strain: B3 (CGMCC 5234) growth phase: Early solventogenesis
Treatment protocol
An 8-mL sample of planktonic culture was withdrawn and the cells were collected by centrifugation at 8,000 × g for 6 min at 4°C. To collect the biofilm cells, several pieces of cotton towel were harvested from the fermentation broth and rinsed twice with PBS buffer (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, pH 7.40) to remove contaminating planktonic cells. Then the cotton towel was submerged in 8 mL PBS buffer and vortexed vigorously to disperse adsorbed cells, after which the cotton towel was removed and the resulting suspension was centrifuged to pallet the biofilm cells. All the cells were frozen immediately using liquid nitrogen and then stored at -80 °C prior to RNA extraction.
Growth protocol
Cultures of C. acetobutylicum B3 were grown in modified P2 medium containing 10 g/L glucose as the sole carbohydrate for seed culture. Fermentation experiments with planktonic cells were performed anaerobically in 500-mL Duran bottles with 300 mL of P2 medium (glucose 60 g/L; K2HPO4 0.5 g/L; KH2PO4 0.5 g/L; CH3COONH4 2.2 g/L; MgSO4•7H2O 0.2 g/L; MnSO4•H2O 0.01 g/L; NaCl 0.01 g/L; FeSO4•7H2O 0.01 g/L; p-aminobenzoic acid 1 mg/L; thiamine 1 mg/L; biotin 0.01 mg/L) at 37 °C with 10% inoculum (v/v). For fermentation with biofilm cells, 60 g/L of dried cotton towel was cut into pieces (approximately 2 cm × 3 cm) and sterilized by autoclaving at 121 °C for 15 min before being added to P2 medium with the bacterial inoculum.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using TRIZOL Reagent (Cat#15596-018，Life technologies, Carlsbad, CA, US) following the manufacturer’s instructions and checked for a RIN number to inspect RNA integration by an Agilent Bioanalyzer 2100 (Agilent technologies, Santa Clara, CA, US).
Label
Cy3
Label protocol
Qualified total RNA was further purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany) and RNase-Free DNase Set (Cat#79254, QIAGEN, GmBH, Germany). Total RNA was amplified and labeled by Low Input Quick Amp Labeling Kit, One-Color (Cat#5190-2305, Agilent technologies, Santa Clara, CA, US), following the manufacturer’s instructions. Labeled cRNA were purified by RNeasy mini kit (Cat#74106, QIAGEN, GmBH, Germany).
Hybridization protocol
each Slide was hybridized with 600 ng Cy3-labeled cRNA using Gene Expression Hybridization Kit (Cat#5188-5242, Agilent technologies, Santa Clara, CA, US) in Hybridization Oven (Cat#G2545A, Agilent technologies, Santa Clara, CA, US). After 17 hours hybridization, slides were washed in staining dishes (Cat#121, Thermo Shandon, Waltham, MA, US) with Gene Expression Wash Buffer Kit (Cat#5188-5327, Agilent technologies, Santa Clara, CA, US).
Scan protocol
Slides were scanned by Agilent Microarray Scanner (Cat#G2565CA, Agilent technologies, Santa Clara, CA, US) with default settings, Dye channel: Green, Scan resolution=5μm, PMT 100%, 10%, 16bit.
Description
Gene expression in 20h-grown C.acetobutylicum B3 planktonic cells
Data processing
Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Raw data were normalized by Quantile algorithm, Gene Spring Software 11.0 (Agilent technologies, Santa Clara, CA, US).
Submission date
May 14, 2015
Last update date
May 15, 2015
Contact name
Dong Liu
Organization name
Nanjing Tech University
Department
National Engineering Research Center for Biotechnology, College of Biotechnology and Pharmaceutical Engineering
