Table of Contents

Volume 3, Issue 1

  January/February 2012

  Pages 1 - 71

View covers archive

HOT TOPIC

Molecular diagnostics: the changing culture of medical microbiology

Susan Bullman, Brigid Lucey and Roy D. Sleator
Pages 1 - 7
http://dx.doi.org/10.4161/bbug.3.1.19011
Abstract | Full Text | PDF

Diagnostic molecular biology is arguably the fastest growing area in current laboratory-based medicine. Growth of the so called ‘omics’ technologies has, over the last decade, led to a gradual migration away from the ‘one test, one pathogen’ paradigm, toward multiplex approaches to infectious disease diagnosis, which have led to significant improvements in clinical diagnostics and ultimately improved patient care.




COMMENTARY

Genetic analysis of Down syndrome facilitated by mouse chromosome engineering

Li Zhang, Dawei Fu, Pavel V. Belichenko, Chunhong Liu, Alexander M. Kleschevnikov, Annie Pao, Ping Liang, Steven J. Clapcote, William C. Mobley and Y. Eugene Yu
Pages 8 - 12
http://dx.doi.org/10.4161/bbug.3.1.17696
Abstract | Full Text | PDF

Human trisomy 21 is the most frequent live-born human aneuploidy and causes a constellation of disease phenotypes classified as Down syndrome, which include heart defects, myeloproliferative disorder, cognitive disabilities and Alzheimer-type neurodegeneration. Because these phenotypes are associated with an extra copy of a human chromosome, the genetic analysis of Down syndrome has been a major challenge. To complement human genetic approaches, mouse models have been generated and analyzed based on evolutionary conservation between the human and mouse genomes. These efforts have been greatly facilitated by Cre/loxP-mediated mouse chromosome engineering, which may result in the establishment of minimal critical genomic regions and eventually new dosage-sensitive genes associated with Down syndrome phenotypes. The success in genetic analysis of Down syndrome will further enhance our understanding of this disorder and lead to better strategies in developing effective therapeutic interventions.




REVIEW

Open Access Article

Evolutionary, ecological and biotechnological perspectives on plasmids resident in the human gut mobile metagenome

Lesley A. Ogilvie, Sepinoud Firouzmand and Brian V. Jones
Pages 13 - 31
http://dx.doi.org/10.4161/bbug.3.1.17883
Abstract | Full Text | PDF

Numerous mobile genetic elements (MGE) are associated with the human gut microbiota and collectively referred to as the gut mobile metagenome. The role of this flexible gene pool in development and functioning of the gut microbial community remains largely unexplored, yet recent evidence suggests that at least some MGE comprising this fraction of the gut microbiome reflect the co-evolution of host and microbe in the gastro-intestinal tract. In conjunction, the high level of novel gene content typical of MGE coupled with their predicted high diversity, suggests that the mobile metagenome constitutes an immense and largely unexplored gene-space likely to encode many novel activities with potential biotechnological or pharmaceutical value, as well as being important to the development and functioning of the gut microbiota. Of the various types of MGE that comprise the gut mobile metagenome, plasmids are of particular importance since these elements are often capable of autonomous transfer between disparate bacterial species, and are known to encode accessory functions that increase bacterial fitness in a given environment facilitating bacterial adaptation. In this article current knowledge regarding plasmids resident in the human gut mobile metagenome is reviewed, and available strategies to access and characterize this portion of the gut microbiome are described. The relative merits of these methods and their present as well as prospective impact on our understanding of the human gut microbiota is discussed.




REPORT

A color-based stable multi-copy integrant selection system for Pichia pastoris using the attenuated ADE1 and ADE2 genes as auxotrophic markers

Min Du, Michael B. Battles and Juergen H. Nett
Pages 32 - 37
http://dx.doi.org/10.4161/bbug.3.1.17936
Abstract | Full Text | PDF

The methylotropic yeast Pichia pastoris has been used for more than two decades to successfully produce a large number of recombinant proteins. Currently, a wide variety of auxotrophic and drug based selection markers are employed to screen for clones expressing the protein of interest. For most proteins an increased copy number of the integrated plasmid results in higher levels of expression, but these multi-copy integrants can be unstable due to the propensity of P. pastoris for homologous recombination. Here we describe a multi-copy selection system based on ade1 and ade2 auxotrophic parent strains and the respective attenuated markers with truncated promoter regions. We show that for all four proteins we tested, the use of the attenuated markers leads to increased protein expression when compared with selection based on the full strength markers. The fact that the adenine auxotrophic strains grow more slowly than the complemented counterparts essentially ensures the stability of multi-copy integration. At the same time, the accumulation of a red dye in the auxotrophic strains also provides an easy, color-based selection for transformants with multiple copies.




RESEARCH NOTES

Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system

Stefan Werner, Carola Engler, Ernst Weber, Ramona Gruetzner and Sylvestre Marillonnet
Pages 38 - 43
http://dx.doi.org/10.4161/bbug.3.1.18223
Abstract | Full Text | PDF

Recent progress in the field of synthetic biology has led to the creation of cells containing synthetic genomes. Although these first synthetic organisms contained copies of natural genomes, future work will be directed toward engineering of organisms with modified genomes and novel phenotypes. Much work, however, remains to be done to be able to routinely engineer novel biological functions. As a tool that will be useful for such purpose, we have recently developed a modular cloning system (MoClo) that allows high throughput assembly of multiple genetic elements. We present here new features of this cloning system that allow to increase the speed of assembly of multigene constructs. As an example, 68 DNA fragments encoding basic genetic elements were assembled using three one-pot cloning steps, resulting in a 50 kb construct containing 17 eukaryotic transcription units. This cloning system should be useful for generating the multiple construct variants that will be required for developing gene networks encoding novel functions, and fine-tuning the expression levels of the various genes involved.

Recombinant D. radiodurans cells for bioremediation of heavy metals from acidic/neutral aqueous wastes

Chitra Seetharam Misra, Deepti Appukuttan, Venkata Siva Satyanarayana Kantamreddi, Amara S. Rao and Shree Kumar Apte
Pages 44 - 48
http://dx.doi.org/10.4161/bbug.3.1.18878
Abstract | Full Text | PDF

The stability and superior metal bioremediation ability of genetically engineered Deinococcus radiodurans cells, expressing a non-specific acid phosphatase, PhoN in high radiation environment has already been established. The lyophilized recombinant DrPhoN cells retained PhoN activity and uranium precipitation ability. Such cells also displayed an extended shelf life of 6 months during storage at room temperature and showed surface associated precipitation of uranium as well as other metals like cadmium. Lyophilized cells, immobilized in polyacrylamide gels could be used for uranium bioprecipitation in a flow through system resulting in 70% removal from 1mM input uranium solution and a loading of 1 g uranium/g dry weight cells. Compared with a batch process which achieved a loading of 5.7 g uranium/g biomass, the efficiency of the column process was low due to clogging of the column by the precipitate.




ADDENDA

Bench-top fermentative production of plant benzylisoquinoline alkaloids using a bacterial platform

Akira Nakagawa, Hiromichi Minami, Ju-Sung Kim, Takashi Koyanagi, Takane Katayama, Fumihiko Sato and Hidehiko Kumagai
Pages 49 - 53
http://dx.doi.org/10.4161/bbug.3.1.18446
Abstract | Full Text | PDF

The plant secondary metabolites benzylisoquinoline alkaloids (BIAs) have diverse pharmaceutical activities, and some are used medicinally (e.g., morphine, codeine, berberine). Recently, we constructed a platform to produce BIAs using bioengineered Escherichia coli, which could be useful for bulk production. The E. coli strain used in this system produces the important intermediate (S)-reticuline from glucose or glycerol. Although the amount produced (40 mg/L) exceeded the amount that can be purified from plants, the conversion efficiency from glycerol was only 0.15%; thus, there was much room for improvement. Our production system was developed in a jar fermenter but it is difficult to work with multiple samples using this system. In contrast, many samples can be cultured in parallel using shake flask cultures, allowing optimization of production conditions. Here, we describe bench-top production of (S)-reticuline and optimization of culture conditions using shake flask cultures. The production of (S)-reticuline reached 33.9 mg/L.

Differentiation of programmed Arabidopsis cells

De-Yu Xie and Ming-Zhu Shi
Pages 54 - 59
http://dx.doi.org/10.4161/bbug.3.1.17786
Abstract | Full Text | PDF

Plants express genes that encode enzymes that catalyse reactions to form plant secondary metabolites in specific cell types. However, the mechanisms of how plants decide their cellular metabolic fate and how cells diversify and specialise their specific secondary metabolites remains largely unknown. Additionally, whether and how an established metabolic program impacts genome-wide reprogramming of plant gene expression is unclear. We recently isolated PAP1-programmed anthocyanin-producing (red) and -free (white) cells from Arabidopsis thaliana; our previous studies have indicated that the PAP1 expression level is similar between these two different cell types. Transcriptional analysis showed that the red cells contain the TTG1-GL3/TT8-PAP1 regulatory complex, which controls anthocyanin biosynthesis; in contrast, the white cells and the wild-type cells lack this entire complex. These data indicate that different regulatory programming underlies the different metabolic states of these cells. In addition, our previous transcriptomic comparison indicated that there is a clear difference in the gene expression profiles of the red and wild-type cells, which is probably a consequence of cell-specific reprogramming. Based on these observations, in this report we discuss the potential mechanisms that underlie the programming and reprogramming of gene expression involved in anthocyanin biosynthesis.

Computational studies on a new cationic peroxidase isoenzyme from artichoke leaves

Angela Cardinali, Isabella D’Antuono, Benny Danilo Belviso and Rocco Caliandro
Pages 60 - 66
http://dx.doi.org/10.4161/bbug.3.1.17937
Abstract | Full Text | PDF

Previously we presented the purification, biochemical characterization, and cloning of a cationic peroxidase isoenzyme (CysPrx) from artichoke (Cynara cardunculus subsp scolymus (L.) Hegi) leaves. The protein was shown to have some interesting properties, suggesting that CysPrx could be a considered as a potential candidate for industrial application. In addition, from the CysPrx sequence, two full-lengh cDNAs: CysPrx1 and CysPrx2, differing for three amino acids, were isolated. A three-dimensional model was predicted from CysPrx1 by homology modeling, using two different computational tools. Herein we discuss the roles of particular amino acid residues and structural motifs or regions of both deduced sequences with the aim to find new understandings between the new plant peroxidase isoenzymes and their physiological substrates. Additionally, the obtained information may lead to new methods for improving the stability of the enzyme in several processes of biotechnological interest for peroxidase applications.

Biomedicals from a soil bug: Expanding scFv production host range

Thorben Dammeyer
Pages 67 - 71
http://dx.doi.org/10.4161/bbug.3.1.17739
Abstract | Full Text | PDF

Recombinant antibody fragments have a wide range of applications from research to diagnostics and therapy. Of special interest are small fragments like fragment antigen binding (Fab) or single chain fragment variables (scFv) fragments as they can be produced inexpensively in bacterial expression systems. However, recombinant production efficiencies from established production hosts vary significantly leading to inadequate yields. Gene sequences that have been synthetically adapted to match the codon preferences and respective genomic tRNA pool of the host have been used to improve yields but cannot resolve the principal problem. The development of inducible broad host range scFv expression plasmid constructs leads the way to an easy and efficient screening method for the identification of the optimal bacterial expression host.