Developments in the tools and methodologies of synthetic biology.

Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a body of knowledge from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community

Synthetic Biology: A Bridge between Artificial and Natural Cells.

Artificial cells are simple cell-like entities that possess certain properties of natural cells. In general, artificial cells are constructed using three parts: (1) biological membranes that serve as protective barriers, while allowing communication between the cells and the environment; (2) transcription and translation machinery that synthesize proteins based on genetic sequences; and (3) genetic modules that control the dynamics of the whole cell. Artificial cells are minimal and well-defined systems that can be more easily engineered and controlled when compared to natural cells. Artificial cells can be used as biomimetic systems to study and understand natural dynamics of cells with minimal interference from cellular complexity. However, there remain significant gaps between artificial and natural cells. How much information can we encode into artificial cells? What is the minimal number of factors that are necessary to achieve robust functioning of artificial cells? Can artificial cells communicate with their environments efficiently? Can artificial cells replicate, divide or even evolve? Here, we review synthetic biological methods that could shrink the gaps between artificial and natural cells. The closure of these gaps will lead to advancement in synthetic biology, cellular biology and biomedical applications

Design principles of cell-free replicators

Der heilige Gral der synthetischen Biologie ist die Erschaffung einer minimalen Zelle, welche sowohl zu autonomer Selbstreplikation als auch zu natürlicher Evolution befähigt ist. Bereits heute ist es möglich das zentrale Dogma der Molekularbiologie, also die Implementierung des genetischen Codes mittels Transkription-Translation, in vitro zu rekonstruieren. Doch die Kopplung dieses Prozesses mit einem vollständigen DNA-Selbstreplikationssystem war bisher nur auf ein paar Kilobasen (kbp) beschränkt, weit entfernt von den vorgeschlagenen 113 kbp die für eine minimale Zelle nötig wären. In dieser Arbeit wird die Entwicklung einer Plattform für die transkriptions-translations-gekoppelte DNA-Replikation vorgestellt, genannt PURErep, welche in der Lage ist Genome mit der vorhergesagten Größe einer Minimalzelle zu replizieren. Als wichtiger Schritt in Richtung natürlicher Evolution kann sich der hier beschriebene Selbstreplikator pREP über mehrere Generationen fortpflanzen, sowohl in vitro als auch in vivo. PURErep ist modular aufgebaut und frei verfügbar, sodass es mit beliebigen Funktionen erweitert werden kann. Neben der DNA gibt es weitere Komponenten, die zum Selbsterhalt einer Zelle vermehrt werden müssen. Es konnte gezeigt werden, dass PURErep die simultane Co-Expression mehrerer seiner Proteinkomponenten ermöglicht. Diese Faktoren waren in der Lage sich aktiv an der Selbst-Regeneration des Systems beteiligen, was einen wichtigen Schritt in Richtung biochemischer Autonomie darstellt. Weiterhin wurden Möglichkeiten zur Selbstreplikation des komplexen Ribosoms erforscht, einem wesentlichen Bestandteil des Translationsapparates. Die de novo Synthese und Assemblierung solcher Ribosomen wird eine entscheidende Rolle für zukünftige Entwicklungen spielen. Ein weiteres Merkmal von Zellen stellt ihre Hülle dar, die Zellmembran. Eine von Grund auf neu geschaffene Minimalzelle müsste in der Lage sein, eine ähnliche Hülle selbst zu produzieren. Es wurde ein effizientes Konzept zur Selbst-Verkapselung des pREP Replikators entwickelt, welches vollkommen ohne zusätzlichen Energiebedarf auskommt. Es konnte gezeigt werden, dass diese sogenannten DNA-Nanoflowers Kernstrukturen bildeten und sich über Generation hinweg vermehren können. Insgesamt dienen die in dieser Arbeit dargelegten Entwürfe der Weiterentwicklung unabhängiger Selbstreplikatoren, welche vielleicht in der Lage sein werden eines Tages natürliche Zellen zu imitieren.The holy grail of bottom-up synthetic biology is the creation of a minimal cell capable of autonomous self-replication and open-ended Darwinian evolution. Reconstituting molecular biology’s central dogma, the implementation of genetic information via transcription-translation, is already feasible in vitro. Yet coupling this process to a DNA self-replication system has so far been limited to only a few kilobases (kbp), a far cry from the proposed 113 kbp proposed for a minimal cell. This work presents the development of a transcription-translation coupled DNA replication platform, called PURErep, which is capable of replicating DNA genomes approaching the proposed size of a minimal cell. As an important step towards Darwinian evolution, the herein described self-replicator pREP can propagate over several generations, both in vitro and in vivo. PURErep is modular and freely available, so that it can be extended with further functions as desired. In addition to DNA, there are other components that need to be replicated for the self-preservation of a cell. It could be shown that PURErep enables the simultaneous co-expression for several of its protein components. These factors were able to actively participate in the self-regeneration of the system, representing an important hallmark of biochemical autonomy. Furthermore, the self-reproduction of the complex ribosome was investigated, an essential component of the translational apparatus. The de novo synthesis and assembly of such ribosomes will be a crucial step towards future developments. Another feature of cells is their envelope, the cell membrane. A minimal cell created from scratch should be able to produce a similar compartment by itself. An efficient concept for the self-compartmentalization of the pREP replicator has been developed, which requires no additional energy and is entirely based on self-organization. It could be shown that these so-called DNA nanoflowers formed nuclear structures and could reproduce over generations. Overall, the designs laid out in this work serve to further develop independent self-replicators, which may one day be able to mimic a natural cell

Cell-free prediction of protein expression costs for growing cells

Translating heterologous proteins places significant burden on host cells, consuming expression resources leading to slower cell growth and productivity. Yet predicting the cost of protein production for any given gene is a major challenge, as multiple processes and factors combine to determine translation efficiency. To enable prediction of the cost of gene expression in bacteria, we describe here a standard cell-free lysate assay that provides a relative measure of resource consumption when a protein coding sequence is expressed. These lysate measurements can then be used with a computational model of translation to predict the in vivo burden placed on growing E. coli cells for a variety of proteins of different functions and lengths. Using this approach, we can predict the burden of expressing multigene operons of different designs and differentiate between the fraction of burden related to gene expression compared to action of a metabolic pathway

A novel programmable lysozyme-based lysis system in Pseudomonas putida for biopolymer production

Indexación: Scopus; Web of Science.Cell lysis is crucial for the microbial production of industrial fatty acids, proteins, biofuels, and biopolymers. In this work, we developed a novel programmable lysis system based on the heterologous expression of lysozyme. The inducible lytic system was tested in two Gram-negative bacterial strains, namely Escherichia coli and Pseudomonas putida KT2440. Before induction, the lytic system did not significantly arrest essential physiological parameters in the recombinant E. coli (ECPi) and P. putida (JBOi) strain such as specific growth rate and biomass yield under standard growth conditions. A different scenario was observed in the recombinant JBOi strain when subjected to PHA-producing conditions, where biomass production was reduced by 25% but the mcl-PHA content was maintained at about 30% of the cell dry weight. Importantly, the genetic construct worked well under PHA-producing conditions (nitrogen-limiting phase), where more than 95% of the cell population presented membrane disruption 16 h post induction, with 75% of the total synthesized biopolymer recovered at the end of the fermentation period. In conclusion, this new lysis system circumvents traditional, costly mechanical and enzymatic cell-disrupting procedures.https://www.nature.com/articles/s41598-017-04741-2.pd

Chem Commun (Camb)

A cell-free expression platform for making bacterial ribosomes encapsulated within giant liposomes was capable of synthesizing sfGFP. The liposomes were prepared using a double emulsion template, and compartmentalized in vitro protein synthesis was analysed using spinning disk confocal microscopy. Two different liposome phospholipid formulations were investigated to characterize their effects on the compartmentalized reaction kinetics. This study was performed as a necessary step towards the synthesis of minimal cells.DP2 HL117748/HL/NHLBI NIH HHS/United StatesT32 EB005582/EB/NIBIB NIH HHS/United StatesDP2 HL117748-01/DP/NCCDPHP CDC HHS/United StatesT32EB005582/EB/NIBIB NIH HHS/United States2017-04-07T00:00:00Z27019994PMC482937

In vitro integration of ribosomal RNA synthesis, ribosome assembly, and translation

Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties

Engineering Transcriptional Control and Synthetic Gene Circuits in Cell Free systems

Engineering gene networks offers an opportunity to harness biological function for biotechnological and biomedical applications. In contrast to cell-based systems, cell free extracts offer a flexible and well-characterized context in which to implement predictable gene circuits. Critical to these efforts is the availability of a library of ligand sensitive gene regulatory systems. Here, I describe efforts to develop molecular tools to control gene expression and implement a negative feedback circuit in E.coli cell extracts. First, a strategy to regulate T7 RNA polymerase using DNA aptamers is detailed. I test the hypothesis that a DNA aptamer, when placed near the transcription start site, interferes with transcription in the presence of the target molecule. A DNA aptamer that binds thrombin is used as a model system for demonstrating feasibility of the approach. I show that for the hybrid T7-aptamer promoter, thrombin addition results in up to a 5-fold reduction in gene expression. I further demonstrate that gene expression be tuned by altering the position of the aptamer relative to the transcription start site. I then devised a mechanism to engineer dual regulation of T7 promoters using LacI and TetR repressor proteins. To achieve this, a LacI binding site (lacO) was positioned 92bp upstream from a T7lacO promoter, which resulted in an increased repression from T7lacO promoters presumably by a looping based mechanism. TetR binding sites were introduced into this framework to disrupt the DNA looping to create T7 promoters that respond to both LacI and TetR. I show that positioning a tetO operator between the upstream lacO and the T7lacO promoter results in relieving lacO mediated repression by TetR. Finally, a negative feedback circuit was realized using T7lacO promoters. To this end, mono-cistronic and bi-cistronic system assembly approaches for system assembly are examined leading to the realization of an inducible negative feedback circuit in cell free systems. Collectively, the tools developed in this work pave the way for expanding the library of ligands that can be used for regulating gene expression, enabling signal integration at T7 promoters and facilitating engineering of gene networks in cell free systems

IST Austria Thesis

Synthesis of proteins – translation – is a fundamental process of life. Quantitative studies anchor translation into the context of bacterial physiology and reveal several mathematical relationships, called “growth laws,” which capture physiological feedbacks between protein synthesis and cell growth. Growth laws describe the dependency of the ribosome abundance as a function of growth rate, which can change depending on the growth conditions. Perturbations of translation reveal that bacteria employ a compensatory strategy in which the reduced translation capability results in increased expression of the translation machinery. Perturbations of translation are achieved in various ways; clinically interesting is the application of translation-targeting antibiotics – translation inhibitors. The antibiotic effects on bacterial physiology are often poorly understood. Bacterial responses to two or more simultaneously applied antibiotics are even more puzzling. The combined antibiotic effect determines the type of drug interaction, which ranges from synergy (the effect is stronger than expected) to antagonism (the effect is weaker) and suppression (one of the drugs loses its potency). In the first part of this work, we systematically measure the pairwise interaction network for translation inhibitors that interfere with different steps in translation. We find that the interactions are surprisingly diverse and tend to be more antagonistic. To explore the underlying mechanisms, we begin with a minimal biophysical model of combined antibiotic action. We base this model on the kinetics of antibiotic uptake and binding together with the physiological response described by the growth laws. The biophysical model explains some drug interactions, but not all; it specifically fails to predict suppression. In the second part of this work, we hypothesize that elusive suppressive drug interactions result from the interplay between ribosomes halted in different stages of translation. To elucidate this putative mechanism of drug interactions between translation inhibitors, we generate translation bottlenecks genetically using in- ducible control of translation factors that regulate well-defined translation cycle steps. These perturbations accurately mimic antibiotic action and drug interactions, supporting that the interplay of different translation bottlenecks partially causes these interactions. We extend this approach by varying two translation bottlenecks simultaneously. This approach reveals the suppression of translocation inhibition by inhibited translation. We rationalize this effect by modeling dense traffic of ribosomes that move on transcripts in a translation factor-mediated manner. This model predicts a dissolution of traffic jams caused by inhibited translocation when the density of ribosome traffic is reduced by lowered initiation. We base this model on the growth laws and quantitative relationships between different translation and growth parameters. In the final part of this work, we describe a set of tools aimed at quantification of physiological and translation parameters. We further develop a simple model that directly connects the abundance of a translation factor with the growth rate, which allows us to extract physiological parameters describing initiation. We demonstrate the development of tools for measuring translation rate. This thesis showcases how a combination of high-throughput growth rate mea- surements, genetics, and modeling can reveal mechanisms of drug interactions. Furthermore, by a gradual transition from combinations of antibiotics to precise genetic interventions, we demonstrated the equivalency between genetic and chemi- cal perturbations of translation. These findings tile the path for quantitative studies of antibiotic combinations and illustrate future approaches towards the quantitative description of translation