these days, I'm doing gene knockout mutation to use Red recombination including pKD119 (having araBAD promoter, exonuclease gene) and pKD3 (TetR) as helper and template (cassette) plasmid, respectively.
For mutatagenesis experiment, first, pKD119 transfer to bacteria (Serratia fonticola) ,then, transformant grow at 30 degree until OD600=0.3.
And, L-arabinose (final concentration=0.35%) add to culture broth and exonuclease induced at 37 degree for 45 min.
Following to homology casstte consist of chloramphenicol resistance gene flanked by 36 bp target gene transfer to S. fonticola having pKD119 plasmid by electroporation (1350 V or 2500 V, 1mm or 2mm cuvette).
After electroporation, bacterial cell recover at 37 degree for 3 hr, maybe this process is recombination step.
Then, recovered cell spread onto LB-chloramphenicol agar and incubate at 37 degree.
But I could not fount colony.. what's the problem?
this protocol refer to gene bridge company and other research paper which mentioned E. coli, Serratia, Yersinia, Salmonella species is possible mutaion to use Red recombination system.
My S. fonticola is not only belonging to Serratia, but also close to Yersinia.
which bacteria species classified as Enterobacteraceae family.
My Cassette concentration is enough (200 ng/uL) and exactly amplified.
And, helper plasmid pKD119 is successfully replication in my bacteria, I checked.
What is the problem in my expriments..
Chloramphenicol concentration is high? or another problems?
Please tell me solution.
When you transform your pKD119, do you plate the transformants and pick a single colony to continue with?
gr wendy
Hi Wendy,
Yes, I use to pick single colony on LB-tetracycline agar plate.
Additionally, I confirmed replication and size of pKD119 by extraction and restriction enzyme digestion.
I have question about RecA gene related homologous recombination.
Quick & Easy E. coli Gene Deletion Kit of Gene Bridge company also use similar plasmid based on lambda red recombinase. This plasmid is different from pKD119 in RecA gene.
If I do to insert RecA gene (from my isolate bacteria) behind AraBAD promoter of pKD119, homologous recombination efficiency is increase or not?
Thank you
I dont know the plasmid pKD119, but I have used pKD46. pKD46 has the Red a b and g gene all in one and basicly is the same as the genebridges plasmid pBAD-abg.
I use the pBAD-abg now that I got as a gift from Francis Stewart from Dresden. If I were you I would write him an email and I am pretty sure he will send it to you. He is a very nice guy.
If he agrees for you to use it, I am also willing to send the plasmid to you to save him the hassle.
I would try that instead of changing the plasmid you use now. The pBAD-abg works for me every time
Gr Wendy
pKD119 plasmid is the same as pKD46 plasmid except tetracyline resistance gene.
Unfortunately, only pKD119 is suitable for my bacterium, because my bacterium is ampicillin resistant.
That kind of reason, i can't use ampicillin resistance plasmid pKD46.
I really appreciate your answer and active support^^
Additionally, do you have detail protocol of this experiment? ( ex, arabinose concentration, incubation time, etc.)
I hope looking for solution through comparing with your protocol.
thank you
Np
the pBAD-abg i use is also tet resistant.
I can email you the detailed protocol, explanation and primer design considerations if you like.
What is your email adres?
gr wendy
My e-mail is [email protected]
thanks wendy, your protocol will be a great help to me^^
Hello,
I am facing the same challenge as Byung, that my bacteria is naturally amp resistant. If you are still checking this thread Wendy, I would really appreciate more information about pBAD-abg as well. I am currently seeking a plasmid carrying lambda red to generate mutants in my bacterial species.
Amber.Rose.Paulson[at]gmail[dot]com
Thanks,
Amber
- Badges
- Science topic