Restriction reactions were preformed using Nde-I and Hind-III restriction enzymes (New England Biolabs, Ipswich, MA) followed by ligation reaction (Takara Bio, Shiga, Japan) to pHis parallel bacterial vector containing ampicillin resistance marker (Novagen, EMD Chemicals Inc. Darmstadt, Germany).
thermoglucosidans: pUCG18P5bgaB Strain harboring pUCG18 with P5::bgaB This study Plasmids pUC19 General E. coli cloning vector; contains ampicillin resistance gene Yanisch-perron et al. (1985) pUC19bgaB plasmidacZα plasmid expressing the bgaB gene under control of the pLac promoter.
The expression vector, containing an ampicillin resistance gene, was transformed into the JM109 strain of Escherichia coli, which was then exposed to 1.0 mM isopropyl-1-thio-β-D-galactoside (IPTG) to induce protein expression.
Furthermore, the skeleton of the intermediate vector, containing an ampicillin-resistance gene for bacterial selection and the ColE1 origin of replication, did not need to be removed, and can be used for plasmid rescue procedures after transformation.
Briefly, E. coli with the appropriate vectors were grown in LB broth containing ampicillin (100 µg/ml) at 37°C overnight.
Briefly, E. coli harboring the appropriate vectors were grown in LB broth containing ampicillin (100 µg/ml) and tetracycline (10 µg/ml) at 37°C overnight.
E. coli M15 cells carrying the recombinant pQE-30 vector were cultured overnight at 37 °C in LB broth containing ampicillin and kanamycin.
E. coli BL21 (DE3) cells harboring the recombinant expression vectors were grown overnight in 50 ml LB broth containing ampicillin at 37 °C.
The pACYC-RIL, pRARE2, and pLysSRARE2-containing NiCo21 DE3) were then transformed with pSA-Hp24-6His vector and the transformants were selected on LB agar plates containing Ampicillin (100 μg mL-1) and Chloramphenicol (25 μg mL-1).
PCR-amplified subtracted cDNA were cloned in pGEM-T vector (Promega), transformed into JM109 competent cells (Promega), and plated on LB plates containing ampicillin (100 μg/ml), X-gal (80 μg/ml), and IPTG (0.5 mM).
E. coli strains BL21 and BL21∆luxS were transformed with the expression vectors pColdTF-lsrB and pColdTF-luxS, respectively, and grown on LB agar plates containing ampicillin (100 μg/ml).
