Characterization of a dual biotin tag for improved single stranded DNA production

Generation of single-stranded DNA plays a key role in many biotechnology applications including production of aptamers, single strand conformation polymorphism, nuclease S1 mapping, pyrosequencing, genosensors, probe preparation and labelling, subtractive hybridization as well as nucleic acid sensing and microarrays.

Sequences required for transcription termination at the intrinsic λtI terminator

The λtI terminator is located approximately 280 bp beyond the λint gene, and it has a typical structure of an intrinsic terminator. To identify sequences required for λtI transcription termination a set of deletion mutants were generated, either from the 5′ or the 3′ end onto the λtI region. The termination efficiency was determined by measuring galactokinase (galK) levels by Northern blot assays and by in vitro transcription termination. The importance of the uridines and the stability of the stem structure in the termination were demonstrated. The nontranscribed DNA beyond the 3′ end also affects termination. Additionally, sequences upstream have a small effect on transcription termination. The in vivo RNA termination sites at λtI were determined by S1 mapping and were located at 8 different positions. Processing of transcripts from the 3′ end confirmed the importance of the hairpin stem in protection against exonuclease.

Transcriptional Response of Escherichia coli to External Zinc

ABSTRACT Transcriptional response of Escherichia coli to extracellular zinc was studied using DNA microarray and S1 mapping assays. Addition of external zinc induced the expression of zinc exporter ZntA and inhibited the expression of zinc importer ZnuC. In the continuous presence of zinc, ZnuC repression took place at lower zinc concentrations than ZntA induction. The microarray assay indicated that the addition of excess external zinc induces the expression of many genes that are organized in the regulon for cysteine biosynthesis, implying that cysteine plays a role in transient trapping of free zinc for maintenance of zinc homeostasis. Besides the RpoE regulon, other genes were also induced by zinc, suggesting that periplasmic proteins denatured by zinc induce the genes for protein repair. The microarray data of the newly identified zinc-responsive promoters were confirmed by S1 mapping.