Hi all,
I'm working on transforming E. coli with plasmid in a mixture of linear and circular form. What happened to the linear form once uptake? Do cells ligate them directly? Or cell DNase degrade them to some extent and then ligate? Or both? Thanks!
Hello Chun-Chen Yao ,
Regarding your questions. When you transform the cells with a vector, with a mixture of linear and circular plasmid form, only the circular form will be useful for the bacteria (for the resistance Ab as an example), and the linear form probably will be degraded. The linear plasmid form ligation inside the cytoplasm is unlikely, is more probably that de linear dsDNA will be degraded outside. You have to think that linear dsDNA transformation is more difficult in comparison to circular dsDNA (which is more compact).
I don´t know if this question comes from a problem with the E. coli transformation. If it was that the case, you can consult the book: Molecular Cloning: A Laboratory Manual by Joseph Sambrook and David William Russell
https://books.google.es/books?id=Bosc5JVxNpkC&printsec=frontcover&dq=molecular+cloning+a+laboratory+manual&hl=es&sa=X&ved=2ahUKEwim552l75vuAhWHsRQKHYkpDjoQ6AEwAHoECAEQAg#v=onepage&q=molecular%20cloning%20a%20laboratory%20manual&f=false
If my answer has been useful, please recommend it.
:) :)
And Good luck!
Hello Chun-Chen Yao ,
Regarding your questions. When you transform the cells with a vector, with a mixture of linear and circular plasmid form, only the circular form will be useful for the bacteria (for the resistance Ab as an example), and the linear form probably will be degraded. The linear plasmid form ligation inside the cytoplasm is unlikely, is more probably that de linear dsDNA will be degraded outside. You have to think that linear dsDNA transformation is more difficult in comparison to circular dsDNA (which is more compact).
I don´t know if this question comes from a problem with the E. coli transformation. If it was that the case, you can consult the book: Molecular Cloning: A Laboratory Manual by Joseph Sambrook and David William Russell
https://books.google.es/books?id=Bosc5JVxNpkC&printsec=frontcover&dq=molecular+cloning+a+laboratory+manual&hl=es&sa=X&ved=2ahUKEwim552l75vuAhWHsRQKHYkpDjoQ6AEwAHoECAEQAg#v=onepage&q=molecular%20cloning%20a%20laboratory%20manual&f=false
If my answer has been useful, please recommend it.
:) :)
And Good luck!
Hello Chun-Chen Yao,
there are indeed strains in which the transformation of linear dsDNA works quite well (e.g. naturally competent strains), but in Ecoli these have to overcome a weakened cell envelope. While circular plasmids can initiate replication immediately after transformation, linear DNA molecules must first religate or recombine to establish themselves in a cell. This must happen very quickly, otherwise degradation by intracellular nucleases will proceed. Therefore, transposons (mobile genetic elements) are often used to integrate heterologous DNA into the genome.
I hope I could help & good luck for your project!
most of the competent cells work for the linear plasmid.
the production of rolling circle amplification that needs to be transformed into cells to get the circle plasmid.
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