Table 1.
Oligonucleotides used to construct vectors used in this study.
Table 2.
Plasmids used in this study.
Fig 1.
Construction of FabV and FabI-containing plasmid vectors.
The bla gene (AmpR) was removed and restriction enzyme (ClaI and SmaI) sites were introduced downstream P3 promoter of β-lactamase by whole-plasmid PCR of pUC19, pSA-HP24, and pBR322 vectors. Inserts were prepared by PCR amplifying fabV and fabI using Vibrio cholerae O1 El Tor or E. coli BL21(DE3) genomic DNA respectively as DNA template. Vectors and inserts were restricted using ClaI and SmaI, gel purified, and ligated at 1:3 vector/insert ratio. These manipulations resulted in the construction of (A) FabV-containing pUC19, (B) FabI-containing pUC19, (C) FabV-containing pSA-HP24, and (D) FabI-containing pBR322 plasmid vectors.
Fig 2.
Effect of Triclosan on bacterial growth.
Bacteria were cultured overnight in LB broth, diluted to 0.05 OD600 in the same medium, and grown in the absence and presence (10–0.325μM) of Triclosan at various temperatures (A) 37°C, (B) 30°C, and (C) 22°C, while shaking at 250rpm for 12h. Bacterial growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from six (6) independent experiments done in triplicate. Triclosan, at the concentration of 1.25–2.5μM, inhibited the growth of all three bacterial strains at various temperatures tested.
Fig 3.
Bacterial growth characteristics.
E. coli DH5α, JM109, and BL21(DE3) were transformed with FabV (pUC19-FabV and pSA-HP24-FabV), FabI (pUC19-FabI, pBR322-FabI), or Bla (pUC19–Bla, pSA-HP24-Bla)-plasmids, and the transformants were selected on LB agar plates. Seed cultures were used to inoculate 25mL LB broth in 250mL baffled flasks and cultures grown at 37, 30, and 22°C for up to 12 hours while shaking at 250rpm. Samples were collected at one hour interval and growth was measured by absorbing the diluted samples at 600nm and graphs plotted. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
Fig 4.
E. coli DH5α and JM109 were transformed with high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL-1 ampicillin (for Bla plasmids). Seed cultures were used to inoculated 5mL LB broth in 50mL flasks and cultured for another 18 hours at 37 or 30°C while shaking at 250rpm. Cell density was then measured by absorbing the diluted samples at 600nm and normalized to 2 OD600. One mL of the normalized culture was then used to extract the plasmid following supplied protocol. The quantity of the extracted plasmid DNA was measured by fluorometry using Qubit Fluorometer and Qubit dsDNA BR Assay Kit (Invitrogen, life technology). Fig 4A shows plasmid DNA yield when bacteria were grown at 37°C, whereas Fig 4B shows the plasmid DNA yield from cultures incubated at 30°C. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
Fig 5.
Bacterial transformation efficiency.
Chemically competent E. coli DH5α and JM109 cells were transformed with 100pg μL-1 of purified high-copy number pUC19-Bla, pUC19-FabV, pUC19-FabI, and medium-copy number pSA-Hp24-Bla, pSA-Hp24-FabV, pBR322-FabI plasmids and selected on LB agar plates containing 1μM Triclosan (for FabV/FabI plasmids) and or 100μg mL-1 ampicillin (for Bla plasmids) after 18 hours of incubation at 30°C and transformation efficiency calculated. Fig 5A and B show transformation efficiency of various plasmids in DH5α and JM109 respectively. Fig 5C and D show the effect of incubation time prior to plating the transformants on selection plates. Error bars show standard deviations calculated from at-least six (6) independent experiments performed in triplicate.
Fig 6.
Morphology of transformed bacteria.
E. coli DH5α and JM109 transformed with (A) high-copy number pUC19-Bla, pUC19-FabV, or pUC19-FabI plasmids or (B) medium–copy number pMXB-p24-Bla, pMXBp24-FabV, or pBR322-FabI plasmids were incubated at 30°C for 18 hours on plates with our without selection agent (Ampicillin or Triclosan) and photographed. No appreciable differences noted when the transformants were plated on Agar plates containing selection agent or not.