I try to use lambda red system (PkD46 and Km cassette) to knockout the targeted gene in Sphingobium (gram negative bacteria) and I only got false positive colonies so far. I'd like to test new method, CRISPR/cas, but i could not find any reliable protocol for bacterial genome engineering using this new method.
I appreciate your consideration
Thanks dear Adam for your answer,
Yes, it is not optimized yet, however there is one reference which applied CRISPR method in bacteria; http://www.nature.com/nbt/journal/v31/n3/full/nbt.2508.html
I already introduced PX-330 (carrying CRISPR-Cas9 genes) with appropriate sgRNA into my targeted bacteria, however it doesn't work and could not cut the designed segment :(
Yap, I agree.
Thanks for replying back, I'm continuing with Lambda red system.
Hi Atefeh, have you solved your false positive problem? What antibiotic resistance were you using as selection marker? Thanks!
I'm experiencing similar problems. Clone looking colonies did grow on 25 ug/ml Kanamycin plate after O/N, but did not seem to grow in Kanamycin LB broth (25 ug/ml). Even for some that did grow, PCR yielded negative result.
Hi Liu,
I also used Km cassette, faced with same problem, I guess it isn't inserted in right place (maybe the homology segment is short). I try to use Tet cassette as well.
i prepared the pure cassettes, but failed to get the competent cells; since my target bacteria isn't stable in growth rate. based on my experience we need fresh competent cells which holding pKD46 (could not get any colony when using competent cell stored in -80)
Exact same problem here (also using Chloramphenicol). I'm working with E. coli and most cells are false positives and/or do not grow. Usually we observe a mix of bands for "wild type" and "edited" genotype after colPCR of a single colony
Hi Jorge,
may I know which protocol are you using and the length of target sequence homology?
I use 500 bp homology on both arms of the fixing template and electroporate it at the same time as the guideRNA using freshly prepared cells following the protocol used to create the KEIO library (Datsenko and Wanner, PNAS, 2000)
huuuum, 500 bp should be enough! I'm using only 85 bp as homology tag.
Thanks Jorge
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Peptides