Scaling up genetic circuit design for cellular computing: advances and prospects.

Synthetic biology aims to engineer and redesign biological systems for useful real-world applications in biomanufacturing, biosensing and biotherapy following a typical design-build-test cycle. Inspired from computer science and electronics, synthetic gene circuits have been designed to exhibit control over the flow of information in biological systems. Two types are Boolean logic inspired TRUE or FALSE digital logic and graded analog computation. Key principles for gene circuit engineering include modularity, orthogonality, predictability and reliability. Initial circuits in the field were small and hampered by a lack of modular and orthogonal components, however in recent years the library of available parts has increased vastly. New tools for high throughput DNA assembly and characterization have been developed enabling rapid prototyping, systematic in situ characterization, as well as automated design and assembly of circuits. Recently implemented computing paradigms in circuit memory and distributed computing using cell consortia will also be discussed. Finally, we will examine existing challenges in building predictable large-scale circuits including modularity, context dependency and metabolic burden as well as tools and methods used to resolve them. These new trends and techniques have the potential to accelerate design of larger gene circuits and result in an increase in our basic understanding of circuit and host behaviour.

Genome-wide identification of the CAD gene family and functional analysis of putative bona fide CAD genes in tobacco (Nicotiana tabacum L.).

Cinnamyl alcohol dehydrogenase (CAD) plays a crucial role in lignin biosynthesis, and the gene family encoding various CAD isozymes has been cloned and characterized in numerous plant species. However, limited information regarding the CAD gene family in tobacco is currently available. In this study, we identified 10 CAD genes in Nicotiana tabacum, four in N. tomentosiformis, and six in N. sylvestris. The nucleotide and amino acid sequences of these tobacco CADs demonstrate high levels of similarity, whereas the putative protein sequences conservatively possessed two Zn2+ binding motifs and an NADP(H) cofactor binding motif. Both NtCAD1 and NtCAD2 had conservative substrate binding sites, similar to those possessed by bona fide CADs, and evidence from phylogenetic analysis as well as expression profiling supported their role as bona fide CADs involved in lignin biosynthesis. NtCAD1 has two paralogous genes, NtCAD1-1 and NtCAD1-2. Enzyme activity analysis revealed that NtCAD1-1 and NtCAD1-2 had a high affinity to coniferyl aldehyde, p-coumaryl aldehyde, and sinapyl aldehyde, whereas NtCAD2 preferred coniferyl aldehyde and p-coumaryl aldehyde as substrates. The kinetic parameter assay revealed that NtCAD1-2 functions as the most efficient enzyme. Downregulation of both NtCAD1-1 and NtCAD1-2 resulted in reddish-brown stems without significant changes in lignin content. Furthermore, NtCAD1-1, NtCAD1-2, and NtCAD2 showed distinct expression patterns in response to biotic and abiotic stresses, as well as different phytohormones. Our findings suggest that NtCAD1-1 and NtCAD1-2 are involved in lignin biosynthesis, with NtCAD1-2 also participating in both biological and abiotic stresses, whereas NtCAD2 plays a distinct role mainly in responding to biological and abiotic stresses in tobacco.

Nutrient supplements boost yeast transformation efficiency.

Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per µg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening.