I have ligated my insert into an expression vector featuring a constitutive promoter. The current size is 11.5 kbp. My insert is a peptide antibiotic that also incorporates genes conferring immunity to the host. My expression host is Lactococcus cremoris. Nonetheless, I am unable to successfully transform the plasmid in the expression host despite multiple attempts. My troubleshooting tasks encompass: - The plasmid was analyzed using gel electrophoresis, PCR, and complete insert sequencing. - Various plasmid masses were utilized: 250 ng, 500 ng, and 800 ng. - Chilled conditions were upheld throughout all procedures. Following the electroporation pulse, cold recovery media was promptly added and maintained on ice for 5 minutes. - The empty plasmid was likewise transformed and exhibited robust growth on antibiotic plates. - Certain colonies manifest beyond 48-72 hours; however, those were identified as false positives. To my knowledge, 11.5 kbp is not so big construct to be transformed. Can you give any suggestions to transform the plasmid? Or any background explanation?
Thank you in advance.
What is the actual CFU/µg DNA transformation efficiency of your competent cells using the empty vector?
Amzad,
What is your, cloning efficiency in your bacteria? How many CFU/ug of DNA did you add? Which competent cell are you using? Your DNA concentration during electroporation might be to low I use 15-40 ug/ml or your pulse time and/or voltage might be to low dependent on cell type. I would recommend reviewing these papers, Chu G, Hayakawa H, Berg P. 1987. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15: 1311–1326 and Chu G, Hayakawa H, Berg P. 1987. Electroporation for the efficient transfection of mammalian cells with DNA. Nucleic Acids Res 15: 1311–1326. High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media. Have you tried other transfection methods/reagents?
If the empty plasmid is transforming well and the recombinant is not, then the problem is either with the DNA prep (but it sounds like you have looked at that) or the insert. Maybe you are still getting toxicity from the antibiotic despite expressing the immunity protein. Perhaps the immunity gene is not expressing well, or maybe you are just making too much of the antibiotic.
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