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Confusion about genetic engineering ?!?

i am really confused about how marker genes are added to recombinant DNA in vectors. One source sasy taht the vector has two anitbitic resistance genes e.g gene for ampicillin and tetracillin. When the gene is inserted into the vector one of the anitbiotic resistance gene is cut and the useful gene like insulin is inserted, then both anitibiotics are appiled to test which vector has the useful gene. The NAS book says how antibiotic resistance genes are added to plasmids and later antibiotic is used to test the vector with the gene. Don't plasmids already have antibiotic resistance or is it inserted. Please help solve this confusion!!!! :s-smilie:

Reply 1

Wangers

OK, you're confused.

The antibiotic resistance gene is added in the usual way with Restriction endonucleases and then the sticky ends are bound with Ligase.

Then the idea is to get the gene you want across this gene. So that its no longer immune to the antibiotic. Then use Replica plating to trace the ones with your desired gene from the master plate.

This is because some bacteria will take up the untransformed plasmid(antibiotic resistance) and some will take up the transformed plasmid (gene, no resistance).

Reply 2

$!Littledevil!$

so does that mean both anitbotic resistance gene and gene of interest is added on the same plasmid and if the gene of interest is in the plasmid the antibiotic resistance gene won't work because its been cut. But if the gene if interest is nt in the plasmid the antibiotic resistance gene added before is unaffected?

Reply 3

iainmacn

my understanding is that the plasmid has resistance to antibiotic A and B and when the new gene is inserted it messes up one of them. Let's say resistance to B survives but A doesn't

So bacteria with a transformed plasmid inserted will have resistance to B but not A, so will die on one antibiotic but not the other

HTH