Flow Cytometry QUERY!

deepika gaddam deepika.gaddam at gmail.com
Mon Jan 29 14:40:22 EST 2007


Hi All,
I am a graduate student in the department of Biology at UNC-Charlotte, NC. We
have a BD FACSCalibur System in our department which I want to use for a
specific purpose for my project. I have also contacted BD Biosciences
service support and also the scientific advisor of the company, they gave an
encouraging reply but again that could not solve the issue I am facing. I
wish to explain very briefly the problem I am facing in achieving a very
crucial step in my research. I will really appreciate if I get any
encouraging suggestions.

*Research Details*
I have performed homologous lambda recombineering to introduce a *gfp* gene
variant into *E.coli* strain. I am not using any antibiotics as selection
markers to select for the recombinant, and I am only screening the
recombinant by a change in the phenotype (green fluorescence emitted by the
GFP). This change in the phenotype will be observed using a Blue LED
attached to an interference filter, the blue light will excite the GFP and
inturn the green fluorescence from the GFP will be observed by an absorbing
glass.

*Problem*
The limitation of homologous lambda recombineering is that it is a very
very rare event and the chance of occurance of recombination is 0-1%
(frequency is 1 in 10e6). Even if I have only  *one* positive recombinant,
the chance of finding it amongst the non-gfp cells (negative background) is
very low therefore for this purpose I want to optimise FACS so that I will
be able to screen and detect for the positive recombinant.

Another limitation is that of the signal intensity of the GFP, the
recombinant will have only one copy of the gfp gene integrated into the
chromosome and I am really not sure / do not know about the intensity if the
fluorescence with one copy of the gene.. Can't I get  around FACS if the
signal intensity is low?
I understand that it works quite well with eukaryotic cells but I want to
know how to optimize the instrument with bacterial cells. I have already
tried using FACS once, the controls worked fine but I faced a couple of
problems when I have actually performed lambda recombineering which I feel
will be worth mentioning.
1. the minimum number of cells (in terms of volume and the number of cells)
that can be injected into the machine at one time. Experimental error and
the chance of missing the fluorescing cell when too many cells are injected
at once are the concerns that I have.

2. the huge amount of negative background (non-fluorescing cells).
3. autofluorescence from the non-fluorescing cells and also interference
that might affect the fluroscence.

I would like to know if the model 'BD FACSCalibur System' can be used for
this pupose and if so how to optimize 'BD FACSCalibur System' for this
purpose (recommended settings) and will it be able to detect a
single fluorescing cell (recombinant) amongst many non-fluorescing cells?
How can I modulate (light source and other technical aspects) the instrument
model for the current purpose?
Thanks for your patience and I would be delighted if FACS will be useful for
my present purpose.

Thanks,
Deepika Gaddam
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