[DIYbio] Kanamycin Stability, & Mysterious Orange Colonies

Hi all,
I have a peculiar problem. I'm trying to select for a plasmid that:
A) Confers Kanamycin resistance
B) Bears a fusion protein containing wildtype GFP

I made up Kanamycin plates with 50ug/ml kan, which had recently been
made & filter-sterilised from kanamycin sulphate powder. The powder is,
I believe, about a year old, and has been stored in the fridge according
to packaging instructions. The stock solution (50ug/ml) was stored at
-20C once filtered into eppies.

The cultures were DH10B, isolated from a Top10 kit, which had been left
in LB in a fridge since February/March. I first broke them out into
fresh broth, then subcultured for transformation to get
exponential-phase cells.

I expected a pretty idiot-proof transformation (with PEG-3350 & MgSO4)
of E.coli DH10B, followed by selection of fluorescent green,
kanamycin-resistance cells. The transformation procedure is as per:
https://github.com/cathalgarvey/biohacking-protocols

Instead, I got growth on transformant-plated plates *and* on negative
control plates, which were treated identically but with only added T.E.
rather than DNA solution. Growth is still as single colonies after
spreading, rather than a lawn, but is pretty equally abundant on both
plates, indicating some background resistance to whatever concentration
of Kanamycin I'm using.

Weirder still, when lit by blue light and filtered with an orange
filter, nothing distinguishes the cells.. but when illuminated with a
cheap handheld UVA torch, many of the colonies on *both* plates are
bright fluorescent orange. The intensity of the orange appears to
increase with intermittant exposure to UV.

To ascertain whether the cells are expressing some orange pigment only
upon UV-induced quorum sensing (as it's very clearly a colony-specific
trait), I streaked an orange colony out beside a non-orange colony
(again on kanamycin TB plates), and the results indicated some genetic
factor: the orange colony lead to orange colonies, and the non-orange
colony lead to almost exclusively non-orange colonies, bar one.. which
might just be contamination from the other side.

So, I'm baffled. On the one hand, why is my kanamycin so terribly
non-selective? Any thoughts on powdered kanamycin stability?

On the other hand, what are these fluorescent orange cells? They are
identical to normal E.coli colonies to the naked eye, barring this
vibrant orange fluorescence.

I'm not even going to ask why my plasmid might be failing to
transform/select. It would seem I have bigger problems.

Thanks,
Cathal

--
www.indiebiotech.com
twitter.com/onetruecathal

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