pet14b vector  (Millipore)

Journal: PLoS ONE

Article Title: B-Cell Epitopes in GroEL of Francisella tularensis

doi: 10.1371/journal.pone.0099847

Figure Lengend Snippet: ( A ) ELISA (data from one of two experiments with similar results are shown). Lysates of E. coli BL21 transformed with pET14b vector containing Ft SchuS4 GroEL DNA, Ft LVS GroEL DNA, or no insert (empty vector) were coated on ELISA plates in the left panels, and greater than 10-fold higher concentrations of SchuS4, LVS, or E. coli TG1 lysates were coated on ELISA plates in the right panels. The coated plates were probed with serial dilutions of the indicated mAbs. ( B ) Western blot. Equivalent concentrations of lysates (based on OD 600 of the bacterial cultures) of SchuS4 or of BL21 transformed with SchuS4 rGroEL-vector or empty vector were electrophoresed in preparative 4–15% polyacrylamide gels under denaturing conditions and, after transfer to nitrocellulose, strips were probed with 10 µg/ml of the indicated mAbs. The positions of prestained molecular weight standards (in kDa) are indicated.

Article Snippet: The NdeI/XhoI-cleaved PCR product was initially cloned into a PCR 2.1-TOPO vector (Life Technologies, Grand Island, NY) and then transferred into the pET14b T7 expression vector (Novagen, Madison, WI) to add DNA encoding an N-terminal sequence of six histidine residues (a His tag) to the GroEL gene. pET14b (empty vector) and pET14b containing the SchuS4 GroEL gene insert or the LVS GroEL gene insert were separately transformed into One Shot BL21(DE3)pLysS chemically competent E. coli (Life Technologies), and induction of protein expression was carried out in LB broth for 4 hours at 37°C in the presence of IPTG (isopropyl-β-d-thiogalactopyranoside) according to manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Transformation Assay, Plasmid Preparation, Western Blot, Molecular Weight