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Summary of the March 2016 Issue of BioTechniques

The March 2016 issue of BioTechniques will feature articles describing new methods for:

  • protein expression
  • mRNA analysis
  • genomic DNA isolation from bacterial species
  • avoiding contamination when working with algae cultures
  • modified enhancer profiling and sequence scanning

This issue will also contain a new Tech News article focusing on software solutions in life science research, including the latest in microscopy software, sequence analysis tools, and mass spectrometry platforms.

Protein Expression

A high rate of plasmid instability is associated to the use of some expression vectors in Escherichia coli, resulting in permanent loss of recombinant protein expression. This is due to sequence alterations involving vector promoter elements, triggered by background expression of the cloned gene, and leading to the selection of fast growing, plasmid containing but non expressing cells. Translational coupling, a mechanism of prokaryotic gene expression regulation, has been exploited to select cells able to express recombinant proteins. In the March 2015 issue of BioTechniques, researchers detail a new expression vector in which the cloned gene and a selective marker are co-expressed, allowing selective growth of expressing cells. This approach has proven to be effective on clones encoding toxic proteins. Keywords: protein expression; protein analysis; translational coupling; gene cloning; expression vectors

mRNA Analysis

The concept of 3′end formation of messenger RNA as a static minimally regulated housekeeping process has undergone a paradigm shift. Data from many recent studies have shown that accurate and efficient 3′end formation of mRNA is highly regulated and dysregulation of this process is a hallmark of several diseases. While, there are many global analyses monitoring altered mRNA processing, methods to investigate the specific RNA 3′end processing events in cells have not undergone significant changes. In this issue, researchers describe a facile, gain-of-function cellular reporter for the analysis of mRNA 3′end formation as an alternative to technically challenging or radioactive approaches and offer considerations for optimization and reproducibility. Keywords: cellular reporters; mRNA analysis; mRNA 3’end analysis; mRNA processing

Genomic DNA Isolation from Bacterial Species

Single-cell genomics (SCG) is a recently developed tool to study genomes of unculturable bacterial species and it relies on multiple-strand displacement amplification, polymerase chain reaction, and next-generation sequencing. But the identification method to obtain sufficient amounts of high-quality DNA from samples has become a major issue in current SCG research. In March researchers detail a modified lysing procedure that combines alkaline buffer and thermal shock (freeze/heat) which achieved higher efficiency and wider application. This procedure leads to higher efficiency in lysing Bacillus subtilis and Synechocystis cells comparing with other two frequently used lysis methods from the literature. Provided that more high-quality genome templates applicable to downstream researches have been obtained, the discovery of the current procedure is a promising improvement in SCG research. Keywords: single cell genomics; PCR; genomic DNA isolation; bacterial DNA isolation

Avoiding Contamination When Working with Algae Cultures

Chlamydomonas reinhardtii is a widely used unicellular green alga for studies in photosynthesis, cell cycle regulation, ciliary biogenesis and other physiological processes. Sterile cultures are needed for these researches. However, contamination from bacteria and fungi is inevitable and occurs frequently. Although One-shot Solution cocktail has been developed for removing these contaminants from algal cultures, it is not always effective. In the March issue of BioTechniques, scientists report two additional antibiotic cocktails to treat contamination of Chlamydomonas cultures. A combination of bactericide nalidixic with fungicide azoxystrobin or tebuconazole is more effective than the previously reported cocktail. Keywords: fungal and bacterial contamination; algal cultures; bactercide

Modified Enhancer Profiling and Sequence Scanning

Enhancer elements in most eukaryotic organisms are often positioned at a great distance away from the transcription start site of the gene they regulate. Complex three-dimensional chromatin organization and insulators usually guide and limit the range of an enhancer’s regulatory activity to a specific genetic locus. Rigorous testing of an entire genomic locus is often required in order to uncover the complete set of cis-regulatory modules (CRMs) regulating a gene, especially those with complex and dynamic expression patterns. In the March issue, scientists from Columbia University report a fast and efficient method for enhancer element identification by scanning large genomic regions using transgenic reporter genes. Keywords: enhancer elements; genomic regulatory elements; chromatin; insulators; transgenic reporters; sequence scanning

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