is not functioning properly. <br>
ii) Basal levels of GFP expression suggesting that the promoter is not functioning properly. <br>
iii) Initial increase in flourescence, followed by a plateauing of the flourescence, as the restriction enzymes destroy the genetic material within the cell.
iii) Initial increase in flourescence, followed by a plateauing of the flourescence, as the restriction enzymes destroy the genetic material within the cell.
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*'''[[User:James Chappell|James Chappell]] 12:53, 3 August 2009 (EDT)''':Great to see you have been thinking in terms of the data produced is defiantly the right way to create these assays. Few bits of feedback, think you need to make more measurements than just fluorescence measurements, typically from this it might be hard to decouple the effect of cell growth fluorescence.
 
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Some measures we use of cell numbers use typically two techniques -> optical density (at 600nm) and dilution plating(colony counting), maybe think about how these could be used to measure the effectiveness of the restriction enzymes. Also how have previous teams assessed the killing mechanisms?
 
==Cell Death Assay==
==Cell Death Assay==

Revision as of 13:00, 3 August 2009

Contents

Assays for Cell Death Strategy

Assay to determine cell death

Media:Cell Death Assay.pdf

Restriction Enzyme Assay

Another Possibility

Using GFP marker

Basic genetic circuit for the restriction enzyme testing construct:

Image:II09_Killtestm3.png

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

Image:II09_m3_restenz.png

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 flourescence, followed by a plateauing of the flourescence, as the restriction enzymes destroy the genetic material within the cell.

Cell Death Assay

By Colony Forming Units

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)

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