Engineering orthogonal dual transcription factors for multi-input synthetic promoters

Synthetic biology has seen an explosive growth in the capability of engineering artificial gene circuits from transcription factors (TFs), particularly in bacteria. However, most artificial networks still employ the same core set of TFs (for example LacI, TetR and cI). The TFs mostly function via repression and it is difficult to integrate multiple inputs in promoter logic. Here we present to our knowledge the first set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants and multi-input promoter architectures. Our toolkit contains 12 TFs, flexibly operating as activators, repressors, dual activator–repressors or dual repressor–repressors, on up to 270 synthetic promoters. To engineer non cross-reacting cI variants, we design a new M13 phagemid-based system for the directed evolution of biomolecules. Because cI is used in so many synthetic biology projects, the new set of variants will easily slot into the existing projects of other groups, greatly expanding current engineering capacities

Superconformal symmetry and maximal supergravity in various dimensions

In this paper we explore the relation between conformal superalgebras with 64 supercharges and maximal supergravity theories in three, four and six dimensions using twistorial oscillator techniques. The massless fields of N=8 supergravity in four dimensions were shown to fit into a CPT-self-conjugate doubleton supermultiplet of the conformal superalgebra SU(2,2|8) a long time ago. We show that the fields of maximal supergravity in three dimensions can similarly be fitted into the super singleton multiplet of the conformal superalgebra OSp(16|4,R), which is related to the doubleton supermultiplet of SU(2,2|8) by dimensional reduction. Moreover, we construct the ultra-short supermultiplet of the six-dimensional conformal superalgebra OSp(8*|8) and show that its component fields can be organized in an on-shell superfield. The ultra-short OSp(8*|8) multiplet reduces to the doubleton supermultiplet of SU(2,2|8) upon dimensional reduction. We discuss the possibility of a chiral maximal (4,0) six-dimensional supergravity theory with USp(8) R-symmetry that reduces to maximal supergravity in four dimensions and is different from six-dimensional (2,2) maximal supergravity, whose fields cannot be fitted into a unitary supermultiplet of a simple conformal superalgebra. Such an interacting theory would be the gravitational analog of the (2,0) theory.Comment: 54 pages, PDFLaTeX, Section 5 and several references added. Version accepted for publication in JHE

Engineering of biomolecules by bacteriophage directed evolution

Conventional in vivo directed evolution methods have primarily linked the biomolecule's activity to bacterial cell growth. Recent developments instead rely on the conditional growth of bacteriophages (phages), viruses that infect and replicate within bacteria. Here we review recent phage-based selection systems for in vivo directed evolution. These approaches have been applied to evolve a wide range of proteins including transcription factors, polymerases, proteases, DNA-binding proteins, and protein–protein interactions. Advances in this field expand the possible applications of protein and RNA engineering. This will ultimately result in new biomolecules with tailor-made properties, as well as giving us a better understanding of basic evolutionary processes