I am trying to develop a binary construct containing a gRNA sequence and a Cas9 coding sequence, to transform Agrobacterium, to subsequently transform tomato plants and knock out a target gene.
I am using the vector pYPQ141 (http://www.addgene.org/69290/) as my gRNA entry vector, pYPQ150 (http://www.addgene.org/69301/) as my Cas9 entry vector and pMDC32 (http://www.addgene.org/32078/) as my binary destination vector.
Using LR Clonase II from ThermoFisher, I created the binary vector with the two inserts from each entry vector. The modified binary vector was used to transform E. coli and plated on LB agar containing Kanamycin as the selective antibiotic. The E. coli was bulked up in liquid LB broth and the plasmids extracted using a GeneJET Plasmid miniprep kit. I ran a PCR on the extracted plasmids using a 35S CaMV forward primer (pMDC vector backbone) and a reverse primer targeting the pcoCas9 sequence (from pYPQ150) and got the correct size amplicons on a gel. I also digested the plasmids with BamHI and got the correct fragment sizes.
This plasmid was then used to transform Agrobacterium tumefaciens strain LBA4404 (which carry resistance to Rifampicin and Streptomycin). When I plated the transformed cells on YEP media containing Rif., Strep. and Kan. I got many positive colonies. However, when I perform colony PCR and PCR (using the same primer set as before) on the plasmids isolated from Agrobacterium using alkaline lysis miniprep, I don't get any successful amplicons. I even tried to screen for the Hygromycin resistance gene encoded by pMDC to see if the Agrobacterium was transformed by background unmodified pMDC and still don't get a successful reaction...
Can anyone suggest a possible solution or approach to this problem. I'm very confused and not sure what my next approach should be.
- Why are the Agrobacteria growing on KanR-YEP media but lacking the inserts; Is the plasmid concentration just too low to detect through PCR?
-Is it possible that the inserts have been lost in Agrobacterium
-Is there a more suitable Agrobacterium strain that would mitigate this?
Dear Lorcan Carlsen
Normally the concentration of plasmid extracted from Agro is very low but even with less than 10 ng/ul extracted plasmid you could also verify through PCR. It is really weird but nothing lost during transformation to Agro. I think most of colonies you got from plated agro are just contamination, and it also may be due to antibiotics problems. The best concentration for Kan is 50 ug/ml and for Rif is 25 ug/ml.
You don't need to extract plasmid from Agro colonies, just perform colony PCR by using a sterile toothpick that has been slightly removed a piece of each colony and shaken in a PCR reaction.
I had used LBA4404 for tomato transformation by CRISPR structure lots of time and never seen such problem. normally after between 24-36 h after plating fine colonies must be seen and could be verified if you use Electroporation method. using temperature shock transformation method has lower efficiency. I had used other strains such as GV3101 and C58, but the best and highly efficient strain for co-cultivation of tomato is LBA4404.
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