how to create an auxotrophic bacteria

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Felixke
Felixke's picture
how to create an auxotrophic bacteria

Hallo all,
I was reading about IVET and I wonder how they mutate a certain bacteria so that it lacks a certain gene (from wich you want to research the promotor).
How can you influence the chromosome of that bacteria so that the gene that you want to delete is removed from that bacteria.
I know they use a plasmid for this but how does this work? Does this plasmid then have all the same dna (chromosome) as the bacteria expect the gene you want to delete or?
greetins

varsha
varsha's picture
Hello Felixke.

Hello Felixke.
 IVET utlilizes an auxotroph (failure to grow in absence of a nutrient such a purine) to design plasmid which would carry the enzyme purA (adenylosuccinate synthetase) and other reporters (lacZ)  with it. You could now attach fragments of genome as promoter and use this library in vivo to look for lacZ expression. Several Salmonella virulence mutants were found this way. In the screen you look for rxpression of lacZ (or other reporter) or replication and survival  in the mouse suggesting a role in virulence. I found this old review to be pretty useful. 
As far as generation of a certain mutantion in a bacterium is concerned, it is usually done in the following manner with some variation. If you were looking for a purine auxotroph, you could use a chemical or UV mutagen or transposon mediated insertion on a bunch of bacteria and plate them on plates containing  purine. You could make a replica of the colnies on a  plate lacking purine. Colonies which fail to gro on purine are auxotrops and can be pickes from purine containing plates.
You could also perform targeted mutagenesis by homologoues replacement of nomal gene with a mutated copy.
Let me know if you need more information on these specific methods.
 
Varsha

Felixke
Felixke's picture
Hallo varsha,

Hallo varsha,
thanks for the information and the text.
However, I have one more question: what do you mean with homologoues replacement?
I know about homologoues recombinations, but how does this fit in this story?

varsha
varsha's picture
Hi Felixxe

Hi Felixxe
Homolous replacement is the exchange of a piece of DNA (e.g in the genome)  with another piece of DNA (e.g on a plasmid) by homolous recombination which is made possible by homology at the two ends of the DNA. It is also called allelic exchance.
If you wanted to remove the wild type allele of gene ABC with a mutated or truncated copy, you would introduce a plasmid with gene AxD where part B of the sequence is missing and replaced by a selection marker such as Amp reistance. If screening through a good numberof  trasformed colonies, a few of them would have had the homologus replacement and a ampR in the genome. This process is made easier by use of other suitable markers. Some of the following papers may make it easier for you to understand.

Gene replacement in Mycobacterium

Gene replacements in Gram positive bacteria

Felixke
Felixke's picture
Ok, thats a good explenation.

Ok, thats a good explenation. I'll check those papers for sure.
thanks a lot.