Antimicrobial Assay for E. coli BW25113 (wild type) mutant pool - DR
There is considerable unmet need for novel antibiotics due to the rise of multi-drug resistant pathogens and the threat of engineered bioweapons. There is also an urgent need for novel antibiotics that can act against slow-growing or dormant pathogens and persisting biofilms. The long term goal of this screening campaign is to develop a novel method to identify antimicrobial prodrugs. ..more
BioActive Compounds: 39
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider: Dr. Kim Lewis, Northeastern University (Boston, MA)
There is considerable unmet need for novel antibiotics due to the rise of multi-drug resistant pathogens and the threat of engineered bioweapons. There is also an urgent need for novel antibiotics that can act against slow-growing or dormant pathogens and persisting biofilms. The long term goal of this screening campaign is to develop a novel method to identify antimicrobial prodrugs.
TolC is an outer membrane channel protein involved in the efflux of several hydrophobic and amphipathic molecules and is a component of several multi-drug efflux systems in bacteria. As a strategy to increase the hit rate to identify novel compounds with antimicrobial activity, we screened the NIH Small Molecule Repository using an E. coli strain with mutations in tolC, referred to as E. coli #tolC. TolC deletion mutant strains are expected to be more sensitive to antimicrobial agents that may otherwise be transported out of the cell. A standard bacterial growth assay using OD600 for determining growth was used for this screen. Compounds were initially screened at a final concentration of 10 uM. Hits from that screen (AID 573) were reordered from the NIH small molecule repository (MLSMR) for confirmation screening in dose response.
A total of 1265 compounds that displayed the highest % inhibition and were available for re-order were re-tested in a dose-response screen against a pooled library of E. coli BW25113 (wild type) deletion strains for in vitro non essential enzymes. A total of 1,275 mutants were present in the pool. The strains originated from the Keio library and were provided by Dr. Hirotada Mori. The pool of strains is a complete, ordered E. coli K12 knockout library of 4,320 genes and predicted ORFs. The knockouts were constructed using the Wanner method with a kanamycin cassette replacing the ORFs. For more information on the Keio library, the following link is available: http://ecoli.naist.jp/gb6/Resources/deletion/deletion.html. The compounds were screened at concentrations ranging from 0.2 - 100uM (1% final DMSO).
Protocol for Antimicrobial Assay involving E. coli BW25113 (wild type) in 384-well plates
Preparation of glycerol stock
-Pick a single colony from a plate of E. coli BW25113 provided by Dr. Kim Lewis (Northeastern University, Boston, MA)
-Streak on Luria Broth (LB) agar plate containing 50 ug/ml Kanamycin; incubate overnight (o/n, 16-18 h) at 37C.
-Pour 15 ml LB broth medium + 50 ug/ml Kanamycin into sterile flask
-Transfer a single colony from the overnight streaked agar plate into the flask and incubate o/n in a shaking incubator at 37C, 250 rpm.
-Store aliquots of the culture in 30% glycerol at -80C.
Preparation of assay
-Inoculate LB broth medium containing 50 ug/ml Kanamycin with a small sample from the frozen stock and shake overnight (16-18 h) in water bath or floor shaker at 250 rpm, 37C.
-Place culture at room temperature until 1.5 h before plating time
-Dilute the culture 1:10 in fresh LB broth medium containing 50 ug/ml Kanamycin and shake for 1.5hrs at 250rpm, 37C.
-After 1.5 h, the culture is then diluted 1:1000 in fresh LB broth medium containing 50 ug/ml Kanamycin and 25 ul is dispensed into 384 well plates (drugs have been added to plates at this point in a 5 ul volume *** ) using a Multidrop micro under a laminar flow hood.
-Place plates in polyethylene bags stacked 2 high.
-Incubate plates at 37C for 20 h.
*** In the case of Z' plates: 5 ul medium/DMSO or Chloramphenicol/DMSO (at 30 uM final) is added with Biomek FX.
-Shake plate for 30 sec. Read optical density at 615 nm on Envision plate reader.
LB agar plate prep (100 ml)
-100 ml MilliQ water
-2 g LB broth (Fisher, BP1427-500)
-1.5 g LB agar (Fisher, BP1425-500)
-Autoclave at 121C for 15 min.
-Cool, add Kanamycin to a final concentration of 50 ug/ml, pour plates.
LB broth media (1L)
-1L MilliQ water
-20 g LB broth
-5 g NaCl (Sigma, S3014-500G)
-Autoclave at 121C for 15 min
-Add 50 mg Kanamycin
Kanamycin (Sigma, K1637-5G)
Agar plate stock: Stock was prepared at 5 mg/ml in MilliQ water and filter sterilized. Vials of 1 ml aliquots were frozen. Use 1 vial for each 100 ml of LB agar plates.
Type of plates used:384-well clear flat bottom w/lid, TCT plates (Fisher Corning#3701).
Data were analyzed using IDBS ActivityBase software. Dose response curves were analyzed using XLfit equation 205 for a four parameter logistic fit; the maximum and minimum values were fixed at 100% and 0%.
Possible artifacts in this assay include, but are not limited to, compounds that absorb light at 615 nm or precipitate.
Outcome: Active compounds were defined as compounds that showed equal or greater than 20% inhibition in the assay at any concentration. Compounds with less than 20% inhibition at all concentrations were defined as inactive.
Compound Activity Score ranks a compound based on potency and was calculated as ten times the negative log (base 10) of the molar IC50.
Data Table (Concise)