Standardized Representation of Parts and Assembly for Build Planning.

The design and construction of genetic systems, in silico, in vitro, or in vivo, often involve the handling of various pieces of DNA that exist in different forms across an assembly process: as a standalone "part" sequence, as an insert into a carrier vector, as a digested fragment, etc. Communication about these different forms of a part and their relationships is often confusing, however, because of a lack of standardized terms. Here, we present a systematic terminology and an associated set of practices for representing genetic parts at various stages of design, synthesis, and assembly. These practices are intended to represent any of the wide array of approaches based on embedding parts in carrier vectors, such as BioBricks or Type IIS methods (e.g., GoldenGate, MoClo, GoldenBraid, and PhytoBricks), and have been successfully used as a basis for cross-institutional coordination and software tooling in the iGEM Engineering Committee.

Functional Synthetic Biology.

Synthetic biologists have made great progress over the past decade in developing methods for modular assembly of genetic sequences and in engineering biological systems with a wide variety of functions in various contexts and organisms. However, current paradigms in the field entangle sequence and functionality in a manner that makes abstraction difficult, reduces engineering flexibility and impairs predictability and design reuse. Functional Synthetic Biology aims to overcome these impediments by focusing the design of biological systems on function, rather than on sequence. This reorientation will decouple the engineering of biological devices from the specifics of how those devices are put to use, requiring both conceptual and organizational change, as well as supporting software tooling. Realizing this vision of Functional Synthetic Biology will allow more flexibility in how devices are used, more opportunity for reuse of devices and data, improvements in predictability and reductions in technical risk and cost.

GeneTargeter: Automated In Silico Design for Genome Editing in the Malaria Parasite, Plasmodium falciparum.

Functional characterization of the multitude of poorly described proteins in the human malarial pathogen, Plasmodium falciparum, requires tools to enable genome-scale perturbation studies. Here, we present GeneTargeter (genetargeter.mit.edu), a software tool for automating the design of homology-directed repair donor vectors to achieve gene knockouts, conditional knockdowns, and epitope tagging of P. falciparum genes. We demonstrate GeneTargeter-facilitated genome-scale design of six different types of knockout and conditional knockdown constructs for the P. falciparum genome and validate the computational design process experimentally with successful donor vector assembly and transfection. The software's modular nature accommodates arbitrary destination vectors and allows customizable designs that extend the genome manipulation outcomes attainable in Plasmodium and other organisms.

The horizontal transfer of Pseudomonas aeruginosa PA14 ICE PAPI-1 is controlled by a transcriptional triad between TprA, NdpA2 and MvaT.

Pseudomonas aeruginosa is a major cause of nosocomial infections, particularly in immunocompromised patients or in individuals with cystic fibrosis. Genome sequences reveal that most P. aeruginosa strains contain a significant number of accessory genes gathered in genomic islands. Those genes are essential for P. aeruginosa to invade new ecological niches with high levels of antibiotic usage, like hospitals, or to survive during host infection by providing pathogenicity determinants. P. aeruginosa pathogenicity island 1 (PAPI-1), one of the largest genomic islands, encodes several putative virulence factors, including toxins, biofilm genes and antibiotic-resistance traits. The integrative and conjugative element (ICE) PAPI-1 is horizontally transferable by conjugation via a specialized GI-T4SS, but the mechanism regulating this transfer is currently unknown. Here, we show that this GI-T4SS conjugative machinery is directly induced by TprA, a regulator encoded within PAPI-1. Our data indicate that the nucleotide associated protein NdpA2 acts in synergy with TprA, removing a repressive mechanism exerted by MvaT. In addition, using a transcriptomic approach, we unravelled the regulon controlled by Ndpa2/TprA and showed that they act as major regulators on the genes belonging to PAPI-1. Moreover, TprA and NdpA2 trigger an atypical biofilm structure and enhance ICE PAPI-1 transfer.

The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa.

In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.

Pseudomonas aeruginosa Genome Evolution in Patients and under the Hospital Environment.

Pseudomonas aeruginosa is a Gram-negative environmental species and an opportunistic microorganism, establishing itself in vulnerable patients, such as those with cystic fibrosis (CF) or those hospitalized in intensive care units (ICU). It has become a major cause of nosocomial infections worldwide and a serious threat to Public Health because of overuse and misuse of antibiotics that have selected highly resistant strains against which very few therapeutic options exist. Herein is illustrated the intraclonal evolution of the genome of sequential isolates collected in a single CF patient from the early phase of pulmonary colonization to the fatal outcome. We also examined at the whole genome scale a pair of genotypically-related strains made of a drug susceptible, environmental isolate recovered from an ICU sink and of its multidrug resistant counterpart found to infect an ICU patient. Multiple genetic changes accumulated in the CF isolates over the disease time course including SNPs, deletion events and reduction of whole genome size. The strain isolated from the ICU patient displayed an increase in the genome size of 4.8% with major genetic rearrangements as compared to the initial environmental strain. The annotated genomes are given in free access in an interactive web application WallGene  designed to facilitate large-scale comparative analysis and thus allowing investigators to explore homologies and syntenies between P. aeruginosa strains, here PAO1 and the five clinical strains described.