Hi everyone,
I am looking for advice on how to swap regions on a plasmid I am working with. Basically I want to remove the Amp resistance gene from under the control of the Amp promoter and move it to another region of the plasmid where it will be under the control of an inducible promoter. If I just wanted to delete the gene I would simply linearize all of the plasmid apart from the region containing that bit. However, I want to delete the amp gene and promoter and clone the amp gene in a different region 1000 BP upstream of that.
Is there an efficient way to do this?
Thanks in advance
Ed
This will end up requiring 3 fragments to be generated by PCR:
1. From upstream of the AmpR cassette (but not including it) to the inducible promoter + ribosome binding site associated with it.
2. The AmpR coding sequence
3. From downstream of the AmpR cassette (again not including it) up to the 3' end of the first fragment.
Then assemble it using a one-pot assembly method.
How you design the primers for this will depend on which assembly method you end up using.
As examples, there's:
1. NEBuilder: https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/nebuilder-hifi-dna-assembly
NEBuilder is basically a modified version of "Gibson Assembly"
2. Golden Gate cloning:
https://www.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly
(Caveat here being that it requires some alterations if your fragments contain any unwanted Type IIS restriction sequences)
There's also a lot of methods that are very similar to NEBuilder in terms of primer design like In-Fusion: https://www.takarabio.com/products/cloning/in-fusion-cloning
I would recommend you go with what suggested by Alexandra otherwise if you do not need to 1000bp that is in the middle you can use Lamda red. linearize the plasmid with 2 restriction enzymes that remove the Amp gene. Amplify the Amp gene with homology to the region downstream of the original amp region (to religate the plasmid) and homology to your inducible promoter (I am assuming this is already on the plasmid?) and cotransform into a lamda red expressing strain, plate on amp plates with induction for your promoter then check for correct colonies.
Technical cloning issues aside, if your goal is to have inducible or regulated amp resistance, I think you are going to find that the basal level of expression is probably sufficiently high in the uninduced state to confer amp resistance. This is especially true if you are using a high copy number plasmid. Just something to be aware of.
Hi Michael J. Benedik thanks for your answer. I'm trying to select for strains with induced Ampr expression. I also have a variety of other antibiotics I could use too. Do you know if the basal expression is similarly high in other prokaryotic antibiotics and if so would it be viable to increase antiobiotic concentration so to select for the strain with the highest induction?
Thanks again
It is more a question of mode of action. Many of the antibiotic resistance genes, being enzymes, do not require very high expression to generate significant resistance. I know for example that a single copy of the amp-r gene (beta-lactamase) on the chromosome is sufficient to generate pretty high level resistance. And since most plasmids are 10-100-fold higher in copy number, you are already at the upper limit.
There are a few antibiotic resistance genes where you can generate higher resistance by amplification to increase expression.
What is the experiment you are trying to do, I wonder if there is a better approach to the experiment you want to do?
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