Construction and Expression of Pet Operon Using Shuttle Vector for Mesophilic and Thermophilic Bacteria

Keuntungan fermentasi etanol pada suhu tinggi mendorong penelitian perakitan bakteri termofilik etalogenik. Selain itu, kemampuan bakteri termofilik dalam penggunaan gula pentosa hasil degradasi biomasa memberi peluang untuk menekan biaya produksi bioetanol. Tujuan dari penelitian ini adalah untuk mengkonstruksi pet (production of ethanol) operon dengan menggunakan shuttle vector pMK18 dan melihat ekspresinya dalam bakteri mesofilik dan termofilik. Konstruksi dan ekspresi pet operon dengan menggunakan adhT dari bakteri termofilik dan pdc dari bakteri mesofilik, dan penggunaan mesofilik-termofilik shuttle vector sebagai backbone-nya baru pertama kali dilaporkan. Pet operon adalah suatu susunan gen penyandi produksi etanol yang terdiri dari gen pdc (pyruvate decarboxylase) dan adh (alcohol dehydrogenase). Konstruksi pet operon menggunakan gen adhT dari bakteri termofilik Geobacillus thermoglucosidasius M10EXG dan pdc (pyruvate dehydrogenase) dari bakteri mesofilik Zymomonas mobilis ZM4 telah dilakukan dengan menggunakan mesofilik-termofilik shuttle vector pMK18. Ekspresi pet operon pada bakteri mesofilik Eschericia coli dapat memproduksi 0,3 g/l etanol dengan aktivitas adhT sekitar 0,02 U/mg protein dan aktivitas pdc sekitar 0,004 U/mg protein. Perlu dilakukan penelitian lanjutan untuk perbaikan konstruksi pet operon untuk sistemtermofik pada Thermus thermophilus HB27, karena konstruksi yang didapat belum optimum untuk sistem termofilik ini. Hasil ini diharapkan akan mengawali pengembanganteknik manipulasi genetik pada bakteri termofilik yang masih sangat terbatas, khususnya pengembangan teknik manipulasi termofilik etanologenik

Screening for the optimal siRNA targeting a novel gene (HA117) and construction and evaluation of a derivative recombinant adenovirus

We found a novel gene named as HA117 in our previous research. At this study, we screened for an optimal siRNA targeting the novel gene HA117 using the pSOS-HUS method, verified the results of pSOS-HUS siRNA screening for optimal affinity for the target gene, and constructed and evaluated a recombinant adenovirus carrying the DNA template for transcription of the optimal HA117 siRNA. The pSOS-HUS vector method was successfully utilized as a rapid and effective screen for an optimal siRNA for a target gene. Among five pairs of DNA templates, siRNA transcribed from HAi5 gave the strongest interference with the novel gene HA117; a HAi5-carrying recombinant adenovirus (Ad-HAi5) was successfully constructed and evaluated, laying a foundation for the further study of HA117 gene function with RNAi technology

Co-expression networks in generation of induced pluripotent stem cells.

We developed an adenoviral vector, in which Yamanaka's four reprogramming factors (RFs) were controlled by individual CMV promoters in a single cassette (Ad-SOcMK). This permitted coordinated expression of RFs (SOX2, OCT3/4, c-MYC and KLF4) in a cell for a transient period of time, synchronizing the reprogramming process with the majority of transduced cells assuming induced pluripotent stem cell (iPSC)-like characteristics as early as three days post-transduction. These reprogrammed cells resembled human embryonic stem cells (ESCs) with regard to morphology, biomarker expression, and could be differentiated into cells of the germ layers in vitro and in vivo. These iPSC-like cells, however, failed to expand into larger iPSC colonies. The short and synchronized reprogramming process allowed us to study global transcription changes within short time intervals. Weighted gene co-expression network analysis (WGCNA) identified sixteen large gene co-expression modules, each including members of gene ontology categories involved in cell differentiation and development. In particular, the brown module contained a significant number of ESC marker genes, whereas the turquoise module contained cell-cycle-related genes that were downregulated in contrast to upregulation in human ESCs. Strong coordinated expression of all four RFs via adenoviral transduction may constrain stochastic processes and lead to silencing of genes important for cellular proliferation

Transcription activator like effector (TALE)-directed piggyBac transposition in human cells.

Insertional therapies have shown great potential for combating genetic disease and safer methods would undoubtedly broaden the variety of possible illness that can be treated. A major challenge that remains is reducing the risk of insertional mutagenesis due to random insertion by both viral and non-viral vectors. Targetable nucleases are capable of inducing double-stranded breaks to enhance homologous recombination for the introduction of transgenes at specific sequences. However, off-target DNA cleavages at unknown sites can lead to mutations that are difficult to detect. Alternatively, the piggyBac transposase is able perform all of the steps required for integration; therefore, cells confirmed to contain a single copy of a targeted transposon, for which its location is known, are likely to be devoid of aberrant genomic modifications. We aimed to retarget transposon insertions by comparing a series of novel hyperactive piggyBac constructs tethered to a custom transcription activator like effector DNA-binding domain designed to bind the first intron of the human CCR5 gene. Multiple targeting strategies were evaluated using combinations of both plasmid-DNA and transposase-protein relocalization to the target sequence. We demonstrated user-defined directed transposition to the CCR5 genomic safe harbor and isolated single-copy clones harboring targeted integrations

Characterization of a broad-based mosquito yeast interfering RNA larvicide with a conserved target site in mosquito semaphorin-1a genes

BACKGROUND: RNA interference (RNAi), which has facilitated functional characterization of mosquito neural development genes such as the axon guidance regulator semaphorin-1a (sema1a), could one day be applied as a new means of vector control. Saccharomyces cerevisiae (baker's yeast) may represent an effective interfering RNA expression system that could be used directly for delivery of RNA pesticides to mosquito larvae. Here we describe characterization of a yeast larvicide developed through bioengineering of S. cerevisiae to express a short hairpin RNA (shRNA) targeting a conserved site in mosquito sema1a genes. RESULTS: Experiments conducted on Aedes aegypti larvae demonstrated that the yeast larvicide effectively silences sema1a expression, generates severe neural defects, and induces high levels of larval mortality in laboratory, simulated-field, and semi-field experiments. The larvicide was also found to induce high levels of Aedes albopictus, Anopheles gambiae and Culex quinquefasciatus mortality. CONCLUSIONS: The results of these studies indicate that use of yeast interfering RNA larvicides targeting mosquito sema1a genes may represent a new biorational tool for mosquito control

A broad distribution of the alternative oxidase in microsporidian parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of ironsulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosome

Review of the environmental effects of the Space Station Freedom photovoltaic power module

An overview is provided of the environment in the low Earth orbit (LEO), the interaction of this environment with the Photovoltaic (PV) Power system of the Space Station Freedom is reviewed, and the environmental programs are described that are designed to investigate the interactions of the LEO environment with the photovoltaic power system. Such programs will support and impact the design of the subsystems of the PV module in order to survive the design lifetime in the LEO natural and induced environment