Well-characterized promoters are essential tools for metabolic engineering and synthetic biology. In Streptomyces coelicolor, the native kasOp is a temporally expressed promoter strictly controlled by two regulators, ScbR and ScbR2. In this work, first, kasOp was engineered to remove a common binding site of ScbR and ScbR2 upstream of its core region, thus generating a stronger promoter, kasOp3. Second, another ScbR binding site internal to the kasOp3 core promoter region was abolished by random mutation and screening of the mutant library to obtain the strongest promoter, kasOp* (where the asterisk is used to distinguish the engineered promoter from the native promoter). The activities of kasOp* were compared with those of two known strong promoters, ermEp* and SF14p, in three Streptomyces species. kasOp* showed the highest activity at the transcription and protein levels in all three hosts. Furthermore, relative to ermEp* and SF14p, kasOp* was shown to confer the highest actinorhodin production level when used to drive the expression of actII-ORF4 in S. coelicolor. Therefore, kasOp* is a simple and well-defined strong promoter useful for gene overexpression in streptomycetes.

An Engineered Strong Promoter for Streptomycetes

High-quality human DNA samples and associated information of individuals are necessary for biomedical research. Biobanks act as a support infrastructure for the scientific community by providing a large number of high-quality biological samples for specific downstream applications. For this purpose, biobank methods for sample preparation must ensure the usefulness and long-term functionality of the products obtained. Quality indicators are the tool to measure these parameters, the purity and integrity determination being those specifically used for DNA. This study analyzes the quality indicators in DNA samples derived from 118 frozen human tissues in optimal cutting temperature (OCT) reactive, 68 formalin-fixed paraffin-embedded (FFPE) tissues, 119 frozen blood samples, and 26 saliva samples. The results obtained for DNA quality are discussed in association with the usefulness for downstream applications and availability of the DNA source in the target study. In brief, if any material is valid, blood is t...

DNA Source Selection for Downstream Applications Based on DNA Quality Indicators Analysis

Dom34:hbs1 plays a general role in quality-control systems by dissociation of a stalled ribosome at the 3' end of aberrant mRNA.

Nascent peptide-dependent translation arrest leads to Not4p-mediated protein degradation by the proteasome.

http://www.mcb.uct.ac.za/sites/default/files/image_tool/images/261/People/Staff/academic/laura_roden/publications/biomaterials%202009%20Roden.pdf

The induction of bone formation by coral-derived calcium carbonate/hydroxyapatite constructs.

1. Using Plasmid Vectors in Molecular Cloning 2. Bacteriophage bb and Its Vectors 3. Working with Bacteriophage M13 Vectors 4. Working with High-capacity Vectors 5. Gel Electrophoresis of DNA and Pulsed-field Agarose Gel Electrophoresis 6. Preparation and Analysis of Eukaryotic Genomic DNA 7. Extraction, Purification, and Analysis of mRNA from Eukaryotic Cells 8. In Vitro Amplification of DNA by the Polymerase Chain Reaction 9. Preparation of Radiolabeled DNA and RNA Probes 10. Working with Synthetic Oligonucleotide Probes 11. Preparation of cDNA Libraries and Gene Identification 12. DNA Sequencing 13. Mutagenesis 14. Screening Expression Libraries 15. Expression of Cloned Genes in Escherichia coli 16. Introducing Cloned Genes into Cultured Mammalian Cells 17. Analysis of Gene Expression in Mammalian Cells 18. Protein Interaction Technologies Appendices Index

The Condensed Protocols from Molecular Cloning: A Laboratory Manual

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The Condensed Protocols from Molecular Cloning: A Laboratory Manual