Identification of residue-residue contacts from primary sequence can be used to guide protein structure prediction. Using Escherichia coli CcdB as the test case, we describe an experimental method termed saturation-suppressor mutagenesis to acquire residue contact information. In this methodology, for each of five inactive CcdB mutants, exhaustive screens for suppressors were performed. Proxima...

We have constructed a novel, nonhomologous end-joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I-SceI generated a double-strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing t...

Recombineering, which is the use of homologous recombination for DNA engineering in Escherichia coli, usually uses antibiotic selection to identify the intended recombinant. When combined in a second step with counterselection using a small molecule toxin, seamless products can be obtained. Here, we report the advantages of a genetic strategy using CcdB as the counterselectable agent. Expressio...

Electron tomography is providing a wealth of 3D structural data on biological components ranging from molecules to cells. We are developing a web-accessible database tailored to high-resolution cellular level structural and protein localization data derived from electron tomography. The Cell Centered Database or CCDB is built on an object-relational framework using Oracle 8i and is housed on a ...

BACKGROUND In high-throughput demanding fields, such as biotechnology and structural biology, molecular cloning is an essential tool in obtaining high yields of recombinant protein. Here, we address recently developed restriction-free methods in cloning, and present a more cost-efficient protocol that has been optimized to improve both cloning and clone screening. RESULTS In our case study, t...

Every residue of the 101-aa Escherichia coli toxin CcdB was substituted with Ala, Asp, Glu, Lys, and Arg by using site-directed mutagenesis. The activity of each mutant in vivo was characterized as a function of Controller of Cell Division or Death B protein (CcdB) transcriptional level. The mutation data suggest that an accessibility value of 5% is an appropriate cutoff for definition of burie...

Most bacterial strains harbor plasmids that are maintained with remarkable stability. A large variety of plasmids encode systems that act when other control mechanisms have failed, i.e., when plasmid-free progeny is generated during replication. The mechanisms that control plasmid maintenance by T/A loci are well known: the antagonistic regulators that neutralize the toxins are metabolically un...

In this chapter, we describe a data integration framework for multi-resolution data and some of the tools we have created for achieving integration of distributed data sources. The chapter is divided into two parts. In the first part, we describe a recently developed database for high resolution 3D data on cell structure and protein localization, the Cell Centered Database or CCDB. In the secon...

Microcin B17 is a 3.1-kDa bactericidal peptide; the putative target of this antibiotic is DNA gyrase. Microcin B17 has no detectable effect on gyrase-catalysed DNA supercoiling or relaxation activities in vitro and is unable to stabilise DNA cleavage in the absence of nucleotides. However, in the presence of ATP, or the non-hydrolysable analogue 5'-adenylyl beta,gamma-imidodiphosphate, microcin...