Re: [DIYbio] Re: Kanamycin Stability, & Mysterious Orange Colonies

From: Cathal Garvey <cathalgarvey@gmail.com>
To: "diybio@googlegroups.com" <diybio@googlegroups.com>
Sent: Fri, Nov 30, 2012 11:32:18 EST
Subject: Re: [DIYbio] Re: Kanamycin Stability, & Mysterious Orange Colonies

If it's straight contamination (which I'm taking as the most likely
answer), then it's not just the orange guys: I've also got "plain
looking" cells which look similarly like E.coli, and turn up on
negative and positive plates.

I'm currently running two plates from an old stock of DH10B: one
without selection, one with. If I get growth on the clear plate and
none on the Kan plate, I'll be happy, and will re-start the culture
from a colony.

You're probably right about the outgrowth: I knew some was necessary
after transformation with Kan and other bacteriocidal antibiotics, but
I was only giving them about 30 mins... and my incubator is only 30C.
Might want to leave them 1:30 hours next time: will be repeating
transformation next week hopefully.

Thanks for the advice guys!

On Fri 30 Nov 2012 15:11:08 GMT, Matt Lawes wrote:
> Maybe your DH10B stock isn't quite so.
> Perhaps a contaminant with low innate KanR is in the LB stock. I would
> make some plates with higher kan concentration .... 100, 150, ug/ml
> and see what kills the orange guys.
>
> I would also reisolate the e coli from your stock on plates without
> kan ..... looking for the not orange colonies - sounds like one more
> round beyond what you've done will give you a pure stock.
>
> Kanamycin also needs time ( an hour of outgrowth without Kan) for
> phenotypic expression after transformation of the plasmid. Didn't look
> at your protocol to see of you have that down.
>
> Best,
>
> >matt
>
> /Sent from my Verizon Wireless 4G LTE DROID/
>
>
> -----Original message-----
>
>     *From: *Cathal Garvey <cathalgarvey@gmail.com>*
>     To: *"diybio@googlegroups.com" <diybio@googlegroups.com>*
>     Cc: *"Xabier Vázquez Campos" <xvazquezc@gmail.com>,
>     "methods@net.bio.net" <methods@net.bio.net>,
>     "methods@magpie.bio.indiana.edu" <methods@magpie.bio.indiana.edu>*
>     Sent: *Fri, Nov 30, 2012 10:01:44 EST*
>     Subject: *Re: [DIYbio] Re: Kanamycin Stability, & Mysterious
>     Orange Colonies
>
>     Sorry, the concentration of the stock was mg/ml, the final
>     concentration
>     was ug/ml.
>
>     On 29/11/12 22:46, Xabier Vázquez Campos wrote:
>     > What is the final Kan concentration? Because 50 ug/mL for a stock is
>     > quite low.
>     >
>     > El viernes, 30 de noviembre de 2012 03:50:48 UTC+11, Cathal
>     escribió:
>     >
>     >     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
>     >     <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 <http://www.indiebiotech.com>
>     <http://www.indiebiotech.com>
>     >     twitter.com/onetruecathal <http://twitter.com/onetruecathal>
>     >
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