Biotechnology Tools Derived from the Bacteriophage/Bacteria Arms Race

The long association and intense competition between bacteria and their viruses have created a fertile ground for evolution to develop numerous tools for DNA modification, assembly and degradation. Many of these tools underpin the past 50 years of molecular biology, and others show great potential in shaping the next 50 years of the field. Here, I present some of the tools that have come out of the bacteria-bacteriophage arms race and discuss some of the concepts that may shape their future use. Molecular biology remains a fast-growing area increasingly limited solely by researcher ingenuity

Structures of an apo and a binary complex of an evolved archeal b family DNA polymerase capable of synthesising highly cy-dye labelled DNA

Thermophilic DNA polymerases of the polB family are of great importance in biotechnological applications including high-fidelity PCR. Of particular interest is the relative promiscuity of engineered versions of the exo- form of polymerases from the Thermo- and Pyrococcales families towards non-canonical substrates, which enables key advances in Next-generation sequencing. Despite this there is a paucity of structural information to guide further engineering of this group of polymerases. Here we report two structures, of the apo form and of a binary complex of a previously described variant (E10) of Pyrococcus furiosus (Pfu) polymerase with an ability to fully replace dCTP with Cyanine dye-labeled dCTP (Cy3-dCTP or Cy5-dCTP) in PCR and synthesise highly fluorescent “CyDNA” densely decorated with cyanine dye heterocycles. The apo form of Pfu-E10 closely matches reported apo form structures of wild-type Pfu. In contrast, the binary complex (in the replicative state with a duplex DNA oligonucleotide) reveals a closing movement of the thumb domain, increasing the contact surface with the nascent DNA duplex strand. Modelling based on the binary complex suggests how bulky fluorophores may be accommodated during processive synthesis and has aided the identification of residues important for the synthesis of unnatural nucleic acid polymers

Synthetic biology approaches to biological containment: pre-emptively tackling potential risks

Biocontainment comprises any strategy applied to ensure that harmful organisms are confined to controlled laboratory conditions and not allowed to escape into the environment. Genetically engineered microorganisms (GEMs), regardless of the nature of the modification and how it was established, have potential human or ecological impact if accidentally leaked or voluntarily released into a natural setting. Although all evidence to date is that GEMs are unable to compete in the environment, the power of synthetic biology to rewrite life requires a pre-emptive strategy to tackle possible unknown risks. Physical containment barriers have proven effective but a number of strategies have been developed to further strengthen biocontainment. Research on complex genetic circuits, lethal genes, alternative nucleic acids, genome recoding and synthetic auxotrophies aim to design more effective routes towards biocontainment. Here, we describe recent advances in synthetic biology that contribute to the ongoing efforts to develop new and improved genetic, semantic, metabolic and mechanistic plans for the containment of GEMs

Directed evolution of colE1 plasmid replication compatibility: a fast tractable tunable model for investigating biological orthogonality

Plasmids of the ColE1 family are among the most frequently used in molecular biology. They were adopted early for many biotechnology applications, and as models to study plasmid biology. Their mechanism of replication is well understood, involving specific interactions between a plasmid encoded sense-antisense gene pair (RNAI and RNAII). Due to such mechanism, two plasmids with the same origin cannot be stably maintained in cells-a process known as incompatibility. While mutations in RNAI and RNAII can make colE1 more compatible, there has been no systematic effort to engineer new compatible colE1 origins, which could bypass technical design constraints for multi-plasmid applications. Here, we show that by diversifying loop regions in RNAI (and RNAII), it is possible to select new viable colE1 origins compatible with the wild-type one. We demonstrate that sequence divergence is not sufficient to enable compatibility and pairwise interactions are not an accurate guide for higher order interactions. We identify potential principles to engineer plasmid copy number independently from other regulatory strategies and we propose plasmid compatibility as a tractable model to study biological orthogonality

Contributions from specific and general factors to unique deficits: two cases of mathematics learning difficulties

Mathematics learning difficulties are a highly comorbid and heterogeneous set of disorders linked to several dissociable mechanisms and endophenotypes. Two of these endophenotypes consist of primary deficits in number sense and verbal numerical representations. However, currently acknowledged endophenotypes are underspecified regarding the role of automatic vs. controlled information processing, and their description should be complemented. Two children with specific deficits in number sense and verbal numerical representations and normal or above-normal intelligence and preserved visuospatial cognition illustrate this point. Child H.V. exhibited deficits in number sense and fact retrieval. Child G.A. presented severe deficits in orally presented problems and transcoding tasks. A partial confirmation of the two endophenotypes that relate to the number sense and verbal processing was obtained, but a much more clear differentiation between the deficits presented by H.V. and G.A. can be reached by looking at differential impairments in modes of processing. H.V. is notably competent in the use of controlled processing but has problems with more automatic processes, such as nonsymbolic magnitude processing, speeded counting and fact retrieval. In contrast, G.A. can retrieve facts and process nonsymbolic magnitudes but exhibits severe impairment in recruiting executive functions and the concentration that is necessary to accomplish transcoding tasks and word problem solving. These results indicate that typical endophenotypes might be insufficient to describe accurately the deficits that are observed in children with mathematics learning abilities. However, by incorporating domain-specificity and modes of processing into the assessment of the endophenotypes, individual deficit profiles can be much more accurately described. This process calls for further specification of the endophenotypes in mathematics learning difficulties

Structural Studies of HNA Substrate Specificity in Mutants of an Archaeal DNA Polymerase Obtained by Directed Evolution.

Archaeal DNA polymerases from the B-family (polB) have found essential applications in biotechnology. In addition, some of their variants can accept a wide range of modified nucleotides or xenobiotic nucleotides, such as 1,5-anhydrohexitol nucleic acid (HNA), which has the unique ability to selectively cross-pair with DNA and RNA. This capacity is essential to allow the transmission of information between different chemistries of nucleic acid molecules. Variants of the archaeal polymerase from Thermococcus gorgonarius, TgoT, that can either generate HNA from DNA (TgoT_6G12) or DNA from HNA (TgoT_RT521) have been previously identified. To understand how DNA and HNA are recognized and selected by these two laboratory-evolved polymerases, we report six X-ray structures of these variants, as well as an in silico model of a ternary complex with HNA. Structural comparisons of the apo form of TgoT_6G12 together with its binary and ternary complexes with a DNA duplex highlight an ensemble of interactions and conformational changes required to promote DNA or HNA synthesis. MD simulations of the ternary complex suggest that the HNA-DNA hybrid duplex remains stable in the A-DNA helical form and help explain the presence of mutations in regions that would normally not be in contact with the DNA if it were not in the A-helical form. One complex with two incorporated HNA nucleotides is surprisingly found in a one nucleotide-backtracked form, which is new for a DNA polymerase. This information can be used for engineering a new generation of more efficient HNA polymerase variants

Validation of TerraClass mapping for the Municipality of Paragominas state of Pará

This work aims to evaluate the TerraClass mapping for the year 2014, in the municipality of Paragominas, State of Pará. The validation was made by comparing the mapping with the observations found in the field. Images of the Satétile Landsat-8, OLI sensor of the year 2014, path/row 222/062, 222/063, 223/062 and 223/063 were used to aid in the field. Using this data it was possible to analyze the main representative classes in the area, including agriculture, urban area, forest, clean pasture, dirty pasture, reforestation, regeneration with pasture and secondary vegetation. The secondary vegetation presented 2,198.16 km², clean pasture with 3,332.29 km², agriculture with 896.75 km² and the forest occupying 54.21% of the total area of Paragominas. The overall concordance index was 86%, corroborating the reliability of the mapping performed. The average error was 6% and the total value of discordance was of 14%. Concerning the secondary vegetation, pasture, agriculture, urban area and forest classes, they presented concordance higher to 50%, while regeneration with pasture and reforestation presented greater intensity of omission with 40,57% and 76,31% respectively. Inclusion errors were less than 40% for the secondary vegetation, pasture regeneration, clean pasture and dirty pasture classes. The field work was essential to validate and analyze the accuracy of the 2014 TerraClass Project for the studied region, which becomes important for the understanding of the dynamics of land use

E. coli surface display of streptavidin for directed evolution of an allylic deallylase

Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin 1. Two rounds of directed evolution afforded the double mutant S112M-K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein

Phosphonomethyl Oligonucleotides as Backbone-Modified Artificial Genetic Polymers

Although several synthetic or xenobiotic nucleic acids (XNAs) have been shown to be viable genetic materials in vitro, major hurdles remain for their in vivo applications, particularly orthogonality. The availability of XNAs that do not interact with natural nucleic acids and are not affected by natural DNA processing enzymes, as well as specialized XNA processing enzymes that do not interact with natural nucleic acids, is essential. Here, we report 3′–2′ phosphonomethyl-threosyl nucleic acid (tPhoNA) as a novel XNA genetic material and a prime candidate for in vivo XNA applications. We established routes for the chemical synthesis of phosphonate nucleic acids and phosphorylated monomeric building blocks, and we demonstrated that DNA duplexes were destabilized upon replacement with tPhoNA. We engineered a novel tPhoNA synthetase enzyme and, with a previously reported XNA reverse transcriptase, demonstrated that tPhoNA is a viable genetic material (with an aggregate error rate of approximately 17 × 10–3 per base) compatible with the isolation of functional XNAs. In vivo experiments to test tPhoNA orthogonality showed that the E. coli cellular machinery had only very limited potential to access genetic information in tPhoNA. Our work is the first report of a synthetic genetic material modified in both sugar and phosphate backbone moieties and represents a significant advance in biorthogonality toward the introduction of XNA systems in vivo

Catalysts from synthetic genetic polymers

The emergence of catalysis in early genetic polymers like RNA is considered a key transition in the origin of life1, predating the appearance of protein enzymes. DNA also demonstrates the capacity to fold into three-dimensional structures and form catalysts in vitro2. However, to what degree these natural biopolymers comprise functionally privileged chemical scaffolds3 for folding or the evolution of catalysis is not known. The ability of synthetic genetic polymers (XNAs) with alternative backbone chemistries not found in nature to fold into defined structures and bind ligands4 raises the possibility that these too might be capable of forming catalysts (XNAzymes). Here we report the discovery of such XNAzymes, elaborated in four different chemistries (ANA (arabino nucleic acids)5, FANA (2′-fluoroarabino nucleic acids)6, HNA (hexitol nucleic acids) and CeNA (cyclohexene nucleic acids)7 directly from random XNA oligomer pools, exhibiting in trans RNA endonuclease and ligase activities. We also describe an XNA-XNA ligase metalloenzyme in the FANA framework, establishing catalysis in an entirely synthetic system and enabling the synthesis of FANA oligomers and an active RNA endonuclease FANAzyme from its constituent parts. These results extend catalysis beyond biopolymers and establish technologies for the discovery of catalysts in a wide range of polymer scaffolds not found in nature8. Evolution of catalysis independent of any natural polymer has implications for the definition of chemical boundary conditions for the emergence of life on earth and elsewhere in the universe9