Hello,
I have recently started working with microalgae C. reinhardtii. I seek to use it for my recombinant protein production. However, I face some problems with its transformation. Even though some colonies grow up on plates with an antibiotic used for selection, I can't find any positive clones using PCR. Has anyone faced similar problems and would have any advice? I use the Ble2A expression cassette linked to my protein gene sequence. I use C. reinhardtii strains of CC124, CC400, and CC4350 and try to transform with glass beads or electroporation methods. I have tried to resuspend cells in 10mM EDTA or Chelex, boil, and centrifuge before transferring it to the PCR mix.
Thank you very much in advance!
Thank you for your response, Lina!
Answering your question, no, I don't use autolysin for my transformations. However, I use instead C. reinhardtii strains which are already cell wall deficient (CC400 and CC4350).
Greetings,
You could try using the method on the attached paper, as Agrobacterium tumefasciens is supposed to make things easier. I hope it helps, good luck.
Best regards
The nuclear transformation has bizarre characteristics in gene editing: 1) many false-positive colonies, and 2) lack of the maintenance of their new phenotypes.
Many publications have demonstrated that the nuclear transformants showed the designed phenotypes by knock-out or overexpression. However, their transient expression or less capability to maintain the edited genome have demonstrated that the nuclear transformation has many things to go through for gene stability.
The only thing to do would be that you should perform the experiment in a short period.
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