The missing plasmid...

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderators: Leonid, amiradm, BioTeam

Post Reply
SororSaudade
Death Adder
Death Adder
Posts: 65
Joined: Thu Nov 16, 2006 3:54 pm
Location: Portugal

The missing plasmid...

Post by SororSaudade » Tue Aug 14, 2007 12:24 pm

Hello all!

I was trying for ages to transform some Agrobacterium cells and never manage to get any colonies. When I finally got one colony on the transformation plate I did a PCR on it and it amplified very well a fragment the size I was expecting.
After this I grew these bacteria on solid and liquid medium with the proper antibiotics and they grew very well. When I did a new PCR (with negative and positive controls) in these new cultures I could never get any kind of amplification.
I thought the antibiotic might be damaged and did a new one, but got the same kind of results (even with higher concentrations of it).

Has this ever happened to anybody? Can bacteria get resistant to some antibiotic without the resistance gene?

If some one could help be with this, I would really appreciate it. :)

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Tue Aug 14, 2007 5:26 pm

Spontaneous resistance is not impossible. It depends on the antibiotic though. I am not familiar with Agrobacterium though, so it is impossible to judge how frequent it is.

Which antibiotic are you using?
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

SororSaudade
Death Adder
Death Adder
Posts: 65
Joined: Thu Nov 16, 2006 3:54 pm
Location: Portugal

Post by SororSaudade » Tue Aug 14, 2007 8:05 pm

I'm using kanamycin.

Agrobacterium are naturally resistant to rifampycin... and I also add this to the medium.

But can you explain me how do they get this spontaneus resistance?

Thanks :)

User avatar
canalon
Inland Taipan
Inland Taipan
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada

Post by canalon » Wed Aug 15, 2007 2:31 am

I was not sure for kanamycine, but if you look up the mode of acion you will see that it binds the ribosome and prevents elongation. Like many aminoglycoside with similar mode of action (and rifampicine, which is not an aminoglycoside, but also blocks the ribosome) resistance can be acquired by some mutations in the target which will keep the function intact.

With E. coli this kind of mutation appear in 1 in 10^9 or 10^10 bacteria, but in case of selection that might be enough for you to have picked the wrong one. as for the false positive for the first PCR, I suppose you had a negative control... but did you re-isolate the colony before doing the PCR? Otherwise remeber that you use quite a lot of your PCR target to make your transformation, and you need very little to get a good amplification...
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

SororSaudade
Death Adder
Death Adder
Posts: 65
Joined: Thu Nov 16, 2006 3:54 pm
Location: Portugal

Post by SororSaudade » Wed Aug 15, 2007 10:08 am

wow... thanks for the explanation :)

I already knew I have to do it all over again... just wanted to know what might have happened

Thank you once again

Post Reply

Who is online

Users browsing this forum: No registered users and 1 guest