[DIYbio] Re: Mechanism of Kanamycin Resistance (Was: Kanamycin Stability, & Mysterious Orange Colonies)

Forgot to include the relevant links. Here's the pUC57-Kan vector:
https://www.ncbi.nlm.nih.gov/nucleotide/387781650?report=genbank&log$=nucltop&blast_rank=1&RID=C3N8D5AG016
..and here's the resistance protein:
https://www.ncbi.nlm.nih.gov/protein/347984620

On 07/12/12 11:43, Cathal Garvey wrote:
> Hey Matt,
> This is late in coming, but while continuing troubleshooting I looked up
> the Kan-resistance gene in pUC57-Kan to determine the mechanism of
> resistance.
>
> Turns out that it's a phosphotransferase rather than a resistant
> ribosome; so, the resistance gene doesn't confer immunity, it instead
> degrades the antibiotic, like Ampicillin.
>
> Still lower chances of satellite colonies or contamination of course, as
> it kills non-resistant cells early in culture.
>
> Still though; there are resistant ribosomes out there, right? So why
> aren't they used for resistance genes instead? Ribosomal genes shouldn't
> be very large, and conferring resistance rather than destroying
> antibiotic could help prevent plasmid loss in later phase cultures?
>
> On 30/11/12 16:49, Matt Lawes wrote:
>> That should set you right.
>>
>> With the antibiotic resistances where the ribosome is modified you need time to make the resistant ribosomes. Kan is a particular classic case.
>>
>>> matt
>>
>> Sent from my Verizon Wireless 4G LTE DROID
>>
>>
>> -----Original message-----
>> 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>
>>> <http://www.indiebiotech.com>
>>> > twitter.com/onetruecathal <http://twitter.com/onetruecathal>
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>

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