Synthetic Biology aims to engineer biological systems for applications in health, biomanufacturing, and the environment. However, challenges persist due to limited well-characterized genetic elements, undesired interactions, and the necessity to explore novel biological functions. To tackle these challenges, the SyBER team employs multidisciplinary approaches, including cutting-edge genome engineering methodologies, adaptive laboratory evolution, in vivo and lysate-based high-throughput phenotyping, and modeling, across three complementary research axes:
Across the three research axes, we also contribute to the identification of novel functions and the development of technologies aimed at serving future applications and projects.
The growth-rate-dependent variation in cellular machinery abundances, along with condition-specific regulatory mechanisms, affects gene expression and noise, posing challenges to the design of synthetic circuits.
Transcription termination in Gram-positive bacteria. The transcription termination factor Rho is known to suppress pervasive, mostly antisense, transcription, and we had shown that it acts as a global regulator determining cell fate in B. subtilis [10.1371/journal.pgen.1006909]. Through the CoNoCo ANR-funded project (ANR-18-CE12-0025; collab. with C Condon, Paris, M Boudevilain, Orléans and P Romby, Strasbourg), we expanded our investigation into Rho’s function across Gram-positive bacteria. Our research revealed that manipulating Rho expression, either through inactivation or overproduction, influences diverse developmental programs, including motility, biofilm formation, and sporulation [10.1101/2023.12.01.569620]. Furthermore, we discovered that wild-type B. subtilis cells require reduced Rho levels during the transition to stationary phase to effectively initiate and carry out the sporulation program, highlighting its critical importance in spore formation, germination, and bacterial viability [10.1371/journal.pgen.1010618].
Gene expression decomposition. Gene expression noise and growth-rate-dependent regulations hinder the predictability of circuit behavior [10.1016/j.bbagrm.2020.194502]. For instance, it is acknowledged that prokaryotes can adjust gene expression noise independently of protein mean abundance by modulating the relative levels of transcription and translation. We decomposed gene expression into well-characterized genetic elements (e.g. loci, -35/-10 regions, +1 transcription sites, RBS sequences, etc.) and assembled a library of a hundred synthetic constructs to control GFP expression. Our analysis revealed that expression noise is equally sensitive to variations in transcription or translation rates due to extrinsic noise prevalence, making independent control of mean abundance and noise challenging. This has significant implications for genome evolution and biological engineering [10.1126/sciadv.abc3478]. The library was analyzed across various growth conditions, and data are currently being integrated into a custom model. Ultimately, we aim to redefine the fundamental architecture of gene expression in bacteria and promote the rational design of regulatory sequences to predict protein abundance across growth conditions through reverse engineering.
Synthetic circuits frequently encounter malfunction due to unintended interactions with the host system. To drive innovation, constructing streamlined bacterial chassis is promising. Essential for this endeavor is the development of novel engineering tools to build the next generation of bacterial chassis.
Synthetic bacterial chassis to revisit the minimal bacterial gene set. Using a targeted approach, we deleted large dispensable regions in the B. subtilis genome, resulting in a library of 298 strains with genomes reduced up to 1.48 Mb. High-throughput phenotyping revealed decreased cell fitness and the emergence of synthetic-sick interactions, alongside novel phenotypes such as significantly increased resistance (>300-fold) to the DNA-damaging agent mitomycin C and reduced spontaneous mutagenesis (by 100-fold) compared to wild type. Although the mechanisms behind these phenotypes remain unclear, we leveraged the low evolvability in an engineering strategy involving cycles of adaptive laboratory evolution under induced mutagenesis [10.1093/nar/gkad145]. However, this genome reduction approach proved time-consuming and unsuitable for rapid developments. Through the Bacillus 2.0 ANR-funded project (ANR-18-CE44-0003; collab. with C Lartigue, Bordeaux), we initiated the construction of SynBsu2.0, a 1 Mb genome derived from B. subtilis, by in vitro assembly of synthetic, essential regions, and in-yeast cloning. We also developed the CReasPy-Fusion method for simultaneous cloning and CRISPR-based engineering of megabase-sized genomes in yeast by the fusion of bacterial cells with yeast spheroplasts [10.1021/acssynbio.3c00248]. These strategies aim to facilitate the rapid design, construction, and engineering of synthetic genomes for subsequent back-transplantation into B. subtilis protoplasts.
Development of multiplex engineering tools. CRISPR systems, such as Cas9, pose challenges when targeting the host bacterium’s chromosome, limiting the development of CRISPR-based multiplex genome engineering strategies. To address this, we engineered strains capable of hosting a controllable CRISPR-based killswitch system, tightly regulated by an inducible anti-CRISPR protein AcrIIA4. This marks the first instance of transformation and counter-selection using a pre-integrated killswitch system targeting the bacterial genome. We also developed and validated a highly efficient in vivo engineering method for phage genomes from Gram-positive bacteria using Cas9.
Orthogonality together with novel functionality is essential for future applications in biotechnology.
Orthogonal transcription for protein production. We implemented two synthetic circuitries in B. subtilis operating synergistically, leading to: i) transcription orthogonal to that of B. subtilis to produce a protein of interest, and ii) an inducible arrest of the native transcription of the host to redirect all available resources to the orthogonal transcription machinery. This orthogonal, invasive transcription resulted in a significant increase in protein production compared to current production systems [Patent PCT/EP2024/055294].
Phage-assisted continuous evolution (PACE) to create novel functions. In nature, biological functions are constantly tuned or created by the combined effects of random mutations and selection. The PACE, described only for E. coli at present, is a suitable approach that mimics this process in the lab (termed directed evolution) to modify a function towards a desired property. It relies on a clever assembly of genetic constructions so that phage fitness becomes linked to the desired property via a trans-complementation effect. Directed evolution is then achieved by propagating the phage population continuously fed by a culture of naive bacteria. Through the BioBrickEvolver ANR-funded project (ANR-18-CE43-0002; collab. with P Nicolas, Jouy-en-Josas and P Tavares, Gif-sur-Yvette), we initiated the development of a PACE system using B. subtilis and the phage SPP1. Firstly, we constructed modular and connected mini-bioreactors for the cultivation of the bacterium and the phage [Invention disclosure], along with a multi-deleted B. subtilis chassis strain incapable of producing biofilm and stable for up to 1000 generations in continuous culture. Secondly, we developed phage genome engineering methodologies based on in vitro assembly and transfection of synthetic DNA to recover SPP1 mutants. Using this methodology, we constructed the phages required for the PACE, as well as single-gene mutant phages. The fitness of 36 mutants was characterized during B. subtilis infection, revealing that approximately 25% of phage genes were (nearly) essential for phage propagation. Thirdly, to speed up evolution, we generated conditional hypermutators displaying a wide range of mutation rates and contrasting mutation profiles. Very interestingly we showed that an inherent drawback of proofreading is to skew the net polymerase error rates by amplifying intrinsic biases in nucleotide selectivity [10.1101/2023.12.29.573609]. The three components are currently being integrated to conduct a first PACE experiment.
All the team publications are available in the SYBER-MICALIS HAL collection.
