Kanamycin Stability, & Mysterious Orange Colonies
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Cathal Garvey
Nov 29, 2012, 11:50:48 AM11/29/12
to met...@net.bio.net, met...@magpie.bio.indiana.edu, diybio
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
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
Xabier Vázquez Campos
Nov 29, 2012, 5:46:58 PM11/29/12
to diy...@googlegroups.com, met...@net.bio.net, met...@magpie.bio.indiana.edu
What is the final Kan concentration? Because 50 ug/mL for a stock is quite low.
Nathan McCorkle
Nov 30, 2012, 12:13:19 AM11/30/12
to diy...@googlegroups.com
streak a non-kanamycin plate and see if they fluoresce
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Nathan McCorkle
Nov 30, 2012, 12:24:56 AM11/30/12
to diy...@googlegroups.com
Also it looks like you didn't select a colony for your exponential growth culture... maybe fridge mutants?
On Nov 29, 2012 11:50 AM, "Cathal Garvey" <cathal...@gmail.com> wrote:
Nathan McCorkle
Nov 30, 2012, 12:33:37 AM11/30/12
to diy...@googlegroups.com
Does a concentrated solution of the kanamycn fluoresce? Maybe it degraded and only some bacteria have the right pump or something
http://www.sciencedirect.com/science/article/pii/0378109790900028
Xabier Vázquez Campos
Nov 30, 2012, 1:45:01 AM11/30/12
to diy...@googlegroups.com
Storage shouldn't be a problem, kanamycin is quite stable and can be stored as stock at fridge temperature for long term storage.
The concentration of the stock is usually 50 mg/mL, or at least in the order of mg/mL. The final concentration should be in ug/mL in the plates. So if your stock concentration was indeed 50 ug/mL, you barely had Kan in the plates.
The concentration of the stock is usually 50 mg/mL, or at least in the order of mg/mL. The final concentration should be in ug/mL in the plates. So if your stock concentration was indeed 50 ug/mL, you barely had Kan in the plates.
shreyans chordia
Nov 30, 2012, 7:32:07 AM11/30/12
to diy...@googlegroups.com, met...@net.bio.net, met...@magpie.bio.indiana.edu
Try to find the UV wavelength when the colonies give maximum Fluorescence and compare that value with already known fluorophores. That could give you a clue to source of that fluorescence (degraded kanmycin or some other chemical which may or may not be influenced by E. coli).
Cathal Garvey
Nov 30, 2012, 10:01:28 AM11/30/12
to diy...@googlegroups.com, Xabier Vázquez Campos, met...@net.bio.net, met...@magpie.bio.indiana.edu
Sorry, the concentration of the stock was mg/ml, the final concentration
was ug/ml.
On 29/11/12 22:46, Xabier V�zquez 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 escribi�:
> www.indiebiotech.com <http://www.indiebiotech.com>
> twitter.com/onetruecathal <http://twitter.com/onetruecathal>
> https://groups.google.com/d/msg/diybio/-/Nin12D8fqTMJ.
was ug/ml.
On 29/11/12 22:46, Xabier V�zquez Campos wrote:
> What is the final Kan concentration? Because 50 ug/mL for a stock is
> quite low.
>
> twitter.com/onetruecathal <http://twitter.com/onetruecathal>
>
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Matt Lawes
Nov 30, 2012, 10:11:08 AM11/30/12
to diy...@googlegroups.com
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
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 <cathal...@gmail.com>
To: "diy...@googlegroups.com" <diy...@googlegroups.com>
Cc: "Xabier Vázquez Campos" <xvaz...@gmail.com>, "met...@net.bio.net" <met...@net.bio.net>, "met...@magpie.bio.indiana.edu" <met...@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.
Cathal Garvey
Nov 30, 2012, 10:13:01 AM11/30/12
to diy...@googlegroups.com, Nathan McCorkle
The plates do fluoresce somewhat with blue or UV light, but it's a
blueish fluorescence. Bare plates have nothing like this vibrant orange
fluorescence.
Also, the fluorescence is limited to the colonies themselves, not the
area around them, suggesting it's not a kanamycin breakdown product.
I'm streaking out my old stocks of DH10B to see at what point could
contamination have occurred, and I'll streak on plain and Kan+ plates to
see the difference. So far, it does look like the stock I was using this
month has contamination built-in, possibly the older stocks are clear..
I'll have to incubate another few days and see.
On 30/11/12 05:33, Nathan McCorkle wrote:
> Does a concentrated solution of the kanamycn fluoresce? Maybe it
> degraded and only some bacteria have the right pump or something
>
> http://www.sciencedirect.com/science/article/pii/0378109790900028
>
> On Nov 30, 2012 12:24 AM, "Nathan McCorkle" <nmz...@gmail.com
> <mailto:diybio%2Bunsu...@googlegroups.com>. For more
> Learn more at www.diybio.org <http://www.diybio.org>
> <mailto:diybio%2Bunsu...@googlegroups.com>.
blueish fluorescence. Bare plates have nothing like this vibrant orange
fluorescence.
Also, the fluorescence is limited to the colonies themselves, not the
area around them, suggesting it's not a kanamycin breakdown product.
I'm streaking out my old stocks of DH10B to see at what point could
contamination have occurred, and I'll streak on plain and Kan+ plates to
see the difference. So far, it does look like the stock I was using this
month has contamination built-in, possibly the older stocks are clear..
I'll have to incubate another few days and see.
On 30/11/12 05:33, Nathan McCorkle wrote:
> Does a concentrated solution of the kanamycn fluoresce? Maybe it
> degraded and only some bacteria have the right pump or something
>
> http://www.sciencedirect.com/science/article/pii/0378109790900028
>
> On Nov 30, 2012 12:24 AM, "Nathan McCorkle" <nmz...@gmail.com
> <mailto:nmz...@gmail.com>> wrote:
>
> Also it looks like you didn't select a colony for your exponential
> growth culture... maybe fridge mutants?
>
> On Nov 29, 2012 11:50 AM, "Cathal Garvey" <cathal...@gmail.com
>
> Also it looks like you didn't select a colony for your exponential
> growth culture... maybe fridge mutants?
>
> On Nov 29, 2012 11:50 AM, "Cathal Garvey" <cathal...@gmail.com
> www.indiebiotech.com <http://www.indiebiotech.com>
> twitter.com/onetruecathal <http://twitter.com/onetruecathal>
>
> twitter.com/onetruecathal <http://twitter.com/onetruecathal>
>
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Cathal Garvey
Nov 30, 2012, 11:31:52 AM11/30/12
to diy...@googlegroups.com
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 <cathal...@gmail.com>*
> To: *"diy...@googlegroups.com" <diy...@googlegroups.com>*
> Cc: *"Xabier Vázquez Campos" <xvaz...@gmail.com>,
> "met...@net.bio.net" <met...@net.bio.net>,
> "met...@magpie.bio.indiana.edu" <met...@magpie.bio.indiana.edu>*
> Sent: *Fri, Nov 30, 2012 10:01:44 EST*
> Subject: *Re: [DIYbio] Re: Kanamycin Stability, & Mysterious
> > Learn more at www.diybio.org <http://www.diybio.org>
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
>
>
>
> -----Original message-----
>
> *From: *Cathal Garvey <cathal...@gmail.com>*
> To: *"diy...@googlegroups.com" <diy...@googlegroups.com>*
> Cc: *"Xabier Vázquez Campos" <xvaz...@gmail.com>,
> "met...@net.bio.net" <met...@net.bio.net>,
> "met...@magpie.bio.indiana.edu" <met...@magpie.bio.indiana.edu>*
> Sent: *Fri, Nov 30, 2012 10:01:44 EST*
> Subject: *Re: [DIYbio] Re: Kanamycin Stability, & Mysterious
> > ---
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Matt Lawes
Nov 30, 2012, 11:49:58 AM11/30/12
to diy...@googlegroups.com
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.
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-----
shamrock
Nov 30, 2012, 12:12:29 PM11/30/12
to diy...@googlegroups.com, met...@net.bio.net, met...@magpie.bio.indiana.edu
Hi Cathal,
I had a problem with Kan selection a year or so ago where I was seeing lots of pappilation (cells mutating to Kan resistance). At the time I was using 50 ug/ml which was/is the recommended dose. I tested several concentrations of Kan and a dose to 100 ug/ml in the plates seemed to solve the problem. Now when transforming I use 100ug/ml in the plates and that seems to provide a strong enough selection that I don't see any spontaneous Kan resistant colonies.
Your DH10B stock seems pretty old, and you may have inadvertantly selected for cells that were resistant to low levels of Kan. I usually only keep cells on LB plates in the fridge for a couple of weeks at most-anerobic stabs last longer, a couple of months at least. For longer term storage you really need -80 or vapor phase liquid Nitrogen, or even lyophilization.
Cells that are metabolicly active change over time - it's the nature of the beast!
Tom
Cathal Garvey
Dec 7, 2012, 6:43:35 AM12/7/12
to diy...@googlegroups.com
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?
>> Cc: *"Xabier V�zquez Campos" <xvaz...@gmail.com>,
>> > Learn more at www.diybio.org<http://www.diybio.org> <http://www.diybio.org>
twitter.com/onetruecathal
PGP Public Key: 3B3DE808 (Verify before serious use)
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?
>> "met...@net.bio.net" <met...@net.bio.net>,
>> "met...@magpie.bio.indiana.edu" <met...@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.
>>
>> "met...@magpie.bio.indiana.edu" <met...@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 V�zquez 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
>> escribi�:
>> > 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
>> > ---
>> > You received this message because you are subscribed to the Google
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>> > To post to this group, send email to diy...@googlegroups.com.
>> > To unsubscribe from this group, send email to
>> > diybio+un...@googlegroups.com.
>> > Visit this group at http://groups.google.com/group/diybio?hl=en.
>> > To view this discussion on the web visit
>> > https://groups.google.com/d/msg/diybio/-/Nin12D8fqTMJ.
>> > For more options, visit https://groups.google.com/groups/opt_out.
>> >
>> >
>>
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PGP Public Key: 3B3DE808 (Verify before serious use)
Cathal Garvey
Dec 7, 2012, 6:44:59 AM12/7/12
to diy...@googlegroups.com
Forgot to include the relevant links. Here's the pUC57-Kan vector:
https://www.ncbi.nlm.nih.gov/nucleotide/387781650?report=genbank&log$=nucltop&blast_rank=1&RID=C3N8D5AG016
..and here's the resistance protein:
https://www.ncbi.nlm.nih.gov/protein/347984620
https://www.ncbi.nlm.nih.gov/nucleotide/387781650?report=genbank&log$=nucltop&blast_rank=1&RID=C3N8D5AG016
..and here's the resistance protein:
https://www.ncbi.nlm.nih.gov/protein/347984620
Matt Lawes
Dec 7, 2012, 8:16:40 AM12/7/12
to diy...@googlegroups.com
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
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
-----Original message-----
>>> Cc: *"Xabier Vázquez Campos" <xvaz...@gmail.com>,
>>> "met...@net.bio.net" <met...@net.bio.net>,
>>> "met...@magpie.bio.indiana.edu" <met...@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:
>> Learn more at www.diybio.org<http://www.diybio.org&g
Cathal Garvey
Dec 7, 2012, 8:21:13 AM12/7/12
to diy...@googlegroups.com
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.
>>>> Cc: *"Xabier V�zquez Campos" <xvaz...@gmail.com>,
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.
>>>> "met...@net.bio.net" <met...@net.bio.net>,
>>>> "met...@magpie.bio.indiana.edu" <met...@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 V�zquez 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
>>>> escribi�:
>>>> "met...@magpie.bio.indiana.edu" <met...@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 V�zquez 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
Matt Lawes
Dec 7, 2012, 8:27:16 AM12/7/12
to diy...@googlegroups.com
Yep I agree with your line of thought. I think if you look at the various resistances that tetracycline has the greatest variety - everything from effluent pumps to modifying enzymes to ribosomal mutants. If I recall correctly (big IF, lol) the level of resistance conferred by ribosomal mutants is lower than pumps / modifying enzymes. So that may be part of the reason for resistance cassette choice for plasmids. I'll dig a bit though to confirm......
-----Original message-----
>>>> Cc: *"Xabier Vázquez Campos" <xvaz...@gmail.com>,
>>>> "met...@net.bio.net" <met...@net.bio.net>,
>>>> "met...@magpie.bio.indiana.edu" <met...@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:
>>>>&n
Matt Lawes
Dec 7, 2012, 8:31:06 AM12/7/12
to diy...@googlegroups.com
Lol .... there's a typo for the ages!. .... that should read EFFLUX pump!
Blushes (stupid autotyper!) and goes back to his coffee......
Blushes (stupid autotyper!) and goes back to his coffee......
>matt
Sent from my Verizon Wireless 4G LTE DROID
-----Original message-----
Nathan McCorkle
Dec 7, 2012, 1:53:03 PM12/7/12
to diybio
On Fri, Dec 7, 2012 at 3:43 AM, Cathal Garvey <cathal...@gmail.com> 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?
Seems like resistant ribosomes would be worse for overall expression levels, since there'd be competing ribosomes, some of which would be generating truncated proteins, effectively wasting cellular resources
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