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.     

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.     

The application of a potential-sensitive cyanine dye to rat small intestinal brush border membrane vesicles

The sensitivity of the fluorescent dye, 3,3′-diethylthiadicarbocyanine (DiS-C₂(5)), was too low for the detection of membrane potential changes in rat small intestinal membrane vesicles. Only after adding LaCl₃ or after fractionation of the intestinal membranes by free-flow electrophoresis could the dye be used to monitor electrogenic Na⁺-dependent transport systems. It is concluded that the response of this potential-sensitive dye is influenced by the negative surface charge density of the vesicles.     

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.     

A general approach to the concept of varietal ability for synthetic varieties

Summary Genetic effects for varietal value are defined at the level of the population of k-parent synthetic varieties. A simple expression for the total variance among synthetics arises directly from these definitions. A general expression for the covariance among related synthetics is given. Genetic effects are also defined in a completely general way so as to allow for any system of testing and used to derive an expression for the genetic advance in recurrent selection for varietal value. Covariances between relatives evaluated in the system of testing and in varietal combination are introduced, allowing a direct expression of the genetic advance in varietal development when parents are selected either individually or in groups. Some general implications for plant breeding are outlined.Dedicated to Professor F.W. Schnell on the occasion of his 65th birthday     

Specificity of chimpanzee antibodies binding a strain-specific HIV-1 neutralization epitope of the external envelope

Sera from three chimpanzees infected with a primary lymphadenopathy-associated virus (LAV-1) or human T-lymphotropic virus type III (HTLV-IIIB) passage, from two chimpanzees infected with blood from the primary infected chimpanzees, and from one chimpanzee infected with blood from a secondary passage animal all bound the peptides 3B and 3B/RF, sharing the sequence IQRGPGR, with equally high titers. Pepscan analysis confirmed the amino acids Q, R, G, P, and G as irreplaceable in order to retain antigenicity.     

Cell age-related differences in the interaction of a potential-sensitive fluorescent dye with nuclear envelopes of Acetabularia mediterranea

Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC₆(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC₆(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.     

Effects of membrane potential on Na cotransports in eel intestinal brush-border membrane vesicles: Studies with a fluorescent dye

Summary The Na-dependent transport of a number of organic molecules (d-glucose,l-proline,l-alanine,l-phenylalanine) in brush-border membrane vesicles isolated from the intestine of the eel (Anguilla anguilla) was monitored by recording the fluorescence quenching of the voltage-sensitive cyanine dye 3,3-diethylthiacarbocyanine iodide (DiS-C₂(5)). The experimental approach consisted of: a) generating an inside-negative membrane potential mimicking in vivo conditions: b) measuring the rate of membrane potential decay (i.e., the rate of fluorescence quenching decay) due to Na-neutral substrate cotransport. Rates of membrane potential decay showed saturation on substrate concentration andK _app values (the substrate concentration giving 50% of the maximal rate) were estimated for Na-dependent transport ofd-glucose (0,099mm),l-alanine (0.516mm),l-proline (0.118mm) andl-phenylalanine (2.04mm). The influence of an inside-negative membrane potential on the affinity of the transporter for glucose and for sodium is discussed.