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outgrowth time andampicillin conc for low-copy numbe plasmid - (Mar/10/2011 )

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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.

-donny-

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.

-pito-

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?

-donny-

donny on Sat Mar 12 13:48:26 2011 said:


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.

-pito-

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

Regards,
p

pito on Sat Mar 12 16:40:33 2011 said:


donny on Sat Mar 12 13:48:26 2011 said:


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.

-pDNA-

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

Regards,
p

-pDNA-

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?

-claritylight-

pDNA on Sat Mar 12 17:37:05 2011 said:


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.


claritylight on Mon Mar 14 00:16:13 2011 said:


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.

-donny-

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

-pito-

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

-donny-
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