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physiological systems questions




Hi, I have 7 physiological systems questions that I need desperate help with. I have attached the document containing the 7 questions thank you. If i'm pleases with the answers I also have another 13 potential questions. Please include all working out for the question.;Attached is also the notes for the experiment it is based on.;Attachments Preview;Biosensors practical notes updated version.docx Download Attachment;BioNanotechnology Practical: Ion Channels in Membranes.;Membrane Conduction and Ion Channels;Key Learning Objectives;1. Bilayer lipid membranes (BLM) are the major constituent of cell membranes.;2. BLMs block the passage of ions such as Na+, K+, Cl- and Ca++.;3. Ion channels penetrate cell membranes permitting the passage of ions across the membrane.;4. A convenient tool for studying the ion transport properties is a tethered membrane that is more;stable and easier to study than many other model systems.;5.;Gramicidin is bacterial polypeptide and is an example of an ion channel. In this practical class;you will be asked to fabricate a tethered membrane, insert gramicidin, and measure its;conductance.;6. You will be instructed to form a tethered membrane and to include in it the ion channel;Gramicidin.;7. You will need to measure the conductivity of Gramicidin in membrane and determine he;membrane thickness.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;Background;Cell Membranes;Cell membrane properties control the behaviour of all plants, bacteria and animals. Cell membranes consist;of self-assembled supramolecular structures formed by amphiphiles, or compounds that have polar segments;that strongly attract water and non-polar segments that do not. This results in the non-polar segments being;excluded from the aqueous phase and assembling into bimolecular sheets which eventually form closed;spheres which are the precursors of biological cells. The amphiphiles we are interested in here are known as;lipids and the cell-like structures they form when dispersed in water are known as liposomes. Liposomes can;be 10 nm to hundreds of micrometres in diameter but all have walls that are approximately 4 nm thick, and;are nearly impermeable to ions such as Na+, K+ and Cl-. The 4 nm thick lipid bilayer, that forms the wall of a;liposome is similar to that found in all cell membranes, whether they are from bacteria, plants or animals.;Alterations in membrane ionic permeability are the basis of;Signalling between neurones in the brain, and between neurones in the sympathetic and;autonomic nervous systems.;The senses of sight, sound, taste touch and smell in animals, and related functions in plants;and bacteria.;Mitochondrial metabolism and bioenergetics.;Cell membrane biochemistry is a core discipline within medical research and a core interest of the;Pharmaceutical Industry when searching for drug targets to address a wide range of medical conditions.;Membrane research is a significant component of a current major international research effort focussed on;replacement antibiotics for penicillin which is becoming increasingly ineffective against methicillin resistant;bacterial strains of Staphylococcus Aureus. Compounds that interact with membranes are also important in;understanding the effects of many types of venom, toxins, and some chemical warfare agents..;Tethered membranes;Traditional techniques used to study transmembrane ion transport require the use very small liposomes or;single cells pieced using fragile microelectrodes. Tethered membranes provide a stable planar phospholipid;bilayer over a relatively large surface area (2-3 mm2) that is a convenient alternative tool to study ion;transport in membrane bound ion channels. The tethering of the membrane is achieved using sulphur;chemistry to gold (gold is not totally unreactive and possesses a chemistry with sulphur). Molecular tethers;are thus molecules that possess a sulphur group, polar linkers and a hydrophobic segment that embeds in the;lipid bilayer. The polar linkers allow the existence of an aqueous layer, between the gold electrode and the;membrane. The assembly of a tethered membrane is shown below.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;(a) Ethanol solutions containing 0.4mM;disulphides are exposed to pure fresh gold;for 30 minutes. The molecules collide with;the gold and sulphur-gold bonds form;causing the self assembly of a lipid-spacer;monolayer. In todays practical class 10%;of the molecules are hydrophobic lipidic;anchor groups, and ninety percent are;hydrophilic spacers. This ratio can be;reduced to below 1% tether molecules or;up to 100% tether molecules. The motive;for reducing the fraction of tethers is to;provide more space to incorporate large;channels or to increase the number of;tethers to fabricate a more stable device.;(b) Following the adsorption of the self;assembled monolayer at the gold surface a;further 8ul of 3mM free lipid in ethanol is;allowed to assemble at the surface and then;rinsed with buffer.;(c) Rinsing with buffer causes the mix of;tethered and free lipids to form into a;tethered bilayer, 4nm thick on a 3nm;hydrophilic cushion. The hydrophilic;cushion mimics the inside of a cell and the;lipid bilayer mimics a cell membrane.;Ion Channels;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;Ion channels are molecules that create hydrophilic pathways across lipid bilayer membranes permiting ions;to cross otherwise impermeable membranes. Common bacteria such as Pneumonia, Diphtheria, Golden;Staphylococcus and Anthrax are pathogenic because the toxins they produce are ion channels that puncture;the cells of target organisms and collapse their transmembrane potentials.;Gramicidin (gA): Another ion channel, found in the soil bacteria, B. brevis is gramicidin A (See Figure;Below). Being much smaller, with molecular weight of 1882 Da, two molecules end-to-end are required to;span the lipid bilayer. Gramicidin is ion selective and is only conducive to monovalent cations (especially;Na+).;The bacterial ion channel gramicidin (gA). Monomers in the inner and outer leaflets of the bilayer;membrane need to align to form a continuous channel to permit ions to cross the membrane.;(a) Schematic figure of gramicidin A in a tethered membrane. An excitation potential of 20mV a.c. is applied;and the current due to ions being driven back and forth across the membrane is measured.;(b) More detail of gramcidin A showing two gramicidin monmers aligning and forming a conductive dimer.;Beneath the image of the dimer is an end view showing the pore through the centre of gramicidin through;which ions pass.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;Membrane Preparation kit;A six-channel electrode is provided in this practical class that is to be assembled into a flow cell cartridge;(Fig 1A and 2A below). The assembled cartridge plugs into a conductance reader (Fig 2B below) [SDx;tethaPod ], that reads both the membrane conductance and capacitance. A cartridge preparation kit is;supplied by which consists of;individually packaged electrodes pre-coated with tethering chemistry (Fig. 3A below);a flow cell cartridge top containing the gold counter electrode (Fig.2A and 3B below);an alignment jig for use when attaching the electrode to the flow-cell cartridge (Fig. 1A and 3C;below);a silicon rubber pressure pad used when attaching the electrode to the flow cell cartridge (Fig. 3D;below);an aluminium pressure plate used when attaching the electrode to the flow cell cartridge (Fig. 3E;below);a pressure clamp is used when attaching the electrode to the flow cell cartridge (Fig. 1B below);FIGURE 1;FIGURE 2;FIGURE 3;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;In addition to the supplied membrane preparation kit you will need;(i) Pair of scissors to open the slide pack;(ii) A 10ul and 100ul pipette and tips to deliver the phospholipid (8l) and rinse with buffer(100l);(iii) Tweezers to remove the slide from the sealed pack.;(iv) Waste bin to collect used tips.;(v) Phosphate buffered saline (100ml).;(vi) Timer to measure 2 minute incubation times for forming the membrane and a one minute delay for;the adhesive to seal.;FIGURE 4;Introduction to practical exercise;Aim;1.;2.;3.;4.;To prepare tethered membranes containing gramicidin A (gA).;To measure the conductance dependence of the membrane on gramicidin concentration.;Use this measurement to calculate the conduction of a dimeric gramicidin channel.;To determine the dependence of conductivity on the bias potential and from this determine the ion;selectivity.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;5. To measure the membrane capacitance.;6. Use this measurement to calculate the thickness of a lipid bilayer.;7.;Note!;Ensure all equipment, instrumentation and chemicals are available when you;start. Timing is critical for proper membrane formation. Read the entire;experiment through before commencing.;Exercise 1. Prepare tethered membranes containing gramicidin;a. Cut open the silver foil pack, and using tweezers remove the slide.;b. (Never touch the gold with fingers as this may damage the lipid coating lipid formation of the;membrane.);c. The electrode is stored in ethanol and you need to stand it on a tissue to dry. This may take 1-2;minutes.;d. Align the dry slide over the alignment jig, ensuring electrode tracks and the SDX logo on the slide;overlay each other. Using tweezers gently push electrode into the slot.;e. Remove top thin protective layer of plastic from the cartridge. (Be sure that it is only the thin;protective layer that is removed and not the entire adhesive laminate.) This will reveal a sticky;surface which will then bind to the electrode upon contact.;f. Position white cartridge over the top and push into position. Once the two surfaces meet do not peel;them apart or attempt to re-locate them as it will damage the electrode.;g. Gently put the cartridge and electrode into the clamp and tighten. Allow to stand for at least 1;minute, before loosening the pressure. The electrode is now ready for membrane formation.;Membranes are formed as follows;Chambe;r;1;2;3;4;5;6;Constituent in Phospholipid;0nM gA;40nM gA;60nM gA;80nM gA;100nM gA;120nM gA;a. Start stop watch.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;b. Add 8L phospholipid solution (0nMgA) to chamber 1.;c. At 15 seconds, add 8 L 40nM gA solution to chamber 2.;d. At 30 seconds, add 8 L 60nM gA solution tochamber 3.;e. At 45 seconds, add 8 L 80nM gA solution to chamber 4.;f. At 60 seconds, add 8 L 100nM gA solution to chamber 5.;g. At 75 seconds, add 8 L 120nM gA solution to chamber 6.;h. At 120 seconds, to chamber 1 add 100 L PBS.;i. At 135 seconds, to chamber 2 add 100 L PBS.;j. At 150 seconds, to chamber 3 add 100 L PBS.;k. At 165 seconds, to chamber 4 add 100 L PBS.;l. At 180 seconds, to chamber 5 add 100 L PBS.;m. At 195 seconds, to chamber 6 add 100 L PBS. (Total 3 minutes, 15 seconds elapsed).;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;Exercise 2. Testing the bilayer using AC impedance spectroscopy;The conductance and capacitance of the tethered membrane may be measured by inserting the assembled;electrode within the flow cell cartridge into a tethaPod reader.;The reader simplifies the interpretation of the AC impedance spectrum and provides a measure of membrane;conductance (S) and capacitance (nF);Typical conduction values for a freshly formed membrane using the proprietary SDx TM AM199 in PBS are;0.35 0.15 S and capacitance values of 182 nF at room temperature.;The conductance is proportional to the ion flux through the membrane and the capacitance is inversely;proportional to the membrane thickness. A significant additional measure using a tethaPod is the;Goodness of Fit (GOF). This indicates the quality of match between the experimental data and a model of;the tethered membrane. GOF values of less than 0.2 indicate a good match of the data to this simple model;and suggest that the membrane is uniform.;Alternating Current (a.c.) Impedance Spectroscopy;A sine wave excitation of 20mV is applied across the tethered membrane between the tethering gold;electrode and the gold counter electrode. The TethaPod device used here fits a three capacitor, one conductor;model to the experimental data and provides a readout of Gm (membrane conductance) and Cm (membrane;capacitance), thus avoiding the need to perform the more complex calculations.;Software;The Setup menu, provides the ability to choose the communuication port to your computer. This is usually;the highest numbered port displayed. Also Setup permits setting a bias voltage (d.c. potential) across the;tethered membrane circuit, (+100mV to -100mV).. Note that this d.c. potential only charges the coupling;capacitors at the tethering gold surface and at the counter electrode. No potential is applied across the;membrane elements Gm and Cm.;The Chart menu, permits a choice of variables to display as a function of time on the graphical trace or a;numerical DVM (digital voltmeter) display. Also;The Table menu, permits a choice of variables to show in the tabulation at the bottom of the display. Once;the display method is chosen click Start and a display will appear in 1-2 minutes.;Note the GoF, GoF is an acronym for goodness of fit which is a measure of the match between the;modelled spectrum for the displayed Gm and Cm and the experimental data. A high GoF means a bad fit. A;low GoF means a good fit. GoF values should be less than 0.2. Should the GoF be greater than 0.2 it means;the tethered membrane is not capable of being modelled by this equivalent circuit and the readings of Gm and;Cm values should be disregarded, e.g. should the conducting channels aggregate into rafts that are farther;apart than the distance ions can flow in the time of the excitation frequency of approximately one hundred;millisecond then a superposition will be seen of some membrane patches that are conductive and other;patches that are sealed. This will result in two impedance spectra being recorded each with different;characteristics but superimposed into a single spectrum. The reader will reject such recordings as not fitting;the single membrane Gm and Cm model. This filter is useful in determining the presence of such channel;aggregation.;Note the state, The state indicates the stage to which the model has been fitted. Wait until at least state 3 is;reached before interpreting the data. State 4 will be a more accurate refinement but states 1&2 are;meaningless intermediates.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;FIGURE 6;The DVM display on the conductance reader.;FIGURE 7;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;The Chart display on the conductance reader.;Measure the conductance and capacitance.;a. Insert the tethaPlate cartridge into the TethaPod.;b. Open TethaPod Software. Select the highest communication port under Setup.;c. A green LED lights on the front panel of the tethaPod when the instrument is working properly.;Examine the menus Table, Setup, Graphs.;(i) Set: GoF (goodness of fit) to 0.20 (Table/Set GoF Threshold);(ii) Set the potential bias to 100mV. (Setup/Set Bias);(iii) Set instrument to show Gm. (Table/Gm).;(iv) Press Start.;d. The instrument will measure the membrane conductance from each chamber sequentially. (The;instrument is actually fitting a complex impedance function from the sample at a range of;frequencies from 1kHz to 0.1Hz. To avoid the user having to deal with complex impedances the;instrument fits capacitance and conductance values to the data.);e. Once all channels read Yes (Ready column) stop recording.;f. Save data into an Excel Spreadsheet. [Edit/copy selection/paste into Excel/save spreadsheet].;g. Set new bias at -100mV.;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;h. Wait until reader stabilises. Repeat measurement. Save data into an Excel Spreadsheet (do not save;the file when asked at step e).;i. Set new bias at +100mV. Repeat measurement. Save data into an Excel Spreadsheet (do not save the;file when asked at step e).;Exercise 3. Writing Report;Tabulation;From the data you recorded, generate a Table of conduction (Gm in S) versus gramicidin concentration;([gA] in nM), for 0, 100mV and -100 mV bias. An example is given as Table 1 below.;Table1;Channel;1;2;3;4;5;6;gA (nM);0;40;60;80;100;120;SDx tethered membranes March 2014.;Gm (0mV bias);0.271;1.042;1.905;4.098;0.271;8.947;Gm (-100mV bias);0.307;1.172;3.126;4.204;0.307;5.637;Gm (+100mV);0.359;0.605;1.347;2.322;0.359;12.04;BioNanotechnology Practical: Ion Channels in Membranes.;Questions;Describe the effect of applying a positive or negative bias potential from the outer to inner;surface of the membrane. Note! positive here is taken from the reader configuration and means;negative on the tethering gold relative to counter electrode.;What is an explanation for this effect?;(i);Calculate Conduction per gA Channel;(ii);1. Estimate the maximum slope obtained for graph of conduction vs gramicidin concentration. Mark on;your graph how this was obtained. i.e. 5S for 50nM gA (+100mV bias).;2.;Calculate the number of lipid molecules we added to each cell to make the tethered membrane.;Molarity of lipid = 3mM;Volume added = 8L;Number of Molecules = Molarity (mol/L) x Volume (L) x Avogadros number (molecules/mol);=? molecules;3. Calculate the number of molecules of lipid in tethered monolayer film on gold.;Area per tethered molecule = 1nm2;Area of gold electrode = 2mm2 =? tethered molecules.;4. Fraction of added plipid incorporated into membrane. Remember it is a bilayer.;=? tethered molecules/? molecules;~? % of the added lipid.;Note: this tells us that most of the added material is flushed away and only;?% remains trapped as a membrane.;5. Calculate the number of molecules of gramicidin in 8l of 50nM.;Molecules moles = Molarity (M) x Volume (L) x Avogadros number (molecules/mole).;=? molecules. Remember it is a bilayer.;6. Assume the same fraction of gramicidin remains as part of the membrane as the fraction of lipids;(they are very similar molecular weights), then the number of gramicidin in the membrane;= ~? Molecules;7. Calculate the approximate conductance generated per gramicidin (in pS) in the membrane..;Siemens per ion channel = Total Siemens generated/number of gramicidin molecules;=? S /? molecules;~? S/molecule;SDx tethered membranes March 2014.;BioNanotechnology Practical: Ion Channels in Membranes.;(iii) Calculate membrane thickness from the relationship between area plate separation and;permittivity of a capacitor;From the Chart menu select Cm and DVM. Read value for each chamber.;For a capacitor of area, A (m2) and thickness, d (m) and capacitance, Cm (F) is given by;Cm = 0 x r x A /d;where 0 is the permittivity of free space = 8.854 x 10-12 F/m and;r is the relative permittivity of membrane lipid ~ 2.3 and;area A = 3mm2;d =? nm;(iv) What changes occur to the membrane thickness when more gA is added? Why might these;changes be occurring?;SDx tethered membranes March 2014.;View Full Attachment;Physiological Systems questions.docx Download Attachment;1.;Calculatethenumberoflipidmoleculesweaddedtoeachcellto;makethetetheredmembrane.(5marks);Molarityoflipid=3mM;Volumeadded=8m;L;2.;Calculatethenumberofmoleculesoflipidinthetethered;monolayerfilmongold.(5marks);Areapertetheredmolecule=1nm2;Areaofgoldelectrode=2mm2;3.;Whatisthefractionofaddedphospholipidthatisincorporatedinto;themembrane?(3marks);4.;5.;6.;Calculatethenumberofmoleculesofgramicidinin8m;lofa50nM;solution.(5marks);Assumethesamefractionofgramicidinremainsaspartofthe;membraneasthefractionoflipids(theyareverysimilarmolecular;weights),thenhowmanymoleculesofgramicidinarethereinthe;membrane(3marks);Calculatetheapproximateconductancegeneratedpergramicidin;(inpS)inthemembrane.(4marks);TotalSiemensgenerated=5S;7.Onecancalculatemembranethicknessfromtherelationshipbelow;betweenareaplateseparationandpermittivityofacapacitor.;Thefollowingresultswereobtainedfromthebiosensor;experiment.;gA(nM);Capacitance(nF);0;17.6;40;21.9;80;26.4;Foracapacitorofarea,A(m2)andthickness,d(m)the;capacitance,Cm(F)isgivenby;Cm=e 0xe rxA/d;wheree 0isthepermittivityoffreespace=8.854x1012F/mand;e ristherelativepermittivityofmembranelipid~2.3and;areaA=3mm2;(a)Calculatethemembranethicknessatthethreedifferent;gramicidinconcentrations.(9marks);(b)WhatchangesoccurtothemembranethicknesswhenmoregA;isadded?Whymightthesechangesbeoccurring?(4marks)


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