Abstracts for parallel session on Friday May 20th

Synthetic nucleoid and gene expression

8.1: Towards a minimal nucleoid in cell-like reactors

Ferdinand Greiss¹, Shirley Daube¹, Vincent Noireaux², Roy Bar-Ziv¹

1: Weizmann Institute of Science, Israel;  2: University of Minnesota, United States of America

As life’s central processing unit, the nucleoid handles a stream of information to maintain homeostasis. The hundreds of required genetic operations are regulated in parallel on a sequence-specific level with DNA-binding molecules and a systemic level with the dynamic organization of the chromosome in space. Therefore, DNA is not only the template to produce biomolecules, but can orchestrate the gene activity on a larger scale. We aspire to a nucleoid with hundreds of parallel reactions in cell-like reactors and close the gap between the multi-scale dynamics of gene expression in reconstituted systems and live bacteria.

As the first step, we showed how to isolate a minimal genetic decision-making circuit and program cell-like reactors with low gene copy numbers to impose different noise levels on the genetic circuits. We found a trade-off in decision-making between accuracy and speed: accurate decisions were completed in several hours at high DNA numbers, while fuzzy decisions acted within a few minutes at five orders of magnitude lower DNA numbers. The trade-off appears as a general property of decision-making reported for bee swarms, the sensing of environmental cues, and DNA replication.

Currently, we are working with a fast fluorescent reporter system to visualize protein production at immobilized single DNA molecules using the coupled transcription and translation system from E. coli. We tested the effects of DNA structure and nascent transcription factors on transcription activity. Moreover, we fabricated µm-sized cell-like reactors to contain single DNAs that produce multiple rounds of mRNAs and proteins. The reactor allowed to study transcription and translation from a single DNA with hair-trigger sensitivity.

8.2: Molecular evolution of a synthetic DNA replicator

Zhanar Abil¹, Ana Restrepo Sierra¹, Ilja Westerlaken¹, Alicia del Prado², Miguel de Vegua², Christophe Danelon¹

1: Delft University of Technology, the Netherlands;  2: Centro de Biología Molecular Severo Ochoa, Spain

DNA replication is an essential process for information continuity during (synthetic) cell proliferation, as well as for the emergence of biodiversity through Darwinian evolution. Inspired by the mode of replication of the phi29 viral genome, we recently reported that a de novo designed linear DNA template flanked by origins of replication can be amplified by self-encoded proteins in PURE system [1]. In the present study, we ask whether the DNA self-replicator is able to undergo adaptive evolution in liposomes. We performed several rounds of in-liposome evolution starting from 10 pM DNA concentration, i.e. the average number of DNA copies per liposome is lower than one, to ensure genotype-phenotype coupling. The initial and final amounts of DNA were quantified at each round. We found that the replicating DNA is capable to self-improve, yielding a 10-times higher amplification fold after 12 rounds compared to the parental template. Sequence analysis revealed a number of beneficial mutations, in particular residues of the DNA polymerase that are located in the vicinity of the template DNA. These results open new horizons for studying evolutionary processes at a molecular level. Moreover, they allow us to explore the co-evolution of multiple functions through self-improvement of the encoding DNA, a process that may lead to the emergence of synthetic life.

References: [1] Van Nies P, Westerlaken I, Blanken D, Salas M, Mencía M, Danelon C. Self-replication of DNA by its encoded proteins in liposome-based synthetic cells. Nat. Commun. 9, 1583, (2018).

8.3: Genetically encoded stoichiometry for cell-free expression of multifunctional protein systems

Barbara Jackova, Mathieu Morel, Damien Baigl

UMR8640 Pasteur, Ecole Normale Supérieure, Paris, France

Cell-free protein synthesis emerged as a powerful bioengineering technology to synthesize soluble proteins in vitro without tedious cell culture and purification steps. This platform, well adapted for simultaneous expression of multiple proteins, has proven particularly useful in prototyping genetic circuits, developing sensors, biomanufacturing and synthetic cell design. To control the level of expression most studies rely on optimization of expression conditions or on engineering regulatory untranslated DNA regions. However, these methods often fail at controlling relative level of proteins expressed simultaneously. Another approach relies on the optimization of the coding sequence, focusing on codon usage, GC content or mRNA structure. While improving expression of hard to produce proteins, such optimizations are made on a case-by-case basis and don’t allow a quantitative control of the expression yield.

Here we show that inserting degenerated DNA tags coding for the same peptide upstream of a gene of interest allows for easy programmability of proteins expression level. Using reconstituted cell-free expression system, we demonstrate the tunability of expression level for different fluorescent proteins, up to a 10-fold increase for the best sequence, and ratiometric control when two proteins are co-expressed. We are currently deciphering the mechanism at play and expanding the method to functional proteins, featuring molecular recognition or enzymatic activity. We anticipate our technique to be a mean to easily program synthetic cellular systems capable of quantitative control of protein expression, allowing precise self-regulation or production of multifunctional protein assemblies.

8.4: The role of incoherent feedback loops in gene circuit design

Ramez Daniel, Rongying Haung

Technion – Israel Institute of Technology, Israel

Biological regulatory networks in nature comprise feedforward and feedback loops, which allow cells to perform sophisticated tasks such as DNA repair, cell division, and apoptosis. The Incoherent feedforward loop (iFFL), in which the input signal negatively and positively controls the output signal, is the second most abundant network in living cells. Recently, iFFLs have been applied in a few synthetic biological systems, e.g., achieving a robust adaptation in Escherichia coli. In this study, we implemented several iFFL designs into Escherichia coli to solve sophisticated computational functions.

First, we implemented a 2-bit analog-to-digital converter (ADC), which divides the dynamic range of a single input for four ranges (low, low-moderate, moderate-high, high) encoded in two discrete-binary outputs {00, 01, 10, 11}. ADC is useful for biosensing applications and for selecting which specific combinations of several genes to express based on administration of a single inducer. The same network can be easily reconfigured to function as a ternary switch with distinct low, medium, and high output states. Second, we used an iFFL to improve the fold change magnitude of target promoters (ON/OFF ratio) – we experimentally demonstrated that such a network can reduce the basal level while maintaining peak activity. We applied iFFL design in six synthetic and native promoters that are either functionally specific (e.g., Arsenic) or systemically involved in complex pathways in cells (e.g., SOS response) into Escherichia col. Lastly, we also focused on control over circuit dynamics; forming a balance between the negative and positive regulators of the iFFL, we stabilized gene expression dynamics for more than 24 hours.

8.5: Gene expression from genomic DNA in E. coli cell-free expression platform

Andrei Sakai, Wilhelm T.S. Huck

Radboud University, the Netherlands

The construction of the synthetic cell relies on the integration of simpler functional blocks to reconstitute more complex cell-like functions. Researchers were able to reconstitute prokaryotic cellular functions such as DNA replication and lipid synthesis using plasmid DNA (pDNA) as a source of genetic information. Despite the versatility of pDNA, it only encodes dozens of genes at most, which can be a limitation to build the synthetic cell. Therefore, genomic DNA (gDNA), which codes for a higher number of genes, can be a more suitable DNA template for the synthetic cell. To date, cell-free expression using gDNA was able to reconstitute infectious bacteriophages (e.g. MS2, T4, T7), which have small genomes. The cell-free expression (CFE) of more complex genomes (e.g. T. thermophilus, E. coli) was reported, but it still lacks in terms of reproducibility and genetic control. Here, we show that in vitro gene expression can be robustly performed from gDNA isolated from E. coli (4.6 Mb). First, we described a method to isolate intact gDNA and our strategy to enable reliable gene expression measurements (from a single gene). For the results part, we show that gDNA expression in batch experiments differs from pDNA in terms of protein yields, protein synthesis rates, and translational onset. We also observed that expression from gDNA can be enhanced by the presence of a nucleoid-associated protein, which was not observed when using pDNA as a template. We believe that gDNA will enable the construction of synthetic systems with higher complexity levels. In the future, we aim to expand the number of genes that can be expressed from the gDNA and improve expression control in a CFE environment.

8.6: Transducing light energy into chemical energy toward the implementation of photo-artificial simplified-autotroph protocells

Albanese Paola¹, Emiliano Altamura², Fabio Mavelli²

1: University of Siena, Italy;  2: University of Bari “Aldo Moro”, Italy

A continuous energy supply is a critical requirement in the implementation of synthetic cells from scratch that can be considered alive or, at least, maintained in homeostatic conditions far from equilibrium. In this contribution, we will examine the approach for the preparation of protocells, i.e. unilamellar lipid vesicles (GUVs), capable of transducing light energy into chemical energy thanks to the photosynthetic machinery extracted from living bacteria [1]. Two different approaches will be presented and discussed: the first foresees that every single enzyme involved in the bacterial photosynthetic process is extracted and reconstituted in the GUV membrane with the desidered physiological orientation [2,3]; the second pursues the extraction of the entire photosynthetic apparatus in form of organelles, nanometric bacterial vesicles called chromatophores, capable of carrying out the phosphorylation of ADP in ATP under continuous light irradiation when trapped in the aqueous lumen of GUVs [4]. Results, potential and limitations of both approaches will be presented and discussed.

References: [1] Altamura E, et al., The Rise of the Nested Multicompartment Model in Synthetic Cell Research, Frontiers in Molecular Biosciences 8, (2021) 750576;  [2] Altamura E. et al., Highly oriented photosynthetic reaction centres generate a proton gradient in synthetic protocells. PNAS 114(15), (2017) 3837-3842;  [3] Altamura E. et al., Optimizing Enzymatic Photo‐Redox Cycles by a Hybrid Protein Complex Chain. Chem. Photo Chem. 5(1), (2021), 26-31;  [4] Altamura E., et al., Chromatophores efficiently promote light-driven ATP synthesis and DNA transcription inside hybrid multi-compartment artificial cells, PNAS 118(7), (2021) e2012170118.

Sponsored by: