Projects
Photobioelectrochemical Cells
Prof. Itamar Willner, Institute of Chemistry
Prof. Rachel Nechushtai, Plant and Environmental Sciences
Substantial progress has been accomplished in understanding the light-harvesting and electron transfer reactions in the photosynthetic reaction centers (RCs), Photosystem I (PSI), and Photosystem II (PSII). We implemented this knowledge to develop photoelectrochemical cells based on the natural photosynthetic reaction centers. The research efforts involve the electrical wiring of the photosynthetic reaction centers with electrodes using nanotechnological tools (see panel A-D in the figure). The modified electrodes are used for the generation of photocurrents and the conversion of light energy into electrical power. Highlights of the project include the development of artificial photoelectrochemical “leafs” that yield upon irradiation of photosystem-modified electrodes in water, the generation of photocurrents. Future research efforts are directed to couple photosynthetic reaction centers to enzymes that through light transform biomass materials into electrical power.
The (A-D) panels schematically illustrate the bio-photo-chemical Electrodes/Cells obtained in our different collaborative projects
MP-SiO2–NP as Novel Delivery Tool for Anti-Cancer Drugs
Prof. Itamar Willner, Institute of Chemistry
Prof. Rachel Nechushtai, Plant and Environmental Sciences
Mesoporous nanoparticles (MP-NP) attract a growing interest as high surface area materials for drug delivery. “Locking” of drugs in the channels of mesoporous nanoparticles by means of stimuli-responsive capping units provides exciting opportunities to develop “smart materials” for the targeted and dose-controlled release of drugs into specific cells. We develop drug-loaded mesoporous nanoparticles that release the drug in response
Step 1 : Introduction of MP-SiO2-NP into liver Cancer (HepG2) cells
Adding anti-FITC quenched fluorescence outside of cells
All green fluorescence imaged (in epi-fluorescent
microscope) is inside HepG2
Step 2 : Analysis of cytotoxicity of MP-SiO2-NP
with/without release of doxorubicin in cells
Alamar blue assay was the tool for detection of cell viability in image plate
RNA-zyme based sensors
Prof. Itamar Willner, Institute of Chemistry
Prof. Shimshon Belkin, Institute of Life Sciences
Whole cell bacterial sensors are being developed that will demonstrate for the first time bioluminescent responses to external stimuli mediated by intracellular activity of an “RNAzyme” molecule. DNAzymes (also known as deoxyribozymes) are synthetic enzymes made of short, single strands of deoxyribonucleic acid with catalytic capabilities. The reporter system constructed in our lab is based on ‘10-23’ DNAzyme which can catalyze sequence-specific cleavage of RNA molecules. Two complementary concepts are being engineered:
- “Lights off”: the chemical target (“inducer”) triggers the synthesis of an RNAzyme that cleaves the mRNA of a functioning enzyme.
- “Lights on”: The inducer drives the synthesis of an RNAzyme that cleaves the mRNA of an inhibitor enzyme
Structure-Function Relationships in NEET Proteins
Prof. Rachel Nechushtai, Plant and Environmental Sciences
Prof. Oded Livnah, Institute of Life Sciences
The newly discovered family of iron-sulfur (Fe-S) proteins coded by cisd genes that are defined by a unique CDGSH amino acid sequence in their Fe-S cluster binding domain. These proteins are involved in neuronal development, maintenance of skeletal muscle and in promoting longevity. Moreover, a transcriptional splicing error of Naf-1 (encoded by cisd2) leads to a rare but serious disease called Wolfram Syndrome 2, which is associated with hearing deficiencies, severe blindness, diabetes and a lower life expectancy. mitoNEET (mNT, encoded by cisd1) is involved in diabetes and obesity. Both Naf-1, mNT mRNA and protein levels, are increased in different cancers.
These proteins are localized to the outer mitochondrial and ER membranes and Naf-1 was shown to be involved in cell autophagy/apoptosis. Using x-ray crystallography and biochemical methodologies we characterize, together with Prof. Oded Livnah, the different structural components important for these novel class of 2Fe-2S proteins.
Structure of wt Naf-1 (left) and H114C Naf-1 mutant (right)
NEET– Pro-apoptotic/Anti-apoptotic Protein-protein Interactions
Prof. Rachel Nechushtai, Plant and Environmental Sciences
Prof. Assaf Friedler, Institute of Chemistry
By using peptide arrays of proteins involved in pro- or anti-apoptosis we identified, together with Prof. Assaf Friedler, the binding of tBid, BCl2 and other proteins to Naf-1. Naf-1 is a homodimeric member of the novel FeS protein NEET family which binds two 2Fe-2S clusters. The protein was shown to be an important partner for Bcl-2 at the ER to functionally antagonize Beclin 1-dependent autophagy. By using a combination of peptide array, hydrogen-deuterium exchange, functional and direct coupling analysis (DCA) we elucidated the molecular determinants and functional consequences of NAF-1-Bcl-2 interactions. Naf-1 binds to both the pro- and anti-apoptotic regions (BH3 and BH4) of Bcl-2 as demonstrated by a nested protein fragment analysis in
Identification of Naf-1 binding areas on BCL2 by peptide array (A) and mapping the identified sequences to the BCL2 3D- structure (B).
Remote detection of buried landmines
Prof. Shimshon Belkin, Institute of Life Sciences
Prof. Aharon Agranat, Institute of Applied Physics
All landmines “leak” minute amounts of explosives vapors that accumulate in the soil above them and serve as potential markers for the presence of the explosive device. The present project aims to take advantage of this phenomenon, by providing a new solution for the remote detection of landmines:
- Bacterial biosensors have been engineered to sensitively detect the chemical signature of TNT-based landmines
- These live sensors, encapsulated in hydrogel beads, will be dispersed over the target areas
- Remote optical scanning of the tested area, pinpointing hotspots with enhanced optical activity (fluorescence /luminescence) will indicate the potential presence of landmines.
In the Belkin lab, the engineered sensors are currently undergoing molecular manipulations that will provide them with the required sensitivity; whereas, in the Agranat lab, the remote optical detection apparatus is being developed.
Whole-cell bacterial sensors based on periplasmic-binding proteins (“Biomonar”)
Prof. Shimshon Belkin, Institute of Life Sciences
BIOMONAR develops a suite of dynamic nanoarray biosensors for monitoring environmental pollutants and pathogens, comprising three complementary platforms incorporating members of a single family of selector proteins, bacterial periplasmic binding proteins (PBPs). The project takes advantage of two of the main characteristics of this group of proteins: their demonstrated sensitivity to the immediate environment and their proven amenability to be “tailored” to the detection of specific target chemicals or groups of chemicals. The PBP protein family currently holds promising prospects for achieving predictable and controllable nanobiotechnological interfaces for biosensing. Using state-of-the-art “test-tube evolution” approaches, combined with a high throughput screening, BIOMONAR generates engineered PBPs, with differing detection specificities and sensitivity ranges, integrated into
a. Functionalized solid surfaces, onto which arrays of the different PBPs will be patterned.
b. Arrays of PBP-containing sets of multifunctional liposomal preparations.
c. Arrays of live bacterial strains, in each of which a different PBP will trigger a bioluminescent signal in response to the presence of the target compound.
SP1 as a biological Nanopore
Prof. Oded Shoseyov, Institute of Plant Sciences and Genetics in Agriculture
Prof. Itamar Willner, Institute of Chemistry
SP1 protein as a new type of biological nanopore is described and is utilized to distinguish single-stranded DNA at the single-molecule level. Using the SP1 nanopore to investigate single molecule detection broadens the existing research areas of pore-forming biomaterials from unsymmetrical biological nanopores to symmetrical biological nanopores. This novel nanopore could provide a good candidate for single-molecule detection and characterization of biomaterial applications.
The characterization of the SP1 protein structure and topography assembled in the lipid bilayer.
(A) Structure and charge distribution of SP1. In experimental pH 8, we expect the acidic residues (red) to be predominantly negatively charged and the basic residues (blue) to be positively charged. The ring-like structure of the dodecamer has an inner pore diameter of ~3 nm and an outside diameter of ~11 nm.
(B) Schematic diagrams corresponding to the open and analyte-blocked nanopore ssDNA was added from the cis chamber. Representative ionic currents observed for the SP1 pore in the absence and the presence of 0.1 µM ssDNA at 100 mV.
(C) Typical blockade current transients: (1) poly(dA)20; (2) poly(dA)45; (3) poly(dT)20; (4) poly(dT)45.
Protein-nanoparticle hybrids nanoelectronics structures
Prof. Oded Shoseyov, Institute of Plant Sciences and Genetics in Agriculture
Prof. Danny Porath, Institute of Chemistry
SP1 protein, hybridized with a nanoparticle, produced by the group of O. Shoseyov, is a building block for alternative structures to realize nanoelectronics applications. These structures can form ultra-dense arrays for memory applications and for “lego-like” constructions of nanowires and networks as seen in the figure below. D. Porath’s group has already demonstrated charging, set/reset and ternary logic operation using these building blocks.
SP1 images and structures: (a) TEM image (averaged) and (b) AFM image of SP1 proteins array. (c) SP1 array with schematic of nanoparticles that will serve for the memory. (d) A suggested nanowire and nanostructure.
Production of Nano Crystalline Cellulose (NCC)-recombinant Resilin-CBD Composites
Prof. Oded Shoseyov, Institute of Plant Sciences and Genetics in Agriculture
In this project we are developing a technology which is based on composite Resilin-NCC which results in dramatic alteration in mechanical behavior including high elasticity, resilience and resistance to repeated cycles of mechanical stress. Resilin-NCC chemically modified version, DOPA-Resilin-NCC, adds adhesive or sealant features to material. This platform technology is a breakthrough in material science. It brings together the toughness of cellulose nano-fibers from the plant kingdom, the remarkable elasticity and resilience of Resilin that enables flees to
jump as high as 400 times their height from the insect kingdom, and the adhesion power of DOPA, the functional molecule of mussels that enables them to bind tightly under water to organic and inorganic matter from the marine kingdom.
Comparison of NCC foams with NCC/resilin-CBD composite foams. NCC foam displays high plastic deformation, low elasticity and resilience (A). Highly elastic and shape memory composite NCC/resilin-CBD composite (B).
DNA translocation in nanopores towards DNA sequencing and investigating other bio-related phenomena
Prof. Danny Porath, Institute of Chemistry
Prof. Oded Shoseyov, Institute of Plant Sciences and Genetics in Agriculture
We investigate translocation of DNA through nanopores. The objective is to develop methods for rapid DNA sequencing or investigating the interaction of the translocated DNA with proteins. We are using solid state nanopores that are prepared by TEM drilling in SiN membrane. In one of our studies we made SP1 protein – solid-state hybrid nanopores, to reduce the DNA translocation speed.
Ultra sensitive cancer biomarker detection
Prof. Danny Porath, Institute of Chemistry
Prof. Assaf Friedler, Institute of Chemistry
To address the need of early cancer diagnosis for sustainable health, we are developing an ultra-sensitive and affordable integrated platform combining surface functionalized nanoprobes and single molecule imaging techniques for detecting cancer biomarkers with superior sensitivity than the common immune-based detection methods.
Monitoring enzyme activity using atomic force microscopy
Prof. Danny Porath, Institute of Chemistry
Prof. Assaf Friedler, Institute of Chemistry
Integration of the HIV cDNA into the host chromosome is a key event in the viral replication cycle. It is mediated by the viral Integrase (IN) enzyme, which is an attractive anti-HIV drug target. We study the integration reaction using AFM imaging in a two-LTR system (Guy et al. Chem Comm 2013).
Minimized high-affinity avidins
Prof. Oded Livnah, Dept. of Biological Chemistry, The Institute of Life Sciences
The strept/avidin-biotin system has gained great importance over the years both as a means to study the biorecognition phenomenon and as a prevailing tool for general application in the biological sciences. The extraordinary high affinity of avidin or streptavidin towards biotin is dependent upon the tetrameric architecture of the proteins. Over the years, attempts to minimize avidins by point mutations or loop permutations to their fundamental components were successful, yet the affinity towards biotin
decreased substantially by 7-9 orders of magnitude. High affinity biotin binding monomeric and dimeric avidins are of technological importance in many fields such as biotechnology, cell biology, and nanotechnology. Using these molecules will control the amount of biotin binding sites per molecule and diminish any ‘unwanted’ cross-linking. Such molecules could be either utilized as soluble protein or linked to an immobilized phase. These exotic avidins will be also employed as a template for further design of minimized high affinity avidins.
Physical properties of ‘two-component’ sensory systems in Escherichia coli
Dr. Ady Vaknin, Racah Institute of Physics HUJI
Prof. Victor Sourjik, Heidelberg University, Germany
Prof. Assaf Friedler, Institute of Chemistry HUJI
Adaptive responses to changes in the environment play an important role in the survival and pathogenicity of microbial cells. In bacteria, two-component systems, which consist of a sensory kinase and a cytoplasmic response regulator, play a central role in mediating such adaptive responses. While the importance of spatial organization of bacterial cells is gaining acknowledgement, the intracellular organization
of these sensory kinases is largely unknown. We study basic properties of these signalling molecules in vivo using a variety of fluorescence-based techniques.
Initial screening using imaging and fluorescence polarization measurements have shown that the sensory histidine kinases TorS and EvgS tend to form distinct clusters in Escherichia coli cells. Such clustering was further confirmed at low levels of proteins expression and also with the untagged receptors (PLoS One, 2013).
We are currently exploring further the self-associations of other receptors in response to external stimuli.
Harnessing the rotation power of the bacterial flagellar motor for controlling usefully chemical processes
Prof. Itamar Willner, Institute of Chemistry
Dr. Ady Vaknin, Racah Institute of Physics
Our aim is to utilize the autonomous movement of the bacterial cells and the ability to control it by external stimuli to manipulate various chemical reactions.