Methods and Results

Cloning and development of an E. coli antibiotic susceptibility Screening strain with inducible LptH expression

Method:

• Amplified gene from pET28a-LptH plasmid using Polymerase Chain Reaction (PCR).

• Ligated the PCR product into the pJET-blunt ligation vector.

• Digested pBAD24 & the PCR product with NheI & HindIII restriction enzymes.

• Ligated product into pBAD24 using T4 DNA ligase.

• Transformed pBAD24 plasmid into E. coli BW25113ΔtolC.

Result:

• E. coli BW25113 ΔtolC strain expressed LptH when induced with arabinose.

Bacterial susceptibility assays assessing the effect of LptH overproduction on the activity of standard antibiotics

Method:

• Performed Minimum Inhibitory Concentration Assay (MIC) under 1% Arabinose (+) and 0.2% Glucose (-) conditions.

Result:

• E. coli cells overexpressing LptH were resistant to membrane-targeting drug Polymyxin B.

Method:

• Performed disk diffusion assay with membrane-targeting drugs Colistin and Polymyxin B (POLY B), with Chloramphenicol (CHLOR) as positive control.

• Suppressed LptH expression with 0.2% Glucose or induced with 0.2% Arabinose

Result:

• The agar-based experiment did not show a significant change in antibiotic susceptibility when expressing LptH with 0.2% arabinose.

Expression and purification of LptH from E. coli

Method:

• Transformed pET28a-LptH plasmid into the BL21 (DE3) E. coli expression strain and induced with 1 mM IPTG.

• LptH protein was purified under native conditions using Ni-NTA affinity chromatography.

Result:

• Successfully isolated LptH (MW ~ 19.2 kDa) from the cell lysate.

Purified Pseudomonas aeruginosa LptH Binds E. coli LPS extracts

Method

• Performed serial dilutions of LPS with initial concentration 10 mg/mL.

• Microscale Thermophoresis using Monolith X instrument (NanoTemper).

Results

• Purified P. aeruginosa LptH bound E. coli LPS extracts LPS with an average dissociation constant (Kd) of 90 mM.