I am unable to clone in E.coli a gene for restriction endonuclease that is supposed to be used in an eukaryotic organism. The reason is that the strain of E.coli (DH5alpha) has no protection from this endonuclease. I have to clone this gene in such a way that it can express the active enzyme in a eukaryotic cell. Can you suggest a way to overcome the toxicity of the endonuclease, or may be a different strategy to clone this gene?
The promoter needs to absolutely silent. Even some eukaryotic promoters are active in E.coli.
Copy number control can also help:
-Tightly regulated, high-level expression from controlled copy number vectors based on the replicon of temperate phage N15.
Mardanov AV, Strakhova TS, Smagin VA, Ravin NV.
Gene. 2007 Jun 15;395(1-2):15-21. Epub 2007 Jan 20.
-Bacterial expression system with tightly regulated gene expression and plasmid copy number.
Bowers LM, Lapoint K, Anthony L, Pluciennik A, Filutowicz M.
Gene. 2004 Sep 29;340(1):11-8.
-Copy-control tightly regulated expression vectors based on pBAC/oriV.
Wild J, Szybalski W.
Methods Mol Biol. 2004;267:155-67.
-Tightly regulated vectors for the cloning and expression of toxic genes.
Anthony LC, Suzuki H, Filutowicz M.
J Microbiol Methods. 2004 Aug;58(2):243-50.
The simplest approach would be to insert a synthetic intron into your cDNA. This should prevent expression in prokaryotes which cannot splice the intron out, especially if the intron is designed to encode several stop codons. Another feature that might help is to include strong transcriptional pause/stop sites 5' to your eukaryotic promoter such as the rrnB sites used in the pENTR/DTOPO vectors.
we have been succesful in cloning toxic gene products using Epicentre's Copycutter E.coli. They have the advantage that you can just use your normal expression plasmids and induce higher numbers as necessary. growing at room temp instead of 37° helps, too. this will both reduce copy number and nonspecific expression
The first method:
The methylase gene of the endonuclese is first cloned in E. coli cells using a plasmid vector, and then the endonuclease gene is cloned in E. coli cells harboring the methylase gene.
The second method:
If there is no site of the endonuclese in the genome of lambda phage, the endonuclease gene can be cloned using a lambda phage vector in E. coli cells. Plaques of clones carrying the gene can be found by DNA-DNA hybridization using a labeled DNA sequence of part of the gene.
I know it's old topic, but maybe somebody will need some other way to clone CDS of toxic protein:
Article A novel method for cloning of coding sequences of highly tox...
Lowering culture temperature does not work for more toxic constructs. Lowering copy number in combination with a richer medium may work.
Introducing an intron at an appropriate site is indeed a good solution. See Article Construction of cloning‐friendly mini‐genes for mammalian ex...
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