Figure 1.
Destination vectors constructed in this study. A variety of destination vectors derived from pDEST17 are shown.
Table 1.
Expression data for S. oneidensis response regulator proteins in E. coli
Figure 2.
SDS-PAGE of S. oneidensis NarP expressed from the destination vector pTP247.
A. Lane 1: protein marker; Lane 2: Soluble protein after lysis of E. coli expressing NarP protein. B. Electrophoretic gel shift assays of NarP and phosphorylated NarP-P. Increasing concentrations (0, 0.65, 1.3, 2.6, and 5 µM) of unphosphorylated NarP and phosphorylated NarP are shown. DNA is stained with CyberGreen. The 100bp ladder was used in the experiments.
Figure 3.
Expression of S. oneidensis ORFs from the pTP248 double-tag destination plasmid.
A. The protein expression levels of ORFs were evaluated from whole cell protein lysates of E. coli by SDS-Page fractionation and immunodetection using anti-GST antibody. The S. oneidensis ORF number is shown above each lane. The red asterisks indicate the position of a band that is consistent with the predicted molecular weight of the fusion protein. Note that for ORFs SO0047 and SO0056, a band is not visible in panel A but is visible in the longer film exposure of panel B. The predicted molecular weights (kDa) of the fusion proteins are: SO0037, 40.7; SO0038, 65.1; SO0040, 61.2; SO0041, 41.4; SO0044, 39.7; SO0047, 75.1; SO0049, 86.9; SO0051, 34.3; SO0053, 66.8; SO0054, 74.2; SO0055, 94.7; SO0056, 59.3; SO0057, 79.9; SO0059, 57.1. B. Identical to A. but with a longer film exposure.
Figure 4.
Phage display destination vector constructed in this study.
M13 filamentous phage display vector pTP262 is a destination plasmid that can be used for insertion of ORFs from the pDONR clone set to create phage display plasmids.
Figure 5.
Briefly, phages were incubated with immobilized anti-E. coli thioredoxin antibody, non-binders were removed by washing, bound phage were eluted, amplified, and the process was repeated. Eluted phage clones from round 3 were DNA sequenced.
Table 2.
Strains, plasmids, and primers used in this study