Theeffect of narrow bandwidth infrared radiation on the growth of Escherichia coli
Shang-Ru Tsai, Tsui-Chin Huang, Chia-Ming Liang, Hsin-Yi Chang, Yi-Tsung Chang, Hsuan-Cheng Huang, Hsueh-Fen Juan, Si-Chen Lee
Supplemental materials
Materials and Methods
PTE fabrication process
A 300 nm thick molybdenum (Mo) film was deposited on the n-type silicon substrate as a heating source. A 200 nm thick silver film was then deposited on the Mo layer to retard the heat radiation of substrate. A 100 nm thick silicon dioxide (SiO2) film was sputtered onto the silver film. Finally, a second 100 nm thick silver film was deposited onto the SiO2 film, and perforated with hexagonally arranged circular hole array photolithographically.
IR exposureon E. coli
The E. coli were cultured from a single colony to ensure the uniformity of population for experiments. After being cultured overnight, the cells were diluted tenfold with Luria Broth medium and dispensed in 1µl droplets on agar to form a six by six square arrangement. Before exposure to IR, each six by six droplet arrangement was incubated for 5 h to allow the colonies to enumerate in the same initial conditions. To avoid the edge effect, the diameters of the central square of 16 droplets were measured after the 24-hIR treatment period, and compared to equivalent droplet diameters measured in non-irradiated controls that had been maintained in the incubator for the same period of 24 h.
Protein extraction
Total proteinwas extracted using lysis buffer containing 7M urea (Boehringer, Mannheim, Germany), 2M thiourea, 4% CHAPS (JT Baker, New Jersey, USA) and 0.002% bromophenol blue (Amersco, Ohio, USA).After centrifugation (15000 xg, 4°C, 30 min) the proteinconcentration of the extract was determined by Bradford method (Bio-Rad, Hercules, California, USA).
2-DE
IEF was performed in an EttanTM IPGphorTM 3 IEF system (GE Healthcare, Buckinghamshire, UK) on 18 cm, immobilized, pH 4-7 linear gradient strips (Bio-Rad, Hercules, CA, USA) at 50 μA/strip for the following condition: 50 V, 12h; 100 V, 2h; 250 V, 1 h; 500 V, 1 h; 1kV, 1 h; 4 kV, 1 h; 8 kV, 65 kVh. SDS-PAGE was performed on a polyacrylamide gel with a linear concentration gradient of 10-18% for 40 mA / gel with cooling system. The gels were fixed with 7 % acetic acid / 10 % methanol for 1 h, and stained with SyproRuby overnight. After destained with 7 % acetic acid / 10 % methanol for 1 h, the gels were scanned using Typhoon 9400 (GE Healthcare) at a fluorescence emission of 610nm (green laser 532 nm, normal sensitivity). The protein expression profiles were showed in Fig. S1.
The searchparameters for mass spectrometry data
The searchparameters allowed for peptidemass tolerance, 50 ppm; MS/MS ion mass tolerance,0.25 Da; allow up to one missed cleavage; variable modificationsconsidered were methionine oxidation and cysteinecarboxyamidomethylation; limited to E. coli. Onlysignificant hits as defined by MASCOT probability analysiswere considered (P<0.05).
RNA extraction
Total RNA was extracted from IR exposed and non-exposedE. colicultures. Briefly, the cells were harvested from agar plates and lysed with Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. RNA concentrations weredetermined using NanoDrop 1000 (NanoDrop Technologies, Wilmington, DE, USA).
Real-time PCR
Total RNA was reverse-transcribed into cDNA by randomhexamer using SuperScript III Reverse Transcriptase (Invitrogen). The specific gene primers for real-time PCR were designed using the Beacon Designer 2.5 program (Premier Biosoft International, Palo Alto, CA, USA). Sequences are shown in Table I. The real-time PCR reaction (25 μl) containing 5 μl first-strand cDNA, 12.5 μl iQ SYBR Green supermix kit (Bio-Rad Laboratories, Hercules, CA) and 0.5 μl (10 μM) of each sense and antisense primer was run for 40 amplification cycles in an iCycler iQ5TM Real-Time PCR Detection System (Bio-Rad Laboratories). Cycling conditions were 95 °C for 3 min, 95 °C for 30 s, and 60 °C for 30 s. Relative transcript quantities were calculated using the ΔΔCt method with 16S rRNA as the reference gene amplified from the samples. All reactions were run in triplicate.
Table I. Primers used for real-time PCR analysis.
Gene / Forward primer (5'→3') / Reverse primer (5'→3')OmpA / CGGTCTTCGCTGGCGGTGTTG / GACGAGTGCCGATGGTGTGTGC
OmpF / ACCTATCGTAACTCCAACTTC / AAGCCTTCGTATTCGTAGC
OppA / TGACCAACATCACCAAGAGAAG / CGACATTCCCTGCCATTAGC
RbsB / GCGAAGAAAGCGGGTGAAGGTG / GCTGGCTGGCTGGCAAGAAC
GrxD / GAACTGCCGAAATATGCTAACTG / ATCACAACCGCCGACCAG
YdfG / TGGCTCAACGGCAGGTAG / GGCTAAACTGACGAACAAACG
DapD / TCAGAGCACCCGTATTTAC / GATAACCGCACAGTAGAGG
Crr / CCGACGACAAGAAGGATACC / GATAGCAATACCATCACCAACG
YjgF / AAGCCTTCTTCACCGAACACAAC / GCAGACGGGCAACTTCAACG
KduD / ATCCGTGTGCCTTCTTATACC / CCGTAGTTGTTGAGTATTGTTGG
GpmA / GCTTTGACTTTGCTTACACTTCTG / CACCTGCTCGTCGCCATAC
Mdh / CTGAAGTGGTTGAAGCGAAGGC / CCAGCAGCAGCGGTTGAGAG
KduI / CGTAAGACAGAGCATCCACAG / CGCCAACAATAATTCGGTCAATG
WrbA / CTGGCGAGCGTCTTTAGTTC / AGGTGGATGTGATGGTTTGTTC
Eno / GGTATCGCTAACTCCATCCTG / TCGCCAGAACGGTGAGAG
RplI / ACTGTTCGGTTCCATCGG / CTCTTAGCCACTTCAACGC
Dps / AAGTTACCCGCTGGACATC / CTTTCGCTTCGCCAATCG
Ssb / TGGCGAGCGAATATCTGC / ACGACTTCTGTGGTGTAGC
16S rRNA / GAATGCCACGGTGAATACGTT / ACCCACTCCCATGGTGTGA
FIG S1. The differential expressed protein profile of IR-exposed (IR) and control (Dark) E. coli. The protein identification was performed by MALDI-Q-TOF and the obtained peak lists were searched against MASCOT database. The searchparameters allowed for peptidemass tolerance, 50 ppm; MS/MS ion mass tolerance,0.25 Da; allow up to one missed cleavage; variable modificationsconsidered were methionine oxidation and cysteinecarboxyamidomethylation; limited to E. coli. Onlysignificant hits as defined by MASCOT probability analysiswere considered (P<0.05).