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outgrowth time andampicillin conc for low-copy numbe plasmid


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15 replies to this topic

#1 donny

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Posted 10 March 2011 - 06:23 PM

I am screening a mutant library of my gene in a low-copy number (~5) plasmid. I'm just wondering if I should reduce the ampicillin concentration for my media (100ug/mL at the moment) and increase the outgrowth time before plating to increase the chance of recovering more mutants.

Edited by donny, 10 March 2011 - 09:23 PM.


#2 pito

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Posted 11 March 2011 - 12:07 PM

I dont know the details of what you are doing, but often they incubate the fresh mutans in fresh medium without any antibiotic at all the first hour or something like that. And then they transfer the cells on medium with antibiotics.

But I dont know what mutants you are speaking of or what you are really doing.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#3 donny

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Posted 12 March 2011 - 05:48 AM

I'm performing directed evolution. So, I clone the mutant genes into a very low-copy number plasmid to get a library. Then, I transform them into competent cells and grow them in SOC for 1h. I'll then plate them out on LB-amp agar plates. I'm just wondering if I should reduce the ampicillin concentration (down from 100ug/mL to 50 or lower) for the second step because since the copy-number is really low (~5), I'm afraid some of the transformed cells will die from insufficient b-lactamase and I'll lose some of my variants which would reduce the diversity of my library.

In short, would you reduce the ampicillin concentration and increase the outgrowth time to recover more transformed cells if you're working with a very low-copy number plasmid?

Edited by donny, 12 March 2011 - 05:50 AM.


#4 pito

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Posted 12 March 2011 - 08:40 AM

I'm performing directed evolution. So, I clone the mutant genes into a very low-copy number plasmid to get a library. Then, I transform them into competent cells and grow them in SOC for 1h. I'll then plate them out on LB-amp agar plates. I'm just wondering if I should reduce the ampicillin concentration (down from 100ug/mL to 50 or lower) for the second step because since the copy-number is really low (~5), I'm afraid some of the transformed cells will die from insufficient b-lactamase and I'll lose some of my variants which would reduce the diversity of my library.

In short, would you reduce the ampicillin concentration and increase the outgrowth time to recover more transformed cells if you're working with a very low-copy number plasmid?


I would grow them longer in the SOC, maybe and hour and a half.

And indeed, since its a really low copy number it might be ok to reduce the concentration a bit.

You will just need to add one extra step in your protocol: putting them on new plates with the appropriate concentration.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#5 pDNA

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Posted 12 March 2011 - 09:34 AM

you can reduce the Amp concentration to 50 g/mL for low copy plasmids.

Regards,
p


I'm performing directed evolution. So, I clone the mutant genes into a very low-copy number plasmid to get a library. Then, I transform them into competent cells and grow them in SOC for 1h. I'll then plate them out on LB-amp agar plates. I'm just wondering if I should reduce the ampicillin concentration (down from 100ug/mL to 50 or lower) for the second step because since the copy-number is really low (~5), I'm afraid some of the transformed cells will die from insufficient b-lactamase and I'll lose some of my variants which would reduce the diversity of my library.

In short, would you reduce the ampicillin concentration and increase the outgrowth time to recover more transformed cells if you're working with a very low-copy number plasmid?


I would grow them longer in the SOC, maybe and hour and a half.

And indeed, since its a really low copy number it might be ok to reduce the concentration a bit.

You will just need to add one extra step in your protocol: putting them on new plates with the appropriate concentration.



#6 pDNA

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Posted 12 March 2011 - 09:37 AM

i'm no specialist on directed evolution ...but will incubation after transformation give you evolved clones in that short timespan?

Regards,
p

#7 claritylight

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Posted 13 March 2011 - 04:16 PM

I am just curious why to use a low-copy number plasmid. What is your reasoning for choosing that over a higher copy-number plasmid?

#8 donny

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Posted 13 March 2011 - 05:58 PM

i'm no specialist on directed evolution ...but will incubation after transformation give you evolved clones in that short timespan?

Regards,
p


What I did was I performed error-prone pcr to generate a library of mutant genes. Then, I clone them into the plasmid and transform them. The transformed cells will then be subjected to screening and/or selection to isolate the evolved clones that meet your requirement. I'm not sure if that answers your question.


I am just curious why to use a low-copy number plasmid. What is your reasoning for choosing that over a higher copy-number plasmid?


I'm not too sure either. I basically followed a published protocol. My guess is that the protein is an integral membrane protein so overexpression might compromise the viability of the cells.

#9 pito

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Posted 14 March 2011 - 10:03 AM

The gene you clone, its on your plasmid only then I hope or?

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#10 donny

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Posted 14 March 2011 - 05:38 PM

Yes, I knocked out the gene from e coli and plasmid-complemented the knock-out strain.

#11 Rsm

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Posted 15 March 2011 - 12:26 AM

You should grow your cells at room temperature for extremely low-copy number plasmids. Otherwise, the growth rate of your cells will be higher than the copy rate of your plasmid, and you'll have problems. I would also use Carbenicillin instead of Ampicillin, it is more stable, and will reduce your background (and thus increase your yield) immensely.
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#12 donny

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Posted 15 March 2011 - 05:53 PM

@Rsm

What kind of problems are you referring to?

#13 Rsm

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Posted 16 March 2011 - 12:29 AM

No growth of bacteria, or growth of bacteria without plasmid (to avoid that you should use Carbenicilin). It would seem like they do not survive Ampicillin or maintain the plasmid.
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#14 donny

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Posted 16 March 2011 - 01:02 AM

Fortunately, no problem of such yet, even with 100ug/mL ampicillin. Plasmid yield has been reasonable with 10mL LB. So, we can simply replace ampicillin with carbenicilin for plasmids with Amp resistance?

#15 Rsm

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Posted 16 March 2011 - 01:41 AM

Yes, you can just replace it, also using the same concentrations (50 or 100mg/l). It is more stable at 37C, and thus your concentration of antibiotic will be maintained for longer time.
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