Engineering genetic circuits: advancements in genetic design automation tools and standards for synthetic biology

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Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Fungal populations of the haulm base of wheat

Abstract In a 2-year field survey, the phylloplane mycoflora on the haulm base of wheat was studied by the dilution plate technique. No significant colonisation was registered before the end of May. Cladosporium spp., 'white' and 'red' yeasts were found to be the most abundant fungi.     

A catalogue of <Emphasis Type="Italic">Triticum monococcum</Emphasis> genes encoding toxic and immunogenic peptides for celiac disease patients

The celiac disease (CD) is an inflammatory condition characterized by injury to the lining of the small-intestine on exposure to the gluten of wheat, barley and rye. The involvement of gluten in the CD syndrome has been studied in detail in bread wheat, where a set of “toxic” and “immunogenic” peptides has been defined. For wheat diploid species, information on CD epitopes is poor. In the present paper, we have adopted a genomic approach in order to understand the potential CD danger represented by storage proteins in diploid wheat and sequenced a sufficiently large number of cDNA clones related to storage protein genes of Triticum monococcum. Four bona fide toxic peptides and 13 immunogenic peptides were found. All the classes of storage proteins were shown to contain harmful sequences. The major conclusion is that einkorn has the full potential to induce the CD syndrome, as already evident for polyploid wheats. In addition, a complete overview of the storage protein gene arsenal in T. monococcum is provided, including a full-length HMW x-type sequence and two partial HMW y-type sequences. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Internal lipid synthesis and vesicle growth as a step toward self-reproduction of the minimal cell

One of the major properties of the semi-synthetic minimal cell, as a model for early living cells, is the ability to self-reproduce itself, and the reproduction of the boundary layer or vesicle compartment is part of this process. A minimal bio-molecular mechanism based on the activity of one single enzyme, the FAS-B (Fatty Acid Synthase) Type I enzyme from Brevibacterium ammoniagenes, is encapsulated in 1-palmitoyl-2oleoyl-sn-glycero-3-phosphatidylcholine (POPC) liposomes to control lipid synthesis. Consequently molecules of palmitic acid released from the FAS catalysis, within the internal lumen, move toward the membrane compartment and become incorporated into the phospholipid bilayer. As a result the vesicle membranes change in lipid composition and liposome growth can be monitored. Here we report the first experiments showing vesicles growth by catalysis of one enzyme only that produces cell boundary from within. This is the prototype of the simplest autopoietic minimal cell.     

Effects of global environmental change on carbon partitioning in vegetative plants of Triticum aestivum and closely related Aegilops species

The use of fossil fuel is predicted to cause an increase of the atmospheric CO₂ concentration, which will affect the global pattern of temperature and precipitation. It is therefore essential to incorporate effects of temperature and water supply on carbon partitioning of plants to predict effects of elevated [CO₂] on growth and yield of Triticum aestivum. Although earlier papers have emphasized that elevated [CO₂] favours investment of biomass in roots relative to that in leaves, it has now become clear that these are indirect effects, due to the more rapid depletion of nutrients in the root environment as a consequence of enhanced growth. Broadly generalized, the effect of temperature on biomass allocation in the vegetative stage is that the relative investment of biomass in roots is lowest at a certain optimum temperature and increases at both higher and lower temperatures. This is found not only when the temperature of the entire plant is varied, but also when only root temperature is changed whilst shoot temperature is kept constant. Effects of temperature on the allocation pattern can be explained largely by the effect of root temperature on the roots' capacity to transport water. Effects of a shortage in water supply on carbon partitioning are unambiguous: roots receive relatively more carbon. The pattern of biomass allocation in the vegetative stage and variation in water-use efficiency are prime factors determining a plant's potential for early growth and yield in different environments. In a comparison of a range of T. aestivum cultivars, a high water-use efficiency at the plant level correlates positively with a large investment in both leaf and root biomass, a low stomatal conductance and a large investment in photosynthetic capacity. We also present evidence that a lower investment of biomass in roots is not only associated with lower respiratory costs for root growth, but also with lower specific costs for ion uptake. We suggest the combination of a number of traits in future wheat cultivars, i.e. a high investment of biomass in leaves, which have a low stomatal conductance and a high photosynthetic capacity, and a low investment of biomass in roots, which have low respiratory costs. Such cultivars are considered highly appropriate in a future world, especially in the dryer regions. Although variation for the desired traits already exists among wheat cultivars, it is much larger among wild Aegilops species, which can readily be crossed with T. aestivum. Such wild relatives may be exploited to develop new wheat cultivars well-adapted to changed climatic conditions.     

The critical weed-free period in organically-grown winter wheat

Two experiments were conducted in central southern England between September 1994 and August 1996 to identify the critical weed-free period in organically grown winter wheat (Triticum aestivum, cv. Mercia). In competition with a mixed weed infestation of predominately Alopecurus myosuroides and Tripleurospermum inodorum it was found that wheat yield decreased as the duration of the weed-infested period increased and that the crop needed to be kept free of weeds from sowing in order to completely avoid any yield loss. Also, weeds emerging in the wheat crop (predominately T. inodorum) during the growing season had a significant and detrimental effect on yield. The existence of the critical period, therefore, depends on the imposition of an acceptable yield loss. If a 5% yield loss gives a marginal benefit compared with the cost of weed control, the critical period will begin at 506°C days after sowing (November) and end at 1023°C days after sowing (February). This information could be used by farmers to target mechanical weeding operations to control weeds at a time that will have maximum benefit to the crop.     

Variation in noxiousness of different wheat species for celiac patients

Abstract

Peptic-tryptic (PT) digested prolamins from spelt wheat Triticum aestivum ssp. spelta and landraces ERSA 6 and ERSA 8 of farro wheat T. turgidum ssp. dicoccum were found to agglutinate K562(S) cells, and exert strong toxic effects on Caco-2/TC7 cells. Cytotoxicity of spelt prolamins against Caco-2/TC7 cells was greatly reduced by 10-mer peptide QQPQDAVQPF. By contrast, the PT digests from monoccum wheat (Triticum monococcum) and farro landraces Prometeo, L5563, L5540 and L5558 did not exhibit any negative effects on K562(S) and Caco-2/TC7 cells. Toxic genotypes ERSA 6 and ERSA 8 were found to share the same gliadin pattern, which was absent in inactive landraces. Monococcum, farro and spelt wheats differed from each other in their responses to antibodies specific for 13-mer cytotoxic sequence FPGQQQPFPPQQP and 10-mer peptide QQPQDAVQPF. This latter sequence was found to occur in high amounts in common wheat line FG, Phaseoulus vulgaris, Ph. coccineus and Lens culinaria.     

Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.