Hi! Anyone working with L. tarentolae LEXSY protein expression system? Despite having used the protocol described by Jena I am having trouble on parasite growth. Any further tips you might have?
We are working with LEXSY. You are having growth problem with the transfected or the wilhype LEXSY? If it is the transfected LEXSY, then is the protein of interest could be toxic to the cell?
I am using pLEXSY to over express everything in Leishmania, whatever the species is. And sometimes I experimented some growth delay but only for some proteins. It is possible that the protein you are trying to produce is toxic or just redusses the fitness of the parasite so you experiment such delay. Can you tell us what kind of proteins you are trying to produce? And if you have more than one, does it happen with all of them?
Hi all and sorry for the delay in my answer, The growth is the same if you transfect with control plasmid (pLEXSY) or the pLEXSY with my protein of interest. According to the protocol within 5-7 days after transfection you should find colonies of about 1-2 mm and I never found such colony size.
I used both methods of plating with and without nitrocellulose and I only saw increased colony size after inducing the colonies grown in the nitrocellulose membrane in a new plate with Tet.
I am trying to see if I can recover the clones from these induced colonies removing the Tet. and indeed they grew, however I don´t know if the fact that I induced once in the plate might influence future inductions, as I tried to induct a 1:10 dilution in 1 mL and saw no color change after 2 days (even though the colony on the nitro. membrane was pink-ish).
Tips?
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