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Expressing Multiple Genes From A Single Transcript In Algae And Plants

Green algae have been promoted as vehicles for the production of biofuels, pharmaceuticals, food additives, vaccines, and for toxic substance remediation, and many plants are the focus of efforts to produce drought tolerant, pest resistant, or more nutritious crops. Many of these engineering efforts rely on expression of multiple transgenes (e.g. in a multistep metabolic pathway to avoid accumulation of a toxic intermediate). It can also be useful to produce two or more proteins in a particular stoichiometry, as in a heterodimer that requires equimolar production of two polypeptides. Whether the goal is to express one transgene, or several, most efforts to transform plants and algae require cotransformation of the gene of interest with a selectable marker, such as a gene that confers resistance to a drug or herbicide, or complements an auxotrophy. Unfortunately, commonly used methods for co-transformation of algae and other plants are very inefficient. UC Berkeley investigators have developed a method for polycistronic gene expression,  and show how to achieve this using the organism's own sequences, without recourse to viral elements or other foreign elements, which is important for any technology where bioproducts are generated, since these may be used on humans (cosmetics) or in humans (food additives), especially crop technology.

Foliar Formulation to Protect Plants from Abiotic Stress

Prof. Juan Pablo Giraldo and his colleagues from the University of California, Riverside have developed a foliar formulation for increasing crop protection and photosynthetic performance when crops are under light, heat, and salinity stress. This is achieved by applying a nanomaterial (poly (acrylic acid) nanoceria, PNC) that interacts with plant chloroplasts to reduce abiotic stress. The nanoparticle formulation uses a novel, scalable and biocompatible approach to protect plant seeds, seedlings, and mature plants from stress.  The emerging field of nano-enabled agriculture has the potential to create crops that are protected from climate change induced stresses and have enhanced photosynthesis.   Fig 1: a, Nanoceria (PNC) increases photosynthesis and biomass in Arabidopsis plants under stress. No nanoparticles (NNP) are shown as control. b, Substantial damage to Arabidopsis plants exposed to excess light was mitigated by PNC.  

Low Cost and Scalable Sap Feeding Insect Rearing and Gene Editing System

Profs. Peter Atkinson and Linda Walling at UCR have developed an in vitro rearing system on 3.5-cm and 6-cm  leaf disc plates that support egg to adult development in as little as 19 days. This system translates to a small-footprint, cost-effective rearing process, which can be industrialized, automated  and applied to other sap-feeding insects. Each plate may be used as an independent experiment or a mini-colony of a new whitefly genetic strain. Creating genetically modified whiteflies and other sap-feeding insects for genetic manipulation involves microinjecting embryos (eggs), which remain attached to excised leaf discs, which have been pretreated to remain viable throughout the whitefly life cycle.  This technology can be used to maintain colonies of whitefly in a more cost-effective way than existing approaches. In addition, this technology has been used to generate the first genetic mutants in the glassy-winged sharpshooter, Homalodisca vitripennis, a significant pest of Californian viticulture and thus opening the possibility of developing new strategies for its control and elimination. Fig. 1A shows a wild-type male whitefly and a mutant white male whitefly, which was generated by CRISPR/Cas9 mutagenesis using the leaf-disc injection and rearing protocols. Fig. 1 B shows a mosaic-eyed glassy-winged sharpshooter that was generated using the same technology.     Fig. 2 Each incubator (left) can hold up to 700 experiments/mini-whitefly colonies compared to the bugdorm (right), which houses one colony/experiment per tent. One incubator would replace ~11 biosafety level 2 (BSL2) greenhouses.    

Decorating Chromatin for Precise Genome Editing Using CRISPR

A novel fusion construct that fuses Cas9 to a truncated version of human PRDM9 with the purpose of improving precise genome editing via homologous direceted repair (HDR). PRDM9 is a protein that deposits histone marks H3K4me3 and H3K36me3 simultaneously during meiosis to mark recombination hot spots where crossover occurs and is resolved by homologous recombination. H3K36me3 has also been demonstrated to be required upstream of homologous recombination repair after double stranded breaks (DSBs) and during V(D)J recombination for adaptive immunity. Recent evidence suggests PRDM9 acts as a pioneer factor opening closed chromatin. The newly engineered PRDM9C-Cas9 fusion construct shows increased HDR and decreased non-homologous end joining mediated insertions and deletions (indels).

Fusion Protein for Treatment of Inflammatory Diseases

Researchers at the University of California, Davis have developed a plant-based, fusion protein for use in the treatment of inflammatory diseases.

FEAST - Fast Expectation-Maximization Microbial Source Tracking

UCLA researchers from the Department of Computer Science have developed a method to analyze large genomic data sets to quickly identify bacteria community imbalances.

Methods for Producing Cultured Meat that has Heterogeneous Composition

UCLA researchers in the Departments of Integrative Biology and Physiology and Molecular, Cellular, and Developmental Biology have developed a novel method for the production of marbled, cultured meat with desirable texture and flavor.

Development Of Biosensors For Drought Stress In Plants

Researchers at the University of California, Davis have developed a prototype biosensor that can monitor detectable levels of hormones present in plants experiencing drought or other environmental stress.

Single Conjugative Vector for Genome Editing by RNA-guided Transposition

The inventors have constructed conjugative plasmids for intra- and inter-species delivery and expression of RNA-guided CRISPR-Cas transposases for organism- and site-specific genome editing by targeted transposon insertion. This invention enables integration of large, customizable DNA segments (encoded within a transposon) into prokaryotic genomes at specific locations and with low rates of off-target integration.

TRM: HIF-1 alpha KO Mice (CRE)

Hypoxia-inducible factor 1-alpha is a transcriptional regulator of the adaptive response to hypoxia. When activated under hypoxic conditions, it can turn on over 40 genes involved in a variety of physiological activities. The dysregulation or alteration by mutation can lead to pathophysiology in areas of energy metabolism, cancer, cell survival and tumor invasion.

Method For Production Of Fatty Acids In Blue-Green Algae

Currently, renewable fatty acids are obtained solely from plant oils. Medium chain fatty acids (C8-C14) are typically sourced from coconut and palm oil, whereas longer chain saturated and unsaturated fatty acids are typically sourced from tallow, soy, corn or sunflower oil. Fatty acids are widely used for food, personal care products, industrial applications (e.g., lubricants, adhesives, detergents and plastics), as well as increasingly as biofuels. The demand for renewable fatty acids is rising and expanding. Given the current understanding of biological pathways it becomes possible to utilize other organisms, especially microorganisms, for the production of renewable chemicals such as fatty acids.

A Wearable Platform for In-Situ Analysis of Hormones

UCLA researchers in the Department of Electrical and Computer Engineering have developed a highly sensitive, wearable hormone monitoring platform.

Modified Enzymes to Improve Crop Yield

Researchers at the University of California have identified new modified versions of the carbon fixing enzyme, Phosphoenolpyruvate carboxylase (PPC).  in planta results show that the modified PPC enzymes confer upwards of a five fold increase in carbon fixation when compared to wild type plants. PPC dependent carbon fixation is key to photosynthesis, production of nutrients, and plants conditioning their growth environment. Plants with modified PPCs that increase carbon fixation and photosynthetic output will have increased plant productivity, which is critical for feeding a growing population. Additionally, by identifying surgical changes that can unleash the full productivity of plant PPC’s, it will be possible to increase the rate of depletion of atmospheric CO2.  The combination of these outcomes represents the opportunity to boost agricultural productivity, increase the amount of agriculturally available land by upwards of 100%, and improve the nutritional quality of plants all of which are dependent on removal of CO2 from our atmosphere.  Fig. 2 in vitro comparison of wild type (wt) and modified versions of maize PPC1, which is key to C4 photosynthesis, in the absence or presence of increasing amounts of the allosteric inhibitor, malate. Whereas version A is less affected by malate than wt, both versions B and C are largely unaffected by malate and have a 2-fold increase in activity compared to the wt version.  

Flavonol Profile as a Sun Exposure Assessor for Grapes

Researchers at the University of California, Davis have developed a solar radiation assessment method for grapes that uses a flavonol profile. This method can be done using either HPLC or through the computer processing of the absorption spectra of a purified flavonol extract via a purification kit.

Biological Control of Pierce's Disease of Grape with a Beneficial Bacterium

The plant pathogenic bacterium Xylella fastidiosa causes very serious diseases of many important crops worldwide, including Pierce's disease (grapes), almond leaf scorch (almonds), citrus variegated chlorosis (Citrus), and others. In California's grape industry alone, Pierce's disease causes more than $100 million in losses annually. The inventors have discovered that injection or topical application of grapevines with the beneficial endophytic bacterium Paraburkholderia phytofirmans (PsJN) causes great reductions in pathogen abundance and disease severity. As there are no other effective therapies for the control of this and other diseases caused by Xylella fastidiosa, PsJN has the potential to revolutionize our control of the very serious diseases caused by Xylella fastidiosa bacterium.

Generation of Non-Transgenic, Heritably Gene-Edited Plants

Researchers at the University of California, Davis have developed a guided nuclease based expression system to introduce genetic modifications into plants without the need for tissue culture.

Novel Steroid Hormone Assay

Researchers at the University of California have identified in insects that the membrane transporter, Ecdysone Importer (EcI), is involved in the cellular uptake of the primary steroid hormone ecdysone. Specifically after transport through Ecl, ecdysone’s active form (20-hydroxyecdysone or 20E and related ecdysteroids) enters its target cells and binds to the ecdysone receptor (EcR), which forms a heterodimer with another nuclear receptor and activates transcription of multiple genes involved in molting and metamorphosis. This new discovery of Ecl’s role counters the prevailing consensus that steroid hormones diffuse through cell membranes.  This will enable the screening of new compounds that interact with Ecl.  Such new compounds may be used for insect pest control. Fig. 1 membrane transporters (blue) guide steroid hormones (blue dots) into cells. This new discovery counters the conventionally held scientific consensus that steroid hormones passively diffuse through cell membranes.   Fig. 2 EcI mutants (bottom) were not able to enter into metamorphosis when compared to the control (top).

The Bic Inhibitor Of Cry-Cry And Cry-Cib Oligomerization/ Clustering

UCLA researchers in the Department of Molecular, Cell, and Developmental Biology have discovered two Arabidopsis proteins, BIC1 and BIC2, that are capable of inhibiting light-dependent dimerization of cryptochrome (CRY) molecules. These BICs can be used as an improved drug screening platform through controlled, titratable, label-free and reversible protein – protein interactions.

Rapid, Sensitive Detection of Nucleic Acid Sequences in Environmental Samples

UCLA Researchers at the California NanoSystems Institute have developed a methodology that permits PCR-based detection of nucleic acid sequences in soil that does not require the isolation of DNA.

Early Diagnosis and Treatment for Citrus Greening Disease

University of California, Riverside researcher, Prof. Hailing Jin, has shown that several citrus small RNAs are induced upon infection by Candidatius Liberibacter asiaticus (Las).  These miRNAs and siRNAs would enable the early diagnosis of HLB in citrus trees and nursery stocks.  In addition to the identification of the miRNA biomarker, Prof. Jin also discovered that treating Las infected trees with phosphorus oxyanion improved fruit production.  These studies of the improvement in yield in HLB infected citrus was demonstrated in a 3-year field trial in Florida.  Fig. 1 shows the relative expression levels of miRNA399 in HLB infected citrus. Infected trees express high levels of miRNA 399. Fig. 2 shows leaves from trees that did or did not receive phosphorus oxyanion treatment over a one year period. Leaves treated with phosphorus oxyanion are healthier than leaves from untreated trees.

Method to Develop a Stable Pluripotent Bovine Embryonic Stem Cell Line

Researchers at the University of California, Davis have developed a method to produce stable pluripotent bovine embryonic stem cells.

Fish Tank Effluent Sampling System

Researchers at the University of California, Davis have developed a valve system to collect effluent waste from fish in a closed recirculating aquaponic system (RAS).

Gene Drive System to Control D. suzukii Flies

Prof. Omar Akbari and his lab at UCR have developed a gene drive system using a synthetic maternal effect dominant embryonic arrest element (Medea) to control D. suzukii.  The engineered Medea element is a maternal toxin coupled to a tightly linked embryonic “antidote”.   Female D. suzukii transformed with the Medea element and antidote deposit a toxin into all oocytes.  Should the embryo inherit a Medea element, it may inhibit the toxin’s lethality by expressing miRNAs as an antidote that targets the toxin.  Embryos without a Medea element are not able to counter the effects of the toxin and do not survive past the embryonic stage.The lab has also tested the transgenic D. suzukii Medea in eight geographically distinct populations and showed that the overall transmission rate of the Medea element in each population was 94.2%.  This suggests that D. suzukii Medea should be able to drive robust population replacement and cause a population crash by spreading Medea through a population and making it infertile.

Bacteria from Medicago Root Nodules as Potentialy Useful PPB (Plant Probiotic Bacteria) for Agriculture

UCLA researchers in the Department of Molecular, Cell, and Developmental Biology have discovered new species of plant probiotic bacteria to enhance plant growth for agricultural purposes.

Breast Milk as a Source, Incubation/Storage Medium, and Delivery System for Infant Mucosal Immunity Bacteriophage

Researchers at the University of California, Davis have developed a method to harvest and enrich symbiotic bacteriophage to promote bacterial immunity.

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