Journal: Genome Biology and Evolution
Article Title: Eukaryotic Components Remodeled Chloroplast Nucleoid Organization during the Green Plant Evolution
Figure Lengend Snippet: CreCNS is a DNA binding protein that compacts nucleoids in E. coli . ( A ) Coomassie Brilliant Blue-stained gel after SDS-PAGE of purified partial CreCNS and CreHLP proteins (80 pmol each). ( B ) Electrophoretic mobility shift assay of CreCNS and CreHLP proteins. The 100-bp DNA ladder was incubated with various amounts (80, 40, 20, and 10 pmol) of purified recombinant protein. ( C ) DH5α E. coli cells were transformed with the pQE80l vector containing the sequence of the CreCNS or CreHLP gene, or with the empty vector. Cells were grown in LB medium containing 50 µg/ml carbenicillin at 37 °C. At an OD600 of 0.4–0.6, CreCNS and CreHLP overexpression was induced with 1 mM IPTG for 4 h. The bacterial nucleoids were stained with DAPI. ( D ) Effect of nucleoid condensation mediated by CreCNS and CreHLP on DH5α E. coli cells. Escherichia coli cells were transformed with the pQE80l vector containing the sequence of the CreCNS or CreHLP gene, or with the empty vector. Cells were grown at 37 °C, and OD600 was measured 0–4 h after induction with 1 mM IPTG. Error bars represent standard deviation. There are three independent data points.
Article Snippet: Antibody Preparation pQE80l (Qiagen, Venlo, Netherlands) vectors harboring the cDNA sequences encoding CreCNS, CreHLP, CreWhirly, CreSWIB2, KfHLP, KfpTAC3, KfWhirly and KfSWIB were prepared and transformed into the E scherichia coli BL21 strain and selected on Luria–Bertani (LB) agar medium containing 50 µg/ml carbenicillin (Nacalai Tesque).
Techniques: Binding Assay, Staining, SDS Page, Purification, Electrophoretic Mobility Shift Assay, Incubation, Recombinant, Transformation Assay, Plasmid Preparation, Sequencing, Over Expression, Standard Deviation