jueves, 9 de abril de 2015

The future of the postdoc

There is a growing number of postdocs and few places in academia for them to go. But change could be on the way.


martes, 24 de marzo de 2015

An Escherichia coli Mutant That Makes Exceptionally Long Cells

Although Escherichia coli is a very small (1- to 2-μm) rod-shaped cell, here we describe an E. coli mutant that forms enormously long cells in rich media such as Luria broth, as long indeed as 750 μm. These extremely elongated (eel) cells are as long as the longest bacteria known and have no internal subdivisions. They are metabolically competent, elongate rapidly, synthesize DNA, and distribute cell contents along this length. They lack only the ability to divide. The concentration of the essential cell division protein FtsZ is reduced in these eel cells, and increasing this concentration restores division.

IMPORTANCE Escherichia coli is usually a very small bacterium, 1 to 2 μm long. We have isolated a mutant that forms enormously long cells, 700 times longer than the usual E. coli cell. E. coli filaments that form under other conditions usually die within a few hours, whereas our mutant is fully viable even when it reaches such lengths. This mutant provides a useful tool for the study of aspects of E. coli physiology that are difficult to investigate with small cells.      

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lunes, 16 de marzo de 2015

Diverse uncultivated ultra-small bacterial cells in groundwater

Bacteria from phyla lacking cultivated representatives are widespread in natural systems and some have very small genomes. Here we test the hypothesis that these cells are small and thus might be enriched by filtration for coupled genomic and ultrastructural characterization. Metagenomic analysis of groundwater that passed through a ~0.2-μm filter reveals a wide diversity of bacteria from the WWE3, OP11 and OD1 candidate phyla. Cryogenic transmission electron microscopy demonstrates that, despite morphological variation, cells consistently have small cell size (0.009±0.002 μm3). Ultrastructural features potentially related to cell and genome size minimization include tightly packed spirals inferred to be DNA, few densely packed ribosomes and a variety of pili-like structures that might enable inter-organism interactions that compensate for biosynthetic capacities inferred to be missing from genomic data. The results suggest that extremely small cell size is associated with these relatively common, yet little known organisms.

Cryo-electron tomography images from 3D reconstructions of ultra-small bacteria.

Cryo-electron tomography images from 3D reconstructions of ultra-small bacteria.

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miércoles, 11 de marzo de 2015

CRISPR/Cas is all the rage—and getting more precise and efficient.

Una reciente revisión:

CRISPR (clustered, regularly interspaced, short palindromic repeats) is named for particular DNA loci that are found in many archaea and bacteria. CRISPR works with associated nucleases, including Cas9, to protect the cells from viral infection by inserting short snippets of viral DNA into the CRISPR cassette. By combining the Cas9 nuclease with a short guide RNA that’s custom-designed to bind a specific target, CRISPR/Cas can easily edit any gene you want. Just in the past year, for example, it has allowed researchers to cure a rare liver disease in mice, to excise HIV-inserted genes from human immune cells, and to block HIV from entering blood stem cells. CRISPR/Cas is easier than the other nuclease-based editing technologies, says John Schimenti of Cornell University; scientists are basically a reagent catalog and a round of PCR away from having everything they need to utilize CRISPR.


viernes, 6 de febrero de 2015

Recoded organisms engineered to depend on synthetic amino acids

Genetically modified organisms (GMOs) are increasingly used in research and industrial systems to produce high-value pharmaceuticals, fuels and chemicals. Genetic isolation and intrinsic biocontainment would provide essential biosafety measures to secure these closed systems and enable safe applications of GMOs in open systems, which include bioremediation and probiotics. Although safeguards have been designed to control cell growth by essential gene regulation, inducible toxin switches and engineered auxotrophies, these approaches are compromised by cross-feeding of essential metabolites, leaked expression of essential genes, or genetic mutations.

Here we describe the construction of a series of genomically recoded organisms (GROs) whose growth is restricted by the expression of multiple essential genes that depend on exogenously supplied synthetic amino acids (sAAs). We introduced a Methanocaldococcus jannaschii tRNA:aminoacyl-tRNA synthetase pair into the chromosome of a GRO derived from Escherichia coli that lacks all TAG codons and release factor 1, endowing this organism with the orthogonal translational components to convert TAG into a dedicated sense codon for sAAs. Using multiplex automated genome engineering, we introduced in-frame TAG codons into 22 essential genes, linking their expression to the incorporation of synthetic phenylalanine-derived amino acids. Of the 60 sAA-dependent variants isolated, a notable strain harbouring three TAG codons in conserved functional residuesof MurG, DnaA and SerS and containing targeted tRNA deletions maintained robust growth and exhibited undetectable escape frequencies upon culturing ~1011 cells on solid media for 7 days or in liquid media for 20 days. This is a significant improvement over existing biocontainment approaches.

We constructed synthetic auxotrophs dependent on sAAs that were not rescued by cross-feeding in environmental growth assays. These auxotrophic GROs possess alternative genetic codes that impart genetic isolation by impeding horizontal gene transfer and now depend on the use of synthetic biochemical building blocks, advancing orthogonal barriers between engineered organisms and the environment.

Nature 518, 89–93 (05 February 2015) doi:10.1038/nature14095

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jueves, 8 de enero de 2015

Un nuevo antibiótico de un microorganismo "no cultivable"

Antibiotic resistance is spreading faster than the introduction of new compounds into clinical practice, causing a public health crisis. Most antibiotics were produced by screening soil microorganisms, but this limited resource of cultivable bacteria was overmined by the 1960s. Synthetic approaches to produce antibiotics have been unable to replace this platform. Uncultured bacteria make up approximately 99% of all species in external environments, and are an untapped source of new antibiotics. We developed several methods to grow uncultured organisms by cultivation in situ or by using specific growth factors. Here we report a new antibiotic that we term teixobactin, discovered in a screen of uncultured bacteria. Teixobactin inhibits cell wall synthesis by binding to a highly conserved motif of lipid II (precursor of peptidoglycan) and lipid III (precursor of cell wall teichoic acid). We did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin. The properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.

The structure of teixobactin and the predicted biosynthetic gene cluster.

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jueves, 25 de septiembre de 2014

A faster Rubisco with potential to increase photosynthesis in crops

In photosynthetic organisms, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere1. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants2, 3. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield4, 5, 6. However, the complex nature of Rubisco’s assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful7, 8. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains9, 10. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial β-carboxysomes11, 12. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the β-carboxysome shell proteins4, 5, 6.

Phenotype of the wild-type and transplastomic tobacco lines.

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