Hi,
I have been working on Bacillus thuringiensis, when i amplify the Cry1 gene using genomic DNA i usually get a positive result. i need to know whether these Cry gene's are Genomic or Plasmid borne ?. I have never tried amplification using Plasmids. Has anyone tried to amplify Cry gene on plasmid isolated from Bacillus thuringiensis.
In all known examples, the cry genes are located on large plasmids, often over 100 kb in size. I have yet to see a single report of a chromosomal cry gene in Bacillus thuringiensis (Bt). Because the plasmids are so large, genomic DNA isolation protocols yield a mix of both chromosomal and plasmid DNA fragments. Isolating purified plasmid DNA is technically difficult, although possible. Most Bt researchers don't worry about it and simply work with genomic DNA preps. Regardless, we would very confidently predict that your gene is located on a megaplasmid.
Dear Manohar
Two solutions can be studied. First of all, get a cured strain from B. thuringenesis by successive transfere of the strain in BHI broth supplemented with 1 ug per 10mL ethidium bromide and make plasmid profile of both mutant and wild strain. You will see no plasmids in mutant strain and in its chromosomal DNA make PCR for cry1 gene.
Second and easier : Extract only chromosomal DNA by Maniatis Protocol and run a PCR using cry1 primer and in another experiment extract only plasmid DNA by certain Maniatis or Promega Protocol and run PCR using a specific primer for cry1 gene and after agarose gel electrophresis,look to bands obtained and compare with standard DNA and good luck
In all known examples, the cry genes are located on large plasmids, often over 100 kb in size. I have yet to see a single report of a chromosomal cry gene in Bacillus thuringiensis (Bt). Because the plasmids are so large, genomic DNA isolation protocols yield a mix of both chromosomal and plasmid DNA fragments. Isolating purified plasmid DNA is technically difficult, although possible. Most Bt researchers don't worry about it and simply work with genomic DNA preps. Regardless, we would very confidently predict that your gene is located on a megaplasmid.
Usually these are genomic DNA borne, because the sigma 54 factor encoded by the CRY genes regulates many nitrogen and carbon metabolic pathways in the bacteria. These are basically not pesticidal or insecticidal and its only after they have been partly digested by enzymes like trypsin in the insect gut, that these show insecticidal properties.
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