Oscillations by minimal bacterial suicide circuits reveal hidden facets of host-circuit physiology.

Synthetic biology seeks to enable programmed control of cellular behavior though engineered biological systems. These systems typically consist of synthetic circuits that function inside, and interact with, complex host cells possessing pre-existing metabolic and regulatory networks. Nevertheless, while designing systems, a simple well-defined interface between the synthetic gene circuit and the host is frequently assumed. We describe the generation of robust but unexpected oscillations in the densities of bacterium Escherichia coli populations by simple synthetic suicide circuits containing quorum components and a lysis gene. Contrary to design expectations, oscillations required neither the quorum sensing genes (luxR and luxI) nor known regulatory elements in the P(luxI) promoter. Instead, oscillations were likely due to density-dependent plasmid amplification that established a population-level negative feedback. A mathematical model based on this mechanism captures the key characteristics of oscillations, and model predictions regarding perturbations to plasmid amplification were experimentally validated. Our results underscore the importance of plasmid copy number and potential impact of "hidden interactions" on the behavior of engineered gene circuits - a major challenge for standardizing biological parts. As synthetic biology grows as a discipline, increasing value may be derived from tools that enable the assessment of parts in their final context.

Production of transgenic rice with rice ragged stunt virus synthetic resistance genes

Rice ragged stunt virus (RRSV) is an important pathogen of rice affecting its cultivation in South and South East Asia. An approach based on pathogen derived resistance (PDR) was used to produce RRSV resistant rice cultivars. Sequences from the coding region of RRSV genome segments 7 and 10 (non-structural genes), and 5, 8 and 9 (structural genes) were placed in sense or antisense orientation behind the plant expression promoters CaMV35S, RolC, Ubil, Actl and RBTV. Rice cultivars Taipei 309 and Chinsurah Boro II were transformed by biolistic and/or Agrobacterium-mediated delivery of one or more of these PDR gene constructs. A large number of transgenic lines were produced from calli derived from mature or immature embryos, co-bombarded with the marker gene hph encoding hygromycin resistance and RRSV PDR genes or co-cultivated with strains having the binary vector containing these two genes. Both Mendelian and non-Mendelian segregations were observed in transgenic progeny, especially with transgenic lines produced by biolistics. Preliminary tests conducted in China on selected transgenic lines indicate that plants with RRSV segment 5 antisense PDR gene confer RRSV resistance.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/ S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking. ©FUNPEC-RP.

Synthetic genes for glycoprotein design and the elucidation of hydroxyproline-O-glycosylation codes

Design of hydroxyproline (Hyp)-rich glycoproteins (HRGPs) offers an approach for the structural and functional analysis of these wall components, which are broadly implicated in plant growth and development. HRGPs consist of multiple small repetitive “glycomodules” extensively O-glycosylated through the Hyp residues. The patterns of Hyp-O-glycosylation are putatively coded by the primary sequence as described by the Hyp contiguity hypothesis, which predicts contiguous Hyp residues to be attachment sites of small arabinooligosaccharides (1–5 Ara residues/Hyp); while clustered, noncontiguous Hyp residues are sites of arabinogalactan polysaccharide attachment. As a test, we designed two simple HRGPs as fusion proteins with green fluorescent protein. The first was a repetitive Ser-Hyp motif that encoded only clustered noncontiguous Hyp residues, predicted polysaccharide addition sites. The resulting glycoprotein had arabinogalactan polysaccharide O-linked to all Hyp residues. The second construct, based on the consensus sequence of a gum arabic HRGP, contained both arabinogalactan and arabinooligosaccharide addition sites and, as predicted, gave a product that contained both saccharide types. These results identify an O-glycosylation code of plants.

Mapping quantitative trait loci for resistance to Pratylenchus thornei from synthetic hexaploid wheat in the International Triticeae Mapping Initiative (ITMI) population

Root-lesion nematode (Pratylenchus thornei) is a serious pathogen of wheat in many countries. The International Triticeae Mapping Initiative (ITMI) population of recombinant inbred lines (RILs) was assessed for resistance to P. thornei to determine the chromosome locations of the resistance genes. The ITMI population is derived from a cross between the resistant synthetic hexaploid wheat W-7984 and a susceptible bread wheat cultivar Opata 85. Two years of phenotypic data for resistance to P. thornei were obtained in replicated glasshouse trials. Quantitative trait locus (QTL) analysis was performed using available segregation and map data for 114 RILs. A QTL on chromosome 6DS showed consistent effects for reduced nematode numbers (partial resistance) across years and accounted for 11% and 23% of the phenotypic variation. A second QTL for P. thornei resistance on chromosome 2BS accounted for an additional 19% and 5%. Restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers associated with the QTLs are physically located in regions rich in major genes at the distal ends of the short chromosome arms of 6D and 2B. SSR markers with potential for marker-assisted selection of P. thornei resistance effective in different genetic backgrounds have been identified.

Synthetic Triterpenoid Cyano Enone of Methyl Boswellate Activates Intrinsic, Extrinsic, and Endoplasmic Reticulum Stress Cell Death Pathways in Tumor Cell Lines

We explored the effect of a novel synthetic triterpenoid compound cyano enone of methyl boswellates (CEMB) on various prostate cancer and glioma cancer cell lines. CEMB displayed concentration-dependent cytotoxic activity with submicromolar lethal dose 50% (LD(50)) values in 10 of 10 tumor cell lines tested. CEMB-induced cytotoxicity is accompanied by activation of downstream effector caspases (caspases 3 and 7) and by upstream initiator caspases involved in both the extrinsic (caspase 8) and intrinsic (caspase 9) apoptotic pathways. By using short interfering RNAs (siRNA), we show evidence that knockdown of caspase 8, DR4, Apaf-1, and Bid impairs CEMB-induced cell death. Similar to other proapoptotic synthetic triterpenoid compounds, CEMB-induced apoptosis involved endoplasmic reticulum stress, as shown by partial rescue of tumor cells by siRNA-mediated knockdown of expression of genes involved in the unfolded protein response such as IRE1 alpha, PERK, and ATF6. Altogether, our results suggest that CEMB stimulates several apoptotic pathways in cancer cells, suggesting that this compound should be evaluated further as a potential agent for cancer therapy. Mol Cancer Ther; 10(9); 1635-43. (C)2011 AACR.

Enhanced non-homologous end joining contributes toward synthetic lethality of pathological RAD51C mutants with poly (ADP-ribose) polymerase

Here, we show that PARP inhibitor-mediated cell death of RAD51C-deficient cells occur by NHEJ-driven illegitimate repair of one-ended double-strand breaks, and the hypomorphic RAD51C pathological mutant cells can be targeted by `synergistic toxicity' induced by low-dose PARP inhibitor and IR.Poly (ADP-ribose) polymerase 1 (PARP1) inhibitors are actively under clinical trials for the treatment of breast and ovarian cancers that arise due to mutations in BRCA1 and BRCA2. The RAD51 paralog RAD51C has been identified as a breast and ovarian cancer susceptibility gene. The pathological RAD51C mutants that were identified in cancer patients are hypomorphic with partial repair function. However, targeting cancer cells that express hypomorphic mutants of RAD51C is highly challenging. Here, we report that RAD51C-deficient cells can be targeted by a `synthetic lethal' approach using PARP inhibitor and this sensitivity was attributed to accumulation of cells in the G(2)/M and chromosomal aberrations. In addition, spontaneous hyperactivation of PARP1 was evident in RAD51C-deficient cells. Interestingly, RAD51C-negative cells exhibited enhanced recruitment of non-homologous end joining (NHEJ) proteins onto chromatin and this accumulation correlated with increased activity of error-prone NHEJ as well as genome instability leading to cell death. Notably, inhibition of DNA-PKcs or depletion of KU70 or Ligase IV rescued this phenotype. Strikingly, stimulation of NHEJ by low dose of ionizing radiation (IR) in the PARP inhibitor-treated RAD51C-deficient cells and cells expressing pathological RAD51C mutants induced enhanced toxicity `synergistically'. These results demonstrate that cancer cells arising due to hypomorphic mutations in RAD51C can be specifically targeted by a `synergistic approach' and imply that this strategy can be potentially applied to cancers with hypomorphic mutations in other homologous recombination pathway genes.

Cell-targeted regulation of gene expression through synthetic RNA devices

The ability to interface with and program cellular function remains a challenging research frontier in biotechnology. Although the emerging field of synthetic biology has recently generated a variety of gene-regulatory strategies based on synthetic RNA molecules, few strategies exist through which to control such regulatory effects in response to specific exogenous or endogenous molecular signals. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to the regulation of genes and thus cellular function.

We describe efforts to develop an RNA-based device framework for regulating endogenous genes in human cells. Previously developed RNA control devices have demonstrated programmable ligand-responsive genetic regulation in diverse cell types, and we attempted to adapt this class of cis-acting control elements to function in trans. We divided the device into two strands that reconstitute activity upon hybridization. Device function was optimized using an in vivo model system, and we found that device sequence is not as flexible as previously reported. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct device stability, structure, and localization. The significant limitations of our platform prevented endogenous gene regulation.

We next describe the development of a protein-responsive RNA-based regulatory platform. Employing various design strategies, we demonstrated functional devices that both up- and downregulate gene expression in response to a heterologous protein in a human cell line. The activity of our platform exceeded that of a similar, small-molecule-responsive platform. We demonstrated the ability of our devices to respond to both cytoplasmic- and nuclear-localized protein, providing insight into the mechanism of action and distinguishing our platform from previously described devices with more restrictive ligand localization requirements. Finally, we demonstrated the versatility of our device platform by developing a regulatory device that responds to an endogenous signaling protein.

The foundational tool we present here possesses unique advantages over previously described RNA-based gene-regulatory platforms. This genetically encoded technology may find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.

Gene function prediction from synthetic lethality networks via ranking on demand.

MOTIVATION: Synthetic lethal interactions represent pairs of genes whose individual mutations are not lethal, while the double mutation of both genes does incur lethality. Several studies have shown a correlation between functional similarity of genes and their distances in networks based on synthetic lethal interactions. However, there is a lack of algorithms for predicting gene function from synthetic lethality interaction networks. RESULTS: In this article, we present a novel technique called kernelROD for gene function prediction from synthetic lethal interaction networks based on kernel machines. We apply our novel algorithm to Gene Ontology functional annotation prediction in yeast. Our experiments show that our method leads to improved gene function prediction compared with state-of-the-art competitors and that combining genetic and congruence networks leads to a further improvement in prediction accuracy.

An association mapping approach to identify flowering time genes in natural populations of Lolium perenne (L.)

Sk?t, L., Humphreys, M. O., Armstead, I. P., Heywood, S., Sk?t, K. P., Sanderson, R., Thomas, I. D., Chorlton, K. H., & Sackville Hamilton, N. R. (2005). An association mapping approach to identify flowering time genes in natural populations of Lolium perenne (L.). Molecular Breeding, 15(3), 233-245. Sponsorship: BBSRC RAE2008

High confidence prediction of essential genes in burkholderia cenocepacia

Essential genes are absolutely required for the survival of an organism. The identification of essential genes, besides being one of the most fundamental questions in biology, is also of interest for the emerging science of synthetic biology and for the development of novel antimicrobials. New antimicrobial therapies are desperately needed to treat multidrug-resistant pathogens, such as members of the Burkholderia cepacia complex.

Suppression of TNF- induced NFB activity by gallic acid and its semi-synthetic esters: possible role in cancer chemoprevention

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo dos mecanismos de resistência e virulência de isolados de Cryptococcus neoformans e Cryptococcus gattii e quantificação de genes de bomba de efluxo pós tratamento com Galatos de Alquila

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Metabolic engineering of beta-oxidation in Penicillium chrysogenum for improved semi-synthetic cephalosporin biosynthesis

Industrial production of semi-synthetic cephalosporins by Penicillium chrysogenum requires supplementation of the growth media with the side-chain precursor adipic acid. In glucose-limited chemostat cultures of P. chrysogenum, up to 88% of the consumed adipic acid was not recovered in cephalosporinrelated products, but used as an additional carbon and energy source for growth. This low efficiency of side-chain precursor incorporation provides an economic incentive for studying and engineering the metabolism of adipic acid in P. cluysogenum. Chemostat-based transcriptome analysis in the presence and absence of adipic acid confirmed that adipic acid metabolism in this fungus occurs via beta-oxidation. A set of 52 adipate-responsive genes included six putative genes for acyl-CoA oxidases and dehydrogenases, enzymes responsible for the first step of beta-oxidation. Subcellular localization of the differentially expressed acyl-CoA oxidases and dehydrogenases revealed that the oxidases were exclusively targeted to peroxisomes, while the dehydrogenases were found either in peroxisomes or in mitochondria. Deletion of the genes encoding the peroxisomal acyl-CoA oxidase Pc20g01800 and the mitochondrial acyl-CoA dehydrogenase Pc20g07920 resulted in a 1.6- and 3.7-fold increase in the production of the semi-synthetic cephalosporin intermediate adipoyl-6-APA, respectively. The deletion strains also showed reduced adipate consumption compared to the reference strain, indicating that engineering of the first step of beta-oxidation successfully redirected a larger fraction of adipic acid towards cephalosporin biosynthesis. (C) 2012 Elsevier Inc. All rights reserved.