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

From: Cathal Garvey <cathalgarvey@gmail.com>
To: "diybio@googlegroups.com" <diybio@googlegroups.com>
Sent: Fri, Dec 7, 2012 06:45:18 EST
Subject: [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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