Name: synthesis of nine-atom deltahedral zintl ions of germanium and their functionalization with organic groups
Uploaded: 2012-02-11T14:00:00
Description: Watch this Scientific Journal Video about Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups at JoVE.com

The authors would like to thank the National Science Foundation for the continuous financial support (CHE-0742365) and for the purchase of a Bruker APEX II diffractometer (CHE-0443233) and a Bruker Microtof-II mass spectrometer (CHE-0741793). The authors would also like to thank CEST facility for their use of the Micromass Quattro-LC mass spectrometer.

1. Preparing Niobium Tubes
<ol>
 <li>Before cutting the niobium (Nb) tubes, prepare Nb-cleaning solution. In a 500 ml plastic bottle, measure out and add via a 100 ml measuring cylinder the following stock solutions as received: 110 ml H2SO4, followed by 50 ml HNO3, followed by 40 ml HF. Mix well and allow reaching room temperature before using.</li>
 <li>Measure the Nb tube, 4.5 cm in length, and cut with a pipe cutter. Avoid kinking the tubing. Repeat 3 more times.</li>
 <li>In a well...

It is important to clean well the partially oxidized Nb tubes. However, if the tubes are left too long in the Nb cleaning solution, this will severely compromise the thickness of the tube. Thus, 10 &#x2013; 15 seconds are imperative and the tubes should be very lustrous at the end (Fig 3). After the tubes are sealed inside the fused silica jacket they should be cleaned again with a dilute Nb acid solution. This should result in mild effervescence, cleaning any oxidized areas on Nb tubes that occurred during welding o...

<table border="1">
<tbody>
<tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>D8-Xray diffractometer</td>
 <td>Bruker</td>
 <td>Bruker APEX II</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Electrospray mass spectrometer</td>
 <td>Bruker</td>
 <td>Microtof-II</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Electrospray mass spectrometer</td>
 <td>Micromass</td>
 <td>Quattro-LC</td>
 <td>triple -quadropole</td>
 </tr>
 <tr>
 <td>Drybox</td>
 <td>Innovative Technology</td>
 <td>S-1-M-DL</td>
 <td>IT-Sys1 model</td>
 </tr>
 <tr>
 <td>Inert Gas/Vacuum Shielded 
 Arc Welding Arrangement</td>
 <td><a href="http://www.ldsvacuum.com" target="_blank">LDS Vacuum Products</a> </td>
 <td>Special Order</td>
 <td></td>
 </tr>
 <tr>
 <td>Arc Welder Power Source</td>
 <td>Miller</td>
 <td>Maxstar-91</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Welding Rubber Gloves</td>
 <td>The Home Depot</td>
 <td>&nbsp;KH643</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Electric Engraver</td>
 <td>Burgess Products</td>
 <td>74</td>
 <td>Vibro-Graver</td>
 </tr>
 <tr>
 <td>Circular Glass Saw</td>
 <td>Pistorius Machine Co. Inc</td>
 <td>GC-12-B</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Tube Furnace</td>
 <td>Lindberg/Blue M</td>
 <td>TF55035</td>
 <td>Minimite Laboratory Tube Furnace, Moldatherm (1100 &deg;C)</td>
 </tr>
 <tr>
 <td>Glass Drying Oven</td>
 <td>Fisher Scientific</td>
 <td>13-247-650G</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>High Vacuum Hg Schlenk-Line</td>
 <td> Special Order</td>
 <td>Univ Of Notre Dame</td>
 <td>Alternative: Edwards E050/60; VWR International; Cat. No. EVB302-07-110</td>
 </tr>
 <tr>
 <td>Large Torch</td>
 <td>Victor</td>
 <td>JT100C</td>
 <td>Welding torch, tip: Victor 5-W-J </td>
 </tr>
 <tr>
 <td>Small Torch</td>
 <td>Veriflo Co.</td>
 <td>3A</td>
 <td>Blow-pipe </td>
 </tr>
 <tr>
 <td>Tesla Coil</td>
 <td>VWR International</td>
 <td>KT691550-0000</td>
 <td>Leak detector</td>
 </tr>
 <tr>
 <td>Stirrer/Hot -Plate</td>
 <td>VWR International</td>
 <td>12620-970</td>
 <td>VWR HOT PLATE STR DY-DUAL120V</td>
 </tr>
 <tr>
 <td>Balance</td>
 <td>Denver Instrument Co.</td>
 <td>100A</td>
 <td>XE Series</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>LW Scientific</td>
 <td>E8C-08AV-1501</td>
 <td>Variable speed</td>
 </tr>
 <tr>
 <td>Graphite Reamer, (flaring)</td>
 <td>ABR Imagery, Inc.</td>
 <td>850-523 B01</td>
 <td>Open holes in Glass Blowing and flaring edges</td>
 </tr>
 <tr>
 <td>Striker</td>
 <td>Fisher Scientific</td>
 <td>12-007</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Vise-Grips</td>
 <td>The Home Depot</td>
 <td>0902L3SM</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Pipe-Cutter</td>
 <td>The Home Depot</td>
 <td>32820</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Cutting Pliers</td>
 <td>The Home Depot</td>
 <td>437</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Plastic Beaker</td>
 <td>VWR International</td>
 <td>13890-046</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Measuring Cylinder</td>
 <td>VWR International</td>
 <td>65000-006</td>
 <td>Careful, HF etches glass (if using a glass one)</td>
 </tr>
 <tr>
 <td>Large Plastic Bottle</td>
 <td>VWR International</td>
 <td>16128-542</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>13 x 100 Test-Tubes</td>
 <td>VWR International</td>
 <td>47729-572</td>
 <td>CULTURE TUBE 13X100 CS1000</td>
 </tr>
 <tr>
 <td>Laboratory (Rubber) Stoppers</td>
 <td>Sigma Chemical Co.</td>
 <td>Z164437-100EA</td>
 <td>Size 00</td>
 </tr>
 <tr>
 <td>Test-Tube Rack</td>
 <td>VWR International</td>
 <td>60196-702</td>
 <td>10-13 mm tube OD</td>
 </tr>
 <tr>
 <td>Stir-Bars</td>
 <td>StirBars.com/Big Science Inc.</td>
 <td>SBM-0803-MIC</td>
 <td>PTFE 8x3 mm Micro</td>
 </tr>
 <tr>
 <td>Glass Pipettes</td>
 <td>VWR International</td>
 <td>14673-043</td>
 <td>VWR PIPET PASTEUR 9IN CS1000</td>
 </tr>
 <tr>
 <td>Rubber Bulbs</td>
 <td>VWR International</td>
 <td>56311-062</td>
 <td>Latex, thin walled</td>
 </tr>
 <tr>
 <td>Glass Wool</td>
 <td>Unifrax I LLC</td>
 <td>6048</td>
 <td>Fiberfrax Bulk Fiber Insulation, Ceramic fiber</td>
 </tr>
 <tr>
 <td>Glass Slides</td>
 <td>VWR International</td>
 <td>16004-422</td>
 <td>75x25x1mm, Microscope Slides</td>
 </tr>
 <tr>
 <td>Paratone-N oil</td>
 <td>Hampton Research</td>
 <td>Parabar 10312</td>
 <td>Known as: Paratone-N, Paratone-8277, Infineum V8512</td>
 </tr>
 <tr>
 <td>High Vacuum Silicone Grease</td>
 <td>VWR International</td>
 <td>59344-055</td>
 <td>Dow Corning</td>
 </tr>
 <tr>
 <td>Liquid Nitrogen</td>
 <td>Univ. of Notre Dame</td>
 <td>&nbsp; </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Argon Gas Cylinder</td>
 <td>Praxair Distribution Inc.</td>
 <td>TARGHP </td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Nitrogen Gas Cylinder</td>
 <td>Praxair Distribution Inc.</td>
 <td>QNITPP</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Oxygen Gas Cylinder</td>
 <td>Praxair Distribution Inc.</td>
 <td>OT</td>
 <td>337 cf CYL</td>
 </tr>
 <tr>
 <td>Hydrogen Gas Cylinder</td>
 <td>Praxair Distribution Inc.</td>
 <td>HK</td>
 <td>195 cf CYL</td>
 </tr>
 <tr>
 <td>Propane Gas Cylinder/source</td>
 <td>Univ. of Notre Dame</td>
 <td>UND</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Quartz tubing, Lg</td>
 <td>Quartz Scientific Inc.</td>
 <td>100020B</td>
 <td>20 mm id x 22mm od x 48" clear fused quart tubing</td>
 </tr>
 <tr>
 <td>Quartz tubing, Md</td>
 <td>Quartz Scientific Inc.</td>
 <td>100007B</td>
 <td>Clear Fused Quartz Tubing,7mm id x 9mm od x 48"</td>
 </tr>
 <tr>
 <td>Round Bottom Quartz Joint</td>
 <td>Quartz Scientific Inc.</td>
 <td>6160189B</td>
 <td>Ball joint</td>
 </tr>
 <tr>
 <td>Quartz Safety Glasses</td>
 <td>Wale Apparatus</td>
 <td>11-1127</td>
 <td>waleapparatus.com</td>
 </tr>
 <tr>
 <td>Pyrex Safety Glasses</td>
 <td>Wale Apparatus</td>
 <td>11-2125-B3</td>
 <td>For clear and color borosilicate glass</td>
 </tr>
 <tr>
 <td>Blow Hose Kit</td>
 <td>Glass House</td>
 <td>BH020</td>
 <td>glasshousesupply.com</td>
 </tr>
 <tr>
 <td>Niobium Tubes</td>
 <td>Shaanxi Tony Metals Co., Ltd</td>
 <td>Niobium Tube, 50 ft</td>
 <td>Seamless Niobium Tube Outside diameter: 0.375 (&plusmn;0.005) inches. 
 Wall thickness: 0.02(&plusmn;0.003) Inches Niobium should be annealed.</td>
 </tr>
 <tr>
 <td>PEEK Starter Kit for Mass Spect</td>
 <td>Waters</td>
 <td>PSL613321</td>
 <td>PEEK (PolyEtherEtherKetone) tubing, nuts, ferrule, fits</td>
 </tr>
 <tr>
 <td>Mass Spect Needle Set</td>
 <td>VWR International</td>
 <td>60373-992</td>
 <td>Hamilton Manufacturer (81165)</td>
 </tr>
 <tr>
 <td>H2SO4</td>
 <td>VWR International</td>
 <td>BDH3072-2.5LG</td>
 <td>ACS Grade</td>
 </tr>
 <tr>
 <td>HNO3</td>
 <td>VWR International</td>
 <td>BDH3046-2.5LPC</td>
 <td>ACS Grade</td>
 </tr>
 <tr>
 <td>HF</td>
 <td>VWR International</td>
 <td>BDH3040-500MLP</td>
 <td>ACS Grade</td>
 </tr>
 <tr>
 <td>Distilled Water</td>
 <td>Univ. of Notre Dame</td>
 <td>UND</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Acetone</td>
 <td>VWR International</td>
 <td>BDH1101-4LP</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Ethylenediamine</td>
 <td>VWR International</td>
 <td>AAA12132-0F</td>
 <td>99% 2.5 L</td>
 </tr>
 <tr>
 <td>Toluene</td>
 <td>VWR International</td>
 <td>200004-418</td>
 <td>99.8 %, anhydrous</td>
 </tr>
 <tr>
 <td>Mercury</td>
 <td>Strem Chemicals, Inc.</td>
 <td>93-8046</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>Potassium (K) metal</td>
 <td>Strem Chemicals, Inc.</td>
 <td>19-1989</td>
 <td>Sealed in glass ampoule under Ar</td>
 </tr>
 <tr>
 <td>Germanium (Ge) powder</td>
 <td>VWR International</td>
 <td>AA10190-18</td>
 <td>GERM PWR -100 MESH 99.999% 50G</td>
 </tr>
 <tr>
 <td>Bistrimetylsilylacetylene, 
 (Me3SiC&equiv;CCSiMe3)</td>
 <td>Fischer Scientific</td>
 <td>AC182010100</td>
 <td>&nbsp; </td>
 </tr>
 <tr>
 <td>18-crown-6 
 (1,4,7,10,13,16-Hexaoxacyclooctadecane)</td>
 <td>VWR International</td>
 <td>200001-954</td>
 <td>99%, 25 gm</td>
 </tr>
 <tr>
 <td>2,2,2-crypt 
 (4,7,13,16,21,24-Hexaoxa-1,10 diazabicyclo[8.8.8]hexacosane)</td>
 <td>Sigma Aldrich </td>
 <td>291110-1G</td>
 <td>98%</td>
 </tr>
 </tbody>
</table>

synthesis of nine-atom deltahedral zintl ions of germanium and their functionalization with organic groups

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 Their redox chemistry is even younger, just about ten years old, but despite this short history these deltahedral clusters ions E9n- (E = Si, Ge, Sn, Pb; n = 2, 3, 4) have already shown interesting and diverse reactivity and have been at the forefront of rapidly developing and exciting new chemistry.3-6 Notable milestones are the oxidative coupling of Ge94- clusters to oligomers and infinite chains,7-19 their metallation,14-16,20-25 capping by transition-metal organometallic fragments,26-34 insertion of a transition-metal atom at the center of the cluster which is sometimes combined with capping and oligomerization,35-47 addition of main-group organometallic fragments as exo-bonded substituents,48-50 and functionalization with various organic residues by reactions with organic halides and alkynes.51-58 
This latter development of attaching organic fragments directly to the clusters has opened up a new field, namely organo-Zintl chemistry, that is potentially fertile for further synthetic explorations, and it is the step-by-step procedure for the synthesis of germanium-divinyl clusters described herein. The initial steps outline the synthesis of an intermetallic precursor of K4Ge9 from which the Ge94- clusters are extracted later in solution. This involves fused-silica glass blowing, arc-welding of niobium containers, and handling of highly air-sensitive materials in a glove box. The air-sensitive K4Ge9 is then dissolved in ethylenediamine in the box and then alkenylated by a reaction with Me3SiC&equiv;CSiMe3. The reaction is followed by electrospray mass spectrometry while the resulting solution is used for obtaining single crystals containing the functionalized clusters [H2C=CH-Ge9-CH=CH2]2-. For this purpose the solution is centrifuged, filtered, and carefully layered with a toluene solution of 18-crown-6. Left undisturbed for a few days, the so-layered solutions produced orange crystalline blocks of [K(18-crown-6)]2[Ge9(HCCH2)2]&bull;en which were characterized by single-crystal X-ray diffraction. 
The process highlights standard reaction techniques, work-up, and analysis towards functionalized deltahedral Zintl clusters. It is hoped that it will help towards further development and understanding of these compounds in the community at large.

Watch this Scientific Journal Video about Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups at JoVE.com

Synthesis of Nine-atom Deltahedral Zintl Ions of Germanium and their Functionalization with Organic Groups

We present the high-temperature synthesis of intermetallic precursors K4Ge9, their dissolution in ethylenediamine to form Ge94- deltahedral Zintl ions, and the reaction of the clusters with alkynes to form organo-Zintl ions. The latter are characterized by electrospray mass spectrometry in solutions and by single-crystal X-ray diffraction in the solid state.

Although the first studies of Zintl ions date between the late 1890's and early 1930's they were not structurally characterized until many years later.1,2 ...

synthesis-nine-atom-deltahedral-zintl-ions-germanium-their

Research

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Biology

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

High-Throughput Measurement and Classification of Organic P in Environmental Samples

Many types of organic phosphorus (P) molecules exist in environmental samples1. Traditional P measurements do not detect these organic P compounds since they do not react with colorimetric reagents2,3. Enzymatic hydrolysis (EH) is an emerging method for accurately characterizing organic P forms in environmental samples4,5. This method is only trumped in accuracy by Phosphorus-31 Nuclear Magnetic Resonance Spectroscopy (31P-NMR) -a method that is expensive and requires specialized technical training6. We have adapted an enzymatic hydrolysis method capable of measuring three classes of phosphorus (monoester P, diester P and inorganic P) to a microplate reader system7. This method provides researchers with a fast, accurate, affordable and user-friendly means to measure P species in soils, sediments, manures and, if concentrated, aquatic samples. This is the only high-throughput method for measuring the forms and enzyme-lability of organic P that can be performed in a standard laboratory. The resulting data provides insight to scientists studying system nutrient content and eutrophication potential.

This protocol describes a high-throughput method of enzymatic hydrolysis that utilizes a microplate reader to measure and classify soil phosphorus as P monoesters, P diesters and inorganic P. Up to 96 samples can be measured at one time in a standard laboratory.

Many types of organic phosphorus (P) molecules exist in environmental samples1.  Traditional P measurements do not detect these organic P compounds since ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Synthesis of Wavelength-shifting DNA Hybridization Probes by Using Photostable Cyanine Dyes

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase synthesis using standard phosphoramidite chemistry. Oligonucleotides are post-synthetically labeled by two new photostable cyanine dyes using copper-catalyzed click-chemistry. The synthesis of both donor and acceptor dye is described and is performed in three consecutive steps. With the DNA as the surrounding architecture, these two dyes undergo an energy transfer when they are brought into close proximity by hybridization. Therefore, annealing of two single stranded DNA strands is visualized by a change of fluorescence color. This color change is characterized by fluorescence spectroscopy but can also be directly observed by using a handheld ultraviolet (UV) lamp. The concept of a dual fluorescence color readout makes these oligonucleotide probes excellent tools for molecular imaging especially when the described photostable dyes are used. Thereby, photobleaching of the imaging probes is prevented, and biological processes can be observed in real time for a longer time period.

Photostable cyanine dyes are attached to oligonucleotides to monitor hybridization by energy transfer.

In this protocol, we demonstrate a method for the synthesis of 2&#39;-alkyne modified deoxyribonucleic acid (DNA) strands by automated solid phase ...

Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately 30 x 106 viable cells per mouse. Primary adult mouse keratinocytes are harvested from the dorsal skin of female mice. Male mice (~6 weeks old) can be used for keratinocyte harvesting depending on the requirements of the experiment. Euthanized mice are shaved and sterilized with serial washes in povidone iodine and ethanol solutions (70% alcohol). After disinfecting the mice, the dorsal skin is removed and the subcutaneous fat and muscle are removed with a scalpel and discarded. The skins are cut into small pieces and treated with a mild, low temperature trypsinization to detach the lower dermis from the epidermis. The scraped epidermises are stirred at low speed, filtered to remove the hairs, counted, and re-suspended in culture medium. This method provides an excellent single cell suspension of highly culturable cells for many downstream applications.

The goal of this protocol is to isolate epidermal keratinocytes from the dorsal skin of adult mice for a variety of downstream applications such as molecular biology, biochemistry, fluorescence activated cell sorting, and primary in vitro uses (e.g., clonogenic keratinocytes).

The protocol described here is a reliable method of harvesting primary keratinocytes from adult female mice (54 &#177; 2 days old) yielding approximately ...