2015/report/debugging/cell-lysis

We drew our inspiration for the protocol from the following paper: « William W Ward of Brighter Ideas, Three-Phase Partitioning for Protein Purification ».  We chose the Three-Phase Partitioning method because it seemed to be an easy and interesting method to separate GFP from bacteria. We were following the protocol from the paper but at the step where we should have seen if the concentration of the starting buffer was good we didn't obtain the good result and each concentration seemed to be higher. We decided to go our way and to try with three different concentrations (1.6 M, 3M and 4M) of starting buffer. Finally the result gave that the GFP was not visible to the naked eye but with fluorescence analysis the GFP was existing in the 3 samples. The 1.6M of starting buffer has the higher green fluorescence value so we decided to perform our protocol with this concentration. We performed again the experiment with more bacteria to obtain more green fluorescence with this following protocol:

MATERIAL:

PROTOCOL:

RESULTS:

absorbance of eGFP bacteria: 1.96 OD
absorbance of control bacteria: 2 OD

so we can put the same volume (50ml) because the OD values are close enough

fluorescence result:

eGFP:                      45083

control bacteria:       873

1.6M starting buffer: 1

CONCLUSION:

We measured the fluorescence of 1.6M starting buffer to compare the green fluorescence between bacteria with GFP, bacteria and sample without bacteria.
The fluorescence of control bacteria of 873 can be explained because lots of things fluoresce in green in biology. The difference between eGFP and control bacteria fluorescence is significant.
We obtained about 4ml of free GFP (from lysis of eGFP bacteria).

REFERENCE: [1] William W Ward of Brighter Ideas, Three-Phase Partitioning for Protein Prufification [pdf]

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