A standard vector for the chromosomal integration and characterization of BioBrick™ parts in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background

The chromosomal integration of biological parts in the host genome enables the engineering of plasmid-free stable strains with single-copy insertions of the desired gene networks. Although different integrative vectors were proposed, no standard pre-assembled genetic tool is available to carry out this task. Synthetic biology concepts can contribute to the development of standardized and user friendly solutions to easily produce engineered strains and to rapidly characterize the desired genetic parts in single-copy context.

Results

In this work we report the design of a novel integrative vector that allows the genomic integration of biological parts compatible with the RFC10, RFC23 and RFC12 BioBrick? standards in Escherichia coli. It can also be specialized by using BioBrick? parts to target the desired integration site in the host genome. The usefulness of this vector has been demonstrated by integrating a set of BioBrick? devices in two different loci of the E. coli chromosome and by characterizing their activity in single-copy. Construct stability has also been evaluated and compared with plasmid-borne solutions.

Conclusions

Physical modularity of biological parts has been successfully applied to construct a ready-to-engineer BioBrick? vector, suitable for a stable chromosomal insertion of standard parts via the desired recombination method, i.e. the bacteriophage integration mechanism or homologous recombination. In contrast with previously proposed solutions, it is a pre-assembled vector containing properly-placed restriction sites for the direct transfer of various formats of BioBrick? parts. This vector can facilitate the characterization of parts avoiding copy number artefacts and the construction of antibiotic resistance-free engineered microbes, suitable for industrial use.

     

Neurochemical Changes in the Mouse Hippocampus Underlying the Antidepressant Effect of Genetic Deletion of P2X7 Receptors

Recent investigations have revealed that the genetic deletion of P2X7 receptors (P2rx7) results in an antidepressant phenotype in mice. However, the link between the deficiency of P2rx7 and changes in behavior has not yet been explored. In the present study, we studied the effect of genetic deletion of P2rx7 on neurochemical changes in the hippocampus that might underlie the antidepressant phenotype. P2X7 receptor deficient mice (P2rx7−/−) displayed decreased immobility in the tail suspension test (TST) and an attenuated anhedonia response in the sucrose preference test (SPT) following bacterial endotoxin (LPS) challenge. The attenuated anhedonia was reproduced through systemic treatments with P2rx7 antagonists. The activation of P2rx7 resulted in the concentration-dependent release of [³H]glutamate in P2rx7+/+ but not P2rx7−/− mice, and the NR2B subunit mRNA and protein was upregulated in the hippocampus of P2rx7−/− mice. The brain-derived neurotrophic factor (BDNF) expression was higher in saline but not LPS-treated P2rx7−/− mice; the P2rx7 antagonist Brilliant blue G elevated and the P2rx7 agonist benzoylbenzoyl ATP (BzATP) reduced BDNF level. This effect was dependent on the activation of NMDA and non-NMDA receptors but not on Group I metabotropic glutamate receptors (mGluR_1,5). An increased 5-bromo-2-deoxyuridine (BrdU) incorporation was also observed in the dentate gyrus derived from P2rx7−/− mice. Basal level of 5-HT was increased, whereas the 5HIAA/5-HT ratio was lower in the hippocampus of P2rx7−/− mice, which accompanied the increased uptake of [³H]5-HT and an elevated number of [³H]citalopram binding sites. The LPS-induced elevation of 5-HT level was absent in P2rx7−/− mice. In conclusion there are several potential mechanisms for the antidepressant phenotype of P2rx7−/− mice, such as the absence of P2rx7-mediated glutamate release, elevated basal BDNF production, enhanced neurogenesis and increased 5-HT bioavailability in the hippocampus.     

Replacement of the Y450 (c234) phenyl ring in the carboxyl-terminal region of coagulation factor IX causes pleiotropic effects on secretion and enzyme activity

The interplay between impaired protein biosynthesis and/or function caused by missense mutations, particularly in relation to specific protein regions, has been poorly investigated. As model we chose the severe p.Y450C mutation in the carboxyl-terminal region of coagulation factor IX (FIX) and, by expression of a panel of recombinant variants, demonstrated the key role of the tyrosine phenyl group for both FIX secretion and coagulant activity. Comparison among highly homologous coagulation serine proteases indicate that additive or compensatory pleiotropic effects on secretion and function by carboxyl-terminal mutations produce life-threatening or mild phenotypes in the presence of similarly reduced protein amounts.     

Alternative splicing of human insulin receptor gene (INSR) in type I and type II skeletal muscle fibers of patients with myotonic dystrophy type 1 and type 2

INSR, one of those genes aberrantly expressed in myotonic dystrophy type 1 (DM1) and type 2 (DM2) due to a toxic RNA effect, encodes for the insulin receptor (IR). Its expression is regulated by alternative splicing generating two isoforms: IR-A, which predominates in embryonic tissue, and IR-B, which is highly expressed in adult, insulin-responsive tissues (skeletal muscle, liver, and adipose tissue). The aberrant INSR expression detected in DM1 and DM2 muscles tissues, characterized by a relative increase of IR-A versus IR-B, was pathogenically related to the insulin resistance occurring in DM patients. To assess if differences in the aberrant splicing of INSR could underlie the distinct fiber type involvement observed in DM1 and DM2 muscle tissues, we have used laser capture microdissection (LCM) and RT-PCR, comparing the alternative splicing of INSR in type I and type II muscle fibers isolated from muscle biopsies of DM1, DM2 patients and controls. In the controls, the relative amounts of IR-A and IR-B showed no obvious differences between type I and type II fibers, as in the whole muscle tissue. In DM1 and DM2 patients, both fiber types showed a similar, relative increase of IR-A versus IR-B, as also evident in the whole muscle tissue. Our data suggest that the distinct fiber type involvement in DM1 and DM2 muscle tissues would not be related to qualitative differences in the expression of INSR. LCM can represent a powerful tool to give a better understanding of the pathogenesis of myotonic dystrophies, as well as other myopathies.     

Effect of tree density on root distribution in Fagus sylvatica stands: a semi-automatic digitising device approach to trench wall method

Knowledge of root profiles is essential for measuring and predicting ecosystem dynamics and function. In the present study, the effects of management practices on root (0.5 ≤ ø < 20 mm) spatial distribution were examined in a 40-year-old coppice stand (CpS 1968) and other two stands converted from coppice to thinned high forest in 1994 (CvS 1994) and 2004 (CvS 2004), respectively. The use of a semi-automatic digitising device approach was compared with a conventional root mapping method in order to estimate the time per person required from fieldwork to the final digital map. In July 2009, six trench walls per stand were established according to tree density, i.e. as equidistant as possible from all surrounding trees. Findings highlighted differences between the stands with CvS 1994 showing a lower number of small roots (2 ≤ ø < 5 mm), a higher mean cross-sectional area (CSA) of coarse roots (5 ≤ ø < 20 mm) and different root depth distribution as compared to CpS 1968 and CvS 2004 whose values were close to each other. The three diameter classes selected in this study showed significant relationships in terms of number of roots, scaling down from coarse- to small- and fine-roots. Forest management practices significantly affected only the number of small roots. The number of fine roots (0.5 ≤ ø < 2 mm) was isometrically related to their root length density (RLD, cm cm^?3). No relationship occurred with RLD of very fine roots (ø < 0.5 mm). In conclusion, forest management practices in terms of conversion thinnings significantly affected belowground biomass distribution of beech forest in space and time. In particular, frequency of coarse roots was related to the stand tree density, frequency of small roots was related to the cutting age. Size of coarse roots was related to tree density but only several years after felling. The allometric relationship occurring between fine- and small-roots highlighted how fine root number and RLD were only indirectly affected by forest management practices. These findings suggest that future investigations on the effect of forest thinning practices on fine-root traits like number, length and biomass several years after felling cannot ignore those on small roots.