Synthetic biology: new strategies for directing design

The advancement of synthetic biology is thanks, in large part, to continuing improvements in DNA synthesis. The expansion of synthetic biology into the realm of metabolic engineering has shifted the focus from simply making novel synthetic biological parts to answering the question of how we employ these biological parts to construct genomes that ultimately give rise to useful phenotypes. Much like protein engineering, the answer to this will be arrived at following the combination of rational design and evolutionary approaches. This review will highlight some of the new DNA synthesis-enabled search methods and discuss the application of such methods to the creation of synthetic gene networks and genomes.     

Predicting antigenic sites on proteins.

The ability to predict antigenic sites on proteins is of major importance for the production of synthetic peptide vaccines and synthetic peptide probes of antibody structure. Many predictive methods, based on various assumptions about the nature of the antigenic response have been proposed and tested. This review will discuss the principles underlying the various approaches to predicting antigenic sites and will attempt to answer the question of how well they work.     

Creating parts that allow for rational design: Synthetic biology and the problem of context-sensitivity

The parts-based engineering approach in synthetic biology aims to create pre-characterised biological parts that can be used for the rational design of novel functional systems. Given the context-sensitivity of biological entities, a key question synthetic biologists have to address is what properties these parts should have so that they give a predictable output even when they are used in different contexts. In the first part of this paper I will analyse some of the answers that synthetic biologists have given to this question and claim that the focus of these answers on parts and their properties does not allow us to tackle the problem of context-sensitivity. In the second part of the paper, I will argue that we might have to abandon the notions of parts and their properties in order to understand how independence in biology could be achieved. Using Robert Cummins’ account of functional analysis, I will then develop the notion of a capacity and its condition space and show how these notions can help to tackle the problem of context-sensitivity in biology.     

Spontaneous transmembrane pore formation by short-chain synthetic peptide

Amphiphilic β-peptides, which are synthetically designed short-chain helical foldamers of β-amino acids, are established potent biomimetic alternatives of natural antimicrobial peptides. An intriguing question is how the distinct molecular architecture of these short-chain and rigid synthetic peptides translates to its potent membrane-disruption ability. Here, we address this question via a combination of all-atom and coarse-grained molecular dynamics simulations of the interaction of mixed phospholipid bilayer with an antimicrobial 10-residue globally amphiphilic helical β-peptide at a wide range of concentrations. The simulation demonstrates that multiple copies of this synthetic peptide, initially placed in aqueous solution, readily self-assemble and adsorb at membrane interface. Subsequently, beyond a threshold peptide/lipid ratio, the surface-adsorbed oligomeric aggregate moves inside the membrane and spontaneously forms stable water-filled transmembrane pores via a cooperative mechanism. The defects induced by these pores lead to the dislocation of interfacial lipid headgroups, membrane thinning, and substantial water leakage inside the hydrophobic core of the membrane. A molecular analysis reveals that despite having a short architecture, these synthetic peptides, once inside the membrane, would stretch themselves toward the distal leaflet in favor of potential contact with polar headgroups and interfacial water layer. The pore formed in coarse-grained simulation was found to be resilient upon structural refinement. Interestingly, the pore-inducing ability was found to be elusive in a non-globally amphiphilic sequence isomer of the same β-peptide, indicating strong sequence dependence. Taken together, this work puts forward key perspectives of membrane activity of minimally designed synthetic biomimetic oligomers relative to the natural antimicrobial peptides.     

Analysis of internal (n-1)mer deletion sequences in synthetic oligodeoxyribonucleotides by hybridization to an immobilized probe array.

The purity of a drug substance can influence its toxicity and potency, so impurities must be specifically determined. In the case of synthetic oligodeoxyribonucleotide drugs, however, product complexity makes complete impurity speciation difficult. The goal of the present work was to develop a new analytical method for speciation of individual internal (n-1)mer impurities arising from formal nucleotide deletion in synthetic oligodeoxyribonucleotides. A complete series of oligodeoxyribonucleotide probes were designed, each complementary to an (n-1)mer deletion sequence of the drug in question. Glass plates were used as a solid support for individually immobilizing the entire probe array. The total mixture of internal (n-1) length impurities was isolated from a synthetic oligodeoxyribonucleotide by PAGE and labeled with 35S. Under stringently optimized conditions, only the perfectly sequence-matched oligodeoxyribonucleotide hybridized to each probe, while all other deletion sequences were removed by washing with buffer. The 35S signal intensity of the bound oligodeoxyribonucleotide was proportional to the concentration of each (n-1)mer deletion sequence in the analyte solution. This method has been applied to a number of synthetic phosphorothioate oligodeoxy-ribonucleotide lots and shown to be reliable for speciation and relative quantitation of the internal (n -1)mer deletion sequences present.     

Discipline-building in synthetic biology

Despite the multidisciplinary dimension of the kinds of research conducted under the umbrella of synthetic biology, the US-based founders of this new research area adopted a disciplinary profile to shape its institutional identity. In so doing they took inspiration from two already established fields with very different disciplinary patterns. The analogy with synthetic chemistry suggested by the term ‘synthetic biology’ is not the only model. Information technology is clearly another source of inspiration. The purpose of the paper, with its focus on the US context, is to emphasize the diversity of views and agendas coexisting under the disciplinary label synthetic biology, as the two models analysed are only presented as two extreme postures in the community. The paper discusses the question: in which directions the two models shape this emerging field? Do they chart two divergent futures for synthetic biology?     

Fast, cheap and somewhat in control

Efforts to manipulate living organisms have raised the question of whether engineering principles of hierarchy, abstraction and design can be applied to biological systems. Here, we consider the practical challenges to controlling living organisms that must be surmounted, or at least managed, if synthetic biology and cellular bioengineering are to be productive.     

On the conformation of the alpha sarcin stem-loop of 28S rRNA.

A synthetic RNA that is a substrate for the cytotoxin alpha sarcin has been examined by NMR. The molecule in question includes the entire sequence of the so-called alpha sarcin loop from rat 28S rRNA (U4316-C4332), and it is cleaved at the residue that corresponds to G4325, the site of alpha sarcin cleavage in 28S rRNA. The data show that the terminal stem designed into the molecule's sequence exists, as expected, and that its loop has a definite structure, which is stable to at least 40 degrees C under ionic conditions compatible with its cleavage by alpha sarcin.     

Accounting for Linear Transformations of EEG and MEG Data in Source Analysis

Analyses of electro- and magnetoencephalography (EEG, MEG) data often involve a linear modification of signals at the sensor level. Examples include re-referencing of the EEG, computation of synthetic gradiometer in MEG, or the removal of artifactual independent components to clean EEG and MEG data. A question of practical relevance is, if such modifications must be accounted for by adapting the physical forward model (leadfield) before subsequent source analysis. Here, we show that two scenarios need to be differentiated. In the first scenario, which corresponds to re-referencing the EEG and synthetic gradiometer computation in MEG, the leadfield must be adapted before source analysis. In the second scenario, which corresponds to removing artifactual components to ‘clean’ the data, the leadfield must not be changed. We demonstrate and discuss the consequences of wrongly modifying the leadfield in the latter case for an example. Future EEG and MEG studies employing source analyses should carefully consider whether and, if so, how the leadfield must be modified as explicated here.     

Mapping antibody binding sites on cytochrome c with synthetic peptides: Are results representative of the antigenic structure of proteins?

Crystallographic work on antigen-antibody complexes has revealed that extensive surface areas of proteins may interact with antibodies. On the other hand, most experimental approaches to locate and define antigenic determinants of protein antigens rely on the linear sequence of the polypeptide chain. Hence the question arises whether mapping of antibody binding sites by analysis of the reactivity of anti-protein antibodies with synthetic peptides can provide a representative picture of the antigenic structure of a protein antigen. We have addressed this question using yeast iso-1 cytochrome c as a protein antigen against which antisera were raised in rabbits. The reaction of the antisera with 103 synthetic hexapeptides covering the entire sequence of cytochrome c was tested by the pepscan procedure in which peptides are coupled to polyethylene rods and tested by ELISA. For the assay, anti-cytochrome c antibodies were fractionated by affinity chromatography on native yeast iso-1 cytochrome c and on apo-cytochrome c; the latter is a random coil. It was found that only antibodies retained by the apo-cytochrome c affinity column react with synthetic peptides. These antibodies comprise a small fraction, probably less than 2%, of all cytochrome c-specific antibodies. The majority of antigenic determinants, which seem to consist of strongly conformation-dependent topographic epitopes, could not be uncovered by the peptide approach. Epitope mapping with short peptides seems of limited usefulness in the case of small, globular, and conformationally stable proteins like cytochrome c.     

Technological biology? Things and kinds in synthetic biology

Social scientific and humanistic research on synthetic biology has focused quite narrowly on questions of epistemology and ELSI. I suggest that to understand this discipline in its full scope, researchers must turn to the objects of the field—synthetic biological artifacts—and study them as the objects in the making of a science yet to be made. I consider one fundamentally important question: how should we understand the material products of synthetic biology? Practitioners in the field, employing a consistent technological optic in the study and construction of biological systems, routinely employ the mantra ‘biology is technology’. I explore this categorization. By employing an established definition of technological artifects drawn from the philosophy of technology, I explore the appropriateness of attributing to synthetic biological artifacts the four criteria of materiality, intentional design, functionality, and normativity. I then explore a variety of accounts of natural kinds. I demonstrate that synthetic biological artifacts fit each kind imperfectly, and display a concomitant ontological ‘messiness’. I argue that this classificatory ambivalence is a product of the field’s own nascence, and posit that further work on kinds might help synthetic biology evaluate its existing commitments and practices.     

Double-blind evaluation of oral L-prolyl-Lleucyl-glycine amide in Parkinson's disease.

A 4-month double-blind study comparing the effect of increasing oral doses (up to 1.0 g daily) of synthetic L-proyl-L-leucyl-glycine amide (PLG) and placebo in 20 parkinsonian patients showed no significant improvement in objective scores of functional disability. However, important trends and some significant results were observed with the lower doses of PLG. These essentially negative results may be attributed to poor intestinal absorption of the compound, a short biologic half-life in the blood, or administration of oral doses that were much higher than required, or a combination of factors. In further studies with this peptide, which are encouraged, the intravenous route should be used until the question of intestinal absorption is resolved.     

Expression of the synthetic phytochelatin gene in tobacco

In the process of obtaining transgenic plants suitable for phytoremediation of heavy metal-contaminated soils, a synthetic pseudophytochelatin gene coding for a phytochelatin analog Met(GluCys)₆Gly was de novo designed and cloned. Contrary to natural enzymatically synthesized phytochelatins, this peptide can be made by a template synthesis. A construct carrying the gene in question under the control of the viral constitutive 35S promoter was made on the basis of a binary vector pCAMBIA 1305.1. This construct was used to express the pseudophytochelatin gene in the model transgenic tobacco plants (Nicotiana tabacum L.), and the above plants acquired additional resistance to various cadmium concentrations.     

蜜蜂性别决定与性比调控机理研究

叙述了 4个主要蜜蜂性别决定机理的假说 :即性位点假说、基因平衡假说、蜜蜂性别决定综合假说和性基因数量决定假说。然后就蜜蜂性比由蜂王操纵 ,或是由工蜂操纵进行了论述 ,并对蜜蜂性比调控机理研究提出了一些建议     

Importance of pause between spider courtship vibrations and general problems using synthetic stimuli in behavioural studies

Effectiveness of natural stimuli (recorded and replayed male vibratory courtship signals) in eliciting a female's vibratory response was compared with that of synthetic stimuli in a plant-dwelling spider (Cupiennius salei). Specifically, the role of interseries (pauses between consecutive male series) was evaluated. Effectiveness was assessed by the number of responding females and the number of responses per female per stimulus. In case of both synthetic and natural stimuli, effectiveness increases with increasing duration of interseries in both measures. Interseries are thus represented in the female's innate releasing mechanism. However, synthetic stimuli are less effective (about 55%) than natural stimuli (set 100%). With decreasing interseries, the number of female responses per unit time sharply increases in case of playback of natural stimuli, but only slightly in case of synthetic stimuli. Since males courting on plants decrease interseries as soon as a female responds, thus facilitating their orientation, the data obtained with natural stimuli much better fit natural male courtship behaviour. This finding brings into question the validity of the synthetic stimulus. In the bioacoustics literature there is neither consensus on the effectiveness of a synthetic stimulus necessary to qualify it for behavioural analysis nor on how to measure effectiveness. We propose three measures : the percentage of responding animals, the action pattern of the response and the number of responses. It may be crucial, particularly when addressing evolutionary questions, to use either natural stimuli or synthetic stimuli (nearly) as effective as natural stimuli to avoid biased sampling of experimental animals and results difficult to interpret in biological terms.     

Microinjection of synthetic Xhox-1A homeobox mRNA disrupts somite formation in developing Xenopus embryos

The structural similarity between Drosophila and vertebrate homeobox genes begs the question of whether the vertebrate gene products affect cell fate and pattern formation. To study the function of the Xenopus homeobox protein, Xhox-1A, we microinjected fertilized Xenopus eggs with an excess of synthetic Xhox-RNA and assayed for effects on development. The predominant phenotype is a disturbance in somite formation. When embryos are injected with Xhox-1A mRNA, but not with control mRNAs, morphogenesis of somites occurs chaotically and individual segments are lost. Histological staining, in situ hybridization, and immunohistochemistry indicate that the disorganized somitic tissue has differentiated into muscle cells. Overall, these results suggest that correct regulation of the Xhox-1A gene may be important for the normal development of the segmented somite pattern in early embryos. Moreover, the inferred role of Xhox-1A in somite formation indicates that there may be molecular parallels between mechanisms of segmentation in flies and vertebrates.     

Contributions of lipid bilayer hosts to structure and activity of multifunctional supramolecular guests

The question of whether or not the surrounding lipid bilayer host contributes to structure and activity of included functional guests is a general topic of current scientific concern. We report that synthetic multifunctional pores are of use to address this elusive question, because the detection of their catalytic activity is membrane independent. According to their salt-rate profiles, unstable multifunctional supramolecules with permanent internal charges show highest membrane sensitivity, and the dependence of membrane sensitivity on the acidity of internal cations exceeds that on supramolecule stability. These results can, with all appropriate caution, be interpreted as indications for the existence of long-range EMP-ICR interactions (EMP: external membrane pressure, ICR: internal charge repulsion) between membrane hosts and functional guests that can, for instance, prevent the 'explosion' and promote the 'implosion' of over- and undercharged transmembrane barrel-stave supramolecules, respectively.     

A Bayesian approach to discriminate between alternative DNA sequence segmentations

MOTIVATION: As a result of recombination or rate variation, a DNA sequence alignment may have a mosaic structure, where different segments correspond to different evolutionary histories. While several methods have been developed to predict DNA mosaic structures, they do not properly address the question of whether the predicted segmentation itself is statistically significant, or whether it is significantly better than an alternative mosaic structure predicted with another method. The objective of the present article is to devise an approximate Bayesian hypothesis test to discriminate between alternative candidate mosaic structures. RESULTS: We have applied the proposed discrimination scheme to various synthetic and real-world DNA sequence alignments. On the synthetic data, the algorithm identified the true mosaic structure in nine out of ten cases. On the real-world sequence alignments, it selected the same mosaic structures as predicted in the literature.