Antimicrobial HTS Assay for E. coli BW25113 (wild type)
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. An initial more ..
BioActive Compounds: 10
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
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. An initial approach is to screen the NIH SMR compounds to identify cidal or static compounds against a wild type E. coli stain. The follow-up screen will then be to test active compounds against a pool of E. coli strains overexpressing potential converting enzymes to identify prodrug candidate hits. A standard bacterial growth assay using OD600 for determining growth was used for this screen. Compounds were screened at a final concentration of 10 uM.
Actives were defined as compounds that showed >=50% inhibition in the assay. Compounds were screened in single dose and there is an inherent uncertainty associated with a single determination. Approximately 10,000 compounds were screened in duplicate and have a higher degree of certainty associated with their results. Because of the extremely low hit rate, no further screening was performed using the wild type E. coli strain.
Antimicrobial Assay for E. coli BW25113 (wild type) Protocol for 384-well HTS
Preparation of glycerol stock
-Select a single colony from the source plate.
-Streak on LB agar plate + 50 ug/mL Kanamycin, incubate o/n (16-18 hrs) 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 (16-18 hrs) in shaking incubator at 37C, 250 rpm.
-Store a few samples of the o/n culture in 30% glycerol at -80C
Note: every time you work again with the strain select a small amount of glycerol stock, place it in LB liquid media and grow at 37C, 250 rpm shaking overnight for 16-18 hrs.
Preparation of assay
-Inoculate LB broth media containing 50 ug/mL Kanamycin with a small piece from the frozen stock (not necessary to thaw) and shake o/n (16-18 hrs) in water bath or floor shaker at 250 rpm, 37C.
-Place o/n culture in fridge at 4C until 1.5 hrs before plating time.
-1.5 hrs before plating, dilute the o/n culture 1:10 in fresh LB broth media containing 50 ug/mL Kanamycin and shake for 1.5 hrs at 250 rpm, 37C.
-Dilute culture 1:1000 in fresh LB broth media containing 50 ug/mL Kanamycin and dispense 25 uL into 384 well plates (drugs have been added to plates at this point***) using Multidrop micro in hood.
-Place plates in PE bag stacked 2 high.
-Incubate plates at 37C for 20 hours.
*** In the case of Z' plates: 5ul media/DMSO or Chloramphenicol/DMSO (at 30uM final) is added using the Biomek FX.
-Read A615 on Envision (Abs folder A615- 30s shake, 384 general).
LB agar plate prep (100mL)
-100mL MQ water
-2g LB broth (Fisher, BP1427-500)
-1.5g LB agar (Fisher, BP1425-500)
-Autoclave at 121C for 15 min.
-Cool, add 5 mg Kanamycin, pour plates.
LB broth media (1L)
1L MQ water
-20g LB broth
-5g NaCl (Sigma, S3014-500G)
-Autoclave at 121C for 15 min.
-Add 50mg Kanamycin.
Kanamycin (Sigma, K1637-5G)
Agar plate stock: Stock was prepared at 5 mg/mL in MilliQ water and filter sterilized. Aliquots of 500 ul are in labeled box in -20oC freezer in the HTS prep lab. Use 1 vial for 100 mL agar plates.
LB broth stock: Stock was prepared at 50mg/ml in MilliQ water and filter sterilized. Aliquots of 1 mL are in labeled box in -20C freezer in the HTS prep lab. Use 1 vial for 1 L broth.
Type of plates used
384 well clear flat bottom w/lid, TCT plates (Fisher Corning#3701).
Possible artifacts in this assay include, but are not limited to, compounds that absorb at OD615 or precipitate.
Activity Outcome: Compounds that exhibited >=50% inhibition in the assay are labeled "Active". Compounds for which replicates were performed, the compound must have shown >=50% inhibition at least twice to be labeled as "Active"; else, labeled "Inconclusive". Compounds that exhibited <50% inhibitory are labeled "Inactives".
Because of the inherent error in all high throughput screens including the fallacy of over-interpreting single dose data, compounds that were active in this single dose screen were assigned a score of 100. All other compounds were assigned a score of 0.
Data Table (Concise)