Hi,
I am just starting to work with S.pombe and facing some problems with
the expression of my genes fused GFP, using the nmt1 promotor: in
thiamine -, my cells are so green that I cannot see anything. But when
I add thiamine, the signal localises to mitochondria, whatever my
genes, and even with the empty GFP-vector.
I made a series of tests to try to understand where the problem was
from:
- I grew some WT cells in EMM + thiamine, and not transformed with any
DNA. I read somewhere that thiamine can autofluoresce if converted into
another chemical form.
- I made some fresh thiamine and made sure to keep it in the dark. It
was ordered from Sigma (T4625) and autoclaved.
- Between T+ and T- conditions, I washed the cells at least 3 times
with EMM + supp
- I tried to grow the cells in 5ml to saturation first, and then in
50-100ml
- I transformed pREP41Ngfp that I received from 2 different labs, just
in case the one that I used for my constructions had a mutation in the
nmt1 promoter.
- I used a range of thiamine concentrations from 0.015 to 15uM
...
And each time, I got the same mitochondrial signal (MitoTracker
co-localises)... unfortunately, I've got no other gfp-construct to use
as positive control that the one that made myself. I assume this must
be a background signal due to thiamine rather that GFP, but despite
spending days on Medline, I could not find any paper describing this in
yeast. I am sure that I am doing something very silly without realising
but I am running out of ideas and get desperate for some help.
Has anyone ever got this type of mitochondrial signal? And if this is a
background, why am I not seeing the real GFP signal anyway?
Thank you for any information you could give me,
Isabelle
Isabelle Jourdain, PhD
Post-doctoral fellow
Private bag 11222
Massey University
Palmerston North
New Zealand
Tel: (00-64)-350-5518
Internal: -2208
