Hello!
we're performing a quorum sensing secretome characterization of a couple of bacterial strains, and we have met a few issues, which we cannot explain.
First, we had our Agrobacterium tumefaciens turn blue in an x-gal plate in absence of any AHLs added or an AHL-producer strain. We can be quite sure that there is no contamination as A. tumefaciens is grown in three antibiotics. The strain that we are using for detection, KYC55, has no HSL production machinery as the wildtype plasmid bearing traI HSL-synthase has been removed in this strain. So no mutations could have led to acquiring (back) production of AHL. So why do we see breakdown of X-gal in the apparent absence of AHLs? Are we missing something?
Second question is about the overlay assay. We got 18C TLC plates silica on glass, and when we poured the LB mixture with the sensor strain on the TLC with AHL bacterial extracts, the silica layer deattached from glass and started floating. We will only see tomorrow morning if this affected the assay anyhow, but we were wondering if this deattachment could be somehow avoided.
Thanks!
Artur
Dear Artur Zaduryan,
Kindly purify the A. tumefaciens again by simple streak plate method, pickup single colony, grow overnight in LB with antibiotics and try the biosensor assay once again.
I have sent the TLC procedure in detail by personal email.
Hope this will help.
Best wishes,
HARSHAD
Dear Harshad,
thank you very much for your answer!
We have done multiple replatings prior to final assay lquid culture inoculation, so that, given high 3-antibiotic plates, supposedly rules out contamination.
Thank you for the personal e-mail too!
Kind regards,
Artur
Dear Harshad S. Lade and Dear Artur Zaduryan can you guys share the TLC protocol . I am also having same problem like Artur had.
Best
Aslam
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