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

Oh it makes great sense, I'm just bothered that non-degrading genes
aren't employed instead.

The issue of plasmid loss in late stage culture isn't as much of an
issue with Kanamycin as with Ampicillin, because in serial culture the
non-plasmid-bearing cells will get killed upon re-introduction to
Kanamycin, and will not survive long enough for their resistant fellows
to destroy the antibiotic.

However, I could see it reducing plasmid yield, which can be
commercially significant, especially if you're making small batches. If
cells were immune, but did not degrade antibiotics, then they couldn't
provide "herd immunity" to their plasmid-free relatives, and the
proportion of plasmid-bearing cells in end stage culture would be higher.

I gather with some popular plasmids, the rate of plasmid loss can be
really significant; easily 20% reductions in a few generations without
selection, and destruction of antibiotics can be total within 5 hours or
less of culture, particularly with beta-lactamase and Ampicillin.

Anyways, just thinking aloud. Would be nice to see if anyone's tried
offering alternative ribosomal genes instead of phosphotransferases, and
what the result was. Possibly, it would result in crippled growth rate
and carry significant disadvantages of its own as the cell wastes energy
on "conventional" ribosomes that promptly get inactivated by Kanamycin.

On 07/12/12 13:16, Matt Lawes wrote:
> Hi Cathal,
>
> Thanks for doing your homework! I'm an old fart of a scientist and my memory isn't what it was ......
> So upon reflection ..... here's a better explanation of the need for phenotypic expression of some drug resistances. Versus others. Look at the mechanism of action of the antibiotic - kanamycin inhibits ribosomes, while ampicillin inhibits cell wall synthesis. So in the first case, you can't make the resistance phosphotransferase if ribosomes are inhibited. Make better sense? >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, 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
>>>> >
>>>> > --
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>>>> <http://www.indiebiotech.com>
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