Cell systems- LABS PDF

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Summary

Cell SignallingFeedback loops- lac operon Taken plasmid DNA and transformed it into E  Bacteria share plasmids naturally through horizontal gene transfer- share info between species, primary ways in which antibiotic resistance can spread in a diverse bacterial community  Antibiotic resistance (fo...

Description

Cell Signalling Feedback loops- lac operon

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Taken plasmid DNA and transformed it into E.coli Bacteria share plasmids naturally through horizontal gene transfer- share info between species, primary ways in which antibiotic resistance can spread in a diverse bacterial community Antibiotic resistance (for ampicillin)-forces the bacteria to take up the plasmid because it has also been exposed to ampicillin- wouldn’t take up plasmid unless there is a reason to not spend energy unnecessarily T7 promoter- recognised by the T7 polymerase, which performs transcription T7 promoter is very specific Lac operon- responds to the environment and will control exposure of the promoter Included gene GST- glutathione S-transferase into the plasmid, an enzymes which detoxifies cellular environments (xenobiotics), but can also inhibit kinases in the MAPK signalling pathway- why it is used in this experiment Xenobiotics- chemicals which shouldn't naturally be found in an organism

Glucose available

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Cells want to use glucose as a primary energy source and will only metabolise lactose when glucose is unavailable=> lac operon comes into play The lac repressor (green) binds to the operator (red), so T7 polymerase (yellow) can't bind to the promoter (orange) No lactose-digestion enzymes are produce

Glucose is not available  

Some of the lactose is being converted to allolactose by β-galactosidase Allolactose binds to the repressor and changes its shape- it cannot bind to the operator anymore and so T7 promoter is now free for T7 polymerase to bind and start transcription

T7 polymerase can now bind and transcribe the instructions for...  β-galactosidase, which cleaves lactose into glucose and galactose  β-galactosidase permease, a membrane protein which allows lactose to be transported into the cell  Galactoside acetyltransferase, whose role in the lac operon is not clear Lactose no longer available 

Once the β-galactosidase has cleaved all of the available lactose, the repressor protein will re-bind to the DNA and turn off production of the unrequired enzymes and conserve resources

Lactose and IPTG Allolactose (6-O-β-D- galactopyranosyl-D-glucose; β-D-Galactopyranosyl (1-->6)--glucose)

IPTG (Isopropyl β-d-1- thiogalactopyranoside) IPTG mimics lactose but cannot be metabolised- will remain at same concentration throughout the experiment In experiment, bacteria is exposed to IPTG rather than allolactose because bacteria would digest allolactose and would not be in the system. IPTG may enter the cell using lactose permease (a symporter)

IPTG enters the bacterium, T7 polymerase an bind to promoter and allows for GST to start getting produced. Can also produce other things, not just GST- can be used to make insulin, proteins such as spider silk proteins

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Have a culture of bacterial cells with a plasmid put in, so they can produce a protein we want them to make- in this case it is enzyme GST- glutathione S-transferase Stimulus- IPTG, in other cases stimuli could be heat, light, toxins Culture: bacterial cells growing in broth- contains nutrients and also ampicillin If exposed to ampicillin, bacteria take up plasmid since they want the ampicillin resistance gene Ampicillin useful as could help kill off potential contaminating bacteria unless they are resistant to ampicillin

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Measure density of bacterial cells in a culture using a spectrophotometer Bacteria have been grown to mid-log phase which means there is enough bacteria so when stimulated with IPTG they will produce enough GST so it can be seen- also in most active phase of growth so will be happy to produce protein and will not be too overgrown – as you start to get too many bacteria, they will start to die off and toxic biproducts of decay could build up

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Take sample before addition of IPTG for baseline results ( 4.5 uL) Can now add IPTG to main flask containing bacteria- only a small amount is required since once IPTG is in, it will not get metabolised Need them to stay in suspension so putting them into a shaking incubator at a temperature of 37° - meaning they won't settle to the bottom of the flask=> not come into maximum contact with nutrients and will grow better

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Washing the pellet  We do not want to put broth on the gel therefore uninduced bacteria (without IPTG) need to be cleaned  Using centrifuge- place duplicate samples opposite each other and turn it on  Pushes bacteria in suspension to bottom to form a pellet at the bottom of the Eppendorf tube- broth is a layer above which can be taken off and replaced with another liquidsample buffer in this case which is navy blue meaning it will be easier to see samples in gel  Also helpful because we want the proteins to be stabilised but not sticking to each other and clumping – blue colour also separates out into different colours as the samples run through gel- can be used to track progress of the sample  Gently pipette up and down to resuspend – get rid of pellet Samples     

Three samples: 0 minute non induced, bacteria exposed to IPTG for 30min and 60min – also have a duplicate for each one Put them into a heat block - 94°- very high temp so cells will rupture, proteins will come out and denatureEverything should end up as primary protein structure- linear amino acid chain This is done so all proteins are in the same format for comparison e.g. protein could have a very large primary structure but really small secondary structure Problems- heating up could cause coagulation- within the navy blue sample buffer, there are additives (e.g. SDS) that mean that once protein is in its primary structure, it should stay in its primary structure

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SDS (sodium dodecyl sulphate) coats the linearised protein chains and makes it so its cant stick back to itself Can also add mercapto-ethanol which can break firmer bonds within a protein e.g. disulphide covalent bond

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Precast gel- comes in plastic bag with buffer in it to make sure it doesn’t dry out Useful as they are quick to repair SDS-PAGE PAGE- polyacrylamide gel electrophoresis Using electric current to separate complex mixture Top of gel- negatively charged, bottom- positively charged When proteins are all in linear structure, part of the sample buffer means that all of the protein chain has a net negative charge- will be dragged through gel towards positive electrode Shorter chains will move through gel more easily- separating things out based on size Acrylamide creates a mesh network that the proteins can move through but is highly toxic and associated with a whole variety of tumours and issues with the nervous system Load a ladder, containing every protein inside the bacteria, not just GST Bubbles rising up from electrode at the bottom means it is working GST is approx 30kDa which correlates with one of the red marker bands- looking for a line present in the 30 and 60 minute samples but not 0 minute

Skeletal Muscle Overview   

Provided with strip of glycinerated skeletal muscle tissue, from which muscle fibres (myofibrils) can be dissected for study Using a compound microscope, we can observe the striated pattern in the fibres and measure the length of the relaxed sarcomeres Then induce muscle contraction by adding ATP, observe the contraction, and measure the post-contraction width of the sarcomeres

Background  Muscle tissue made of fibres formed by fusion of cells during development  Single muscle fiber has many nuclei that lie close to its outer membrane  Each fiber contains hundreds of long, threadlike structures called myofibrils, arranged in parallel  Myofibrils make up about 75% of a muscles total volume and are the structures that carry out muscle contraction  Under a microscope, myofibers look striated (Striped) with a repeating pattern of bands and lines perpendicular to the length of the fiber  Muscle contraction occurs through the interaction of the actin and myosin filaments in the sarcomeres  When a muscle contracts, the myosin crossbridges bind to the actin filaments in a manner that causes the actin filaments to be pulled together across the H zone  Under the microscope, the A and I bands are seen to become narrower and the overall width of the sarcomere decreases Mechanism of muscle contraction

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At the molecular level, thin filaments are composed of two chains of identical actin monomers twisted around each other in a double helix, like two twisted strands of pearls Thick filaments are composed of hundreds of myosin molecules, each one a long rod with a globular head In the fiber, the myosin molecules are arranged so that the rods lie alongside one another and the globular heads protrude away from the fiber For a fiber to contract, the myosin heads must first be activated by ATP- one molecule of ATP binds to a myosin head and is hydrolysed to ADP and Pi Both ADP and Pi remain bound to the myosin head and the energy released from ATP hydrolysis is transferred to the myosin head as well- myosin head is now activated When the myosin head binds to the actin filament, its energy is released and the myosin head springs back, carrying the bound actin filament with it- this movement causes the muscle fiber to contract Each one of the actin monomers has a binding site for myosin After the myosin head has sprung, it can interact with a new molecule of ATP- when ATP binds to the myosin head, it released the actin fiber, ADP and Pi The myosin head is now re-activated, and the cycle can begin again If no ATP is available to reactivate the myosin, the atin/myosin complex remains locked together and the muscle cannot relax When an animal dies, its cellular ATP stores are depleted and all its muscles lock- called rigor mortis Muscle fibres do not normally contract without appropriate nerve signals because a control mechanism precents them from doing so The control mechanism works through two regulatory proteins, tropomyosin and troponin, which forms a complex lying along the actin filament The troponin/tropomyosin complex blocks the myosin binding site on the actin fiber, preventing myosin from binding to actin and causing contraction The signal for muscle contraction is a nerve impulse to the muscle fiber that results in intracellular release in calcium ions The calcium ions induce contraction by binding to troponin When Ca2+ binds to troponin, the shape of the troponin/tropomyosin complex is altered. This change in shape allowed activated myosin crossbridges to bind to the actin and release their energy as motion Therefore in a normal muscle fiber rich in ATP, the myosin heads are activated and ready to cause a contraction, but they cannot until a nerve impulse releases Ca2+. the contraction cycles will continue as long as the intracellular Ca2+ concentration is high and as long as ATP is available When Ca2+ levels fall, Ca2+ is released from the troponin molecules, and the troponin/tropomyosin complex again blocks the binding sites on the actin fiber

The glycinerated muscle system  The glycinerated muscle system is different from muscle in living tissue  The glycineration process removes ions and ATP from the tissue and disrupts the troponin/tropomyosin complex so that the binding sites on the actin fibers are no longer blocked  No Ca2+ is needed to induce contraction  However, no ATP is present in the glycinerated tissue, so the myosin heads are not activated  Experiment looks at adding ATP and ions to glycinerated tissue to initiate contraction

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When contraction occurs, you will be able to see the change in length of the sarcomeres and measure the overall shortening in the length of the dissected muscle tissue After the muscle is contracted it will not relax, because there is no opposing muscle to stretch it out Using chicken muscle tissue and rabbit muscle tissue- both of which have been glycinerated (soaked in 50% glycerol solution and -20°C for ~a week) Glycerol wont have been frozen at –20 but will be very cold The glycineration process removes ions from the muscle fibre and disrupts the troponintropomyosin complex

Two different tissues  Rabbits and tissues lead different lifestyles  Shape of samples are reasonably different- keep in mind, we breed chickens for food but not muscle Chicken muscle  Chicken muscle suspended in rigor solution  Muscle was glycinerated at a slight tension- Sample is in liquid (called rigour solution)  In a state of rigour/tension  Rigour means muscle is in a state of tension  There is a specific tissue type (connective tissue) present in this sample making it difficult to separate muscle fibres  Some tissue looks feathery because its made up of individual nerve fibres – trying to get one bundle on its own Rabbit muscle  Rabbit psoas muscle suspended in rigour solution  Muscle was glycinerated at a slight tension  In a state of rigour/tension  Since this sample has less connective tissue, it should be easier to separate out individual strands and also should be able to get longer muscle strands  Teasing out individual fibres with tweezers    

Now, exposing the rabbit muscle to solution B (rigour solution plus Ca2+ ions) and then solution C (rigour solution plus ATP) Use the blotting paper to draw each solution under the coverslip Solution A (regular rigour solution) To replace solution A with new solution (B), we need to put a droplet on solution B on opposite side of coverslip and then use blotting paper to suck out solution A and then solution B will be sucked under the slide

Slides under microscope  Showing a bundle of muscle fibres- we want single fibres  Showing physical trauma- possibly from forceps  Moving through focal place, shows bands on muscle fibres- need focus on internal edge of fibre When solution C comes into contact with fibres- bands on fibres are moving and shifting and also shape of fibres is changing- becoming more brown because they are becoming denser

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When glycinerated, myosin binding sites on actin will be ready for binding These muscles were glycinerated in a state of rigour => the myosin heads should be bound to the actin when a muscle is in rigour There was no change observed from solution B on the muscle fibres Calcium ions change the conformation of the troponin-tropomyosin complex to show the myosin binding sites on the actin strands This muscle is glycinerated, so the actin-myosin binding sites do not require calcium to be revealed In living muscle, contraction cannot occur without calcium ions Solution C contatins ATP, ATP will release the actin-myosin crossbridges in a living muscle In order for a living muscle to complete a muscle "power stroke", ATP needs to be hydrolyzed into ADP and pi You could see the muscle fibres becoming wider when solution C was added Muscle fibres contracted when solution C was added because the muscle are dead and have no ATPase activity so the ATP cant be hydrolysed Adding ATP would allow live muscles to contract but only if calcium ions were also present

Blood slides and ELISA Patient blood films  Recognising red and white blood cells in a "standard" blood film  Looking at slides from patients with various medical concerns How are the blood films made 1. Place a small drop of whole blood on a clean slide- hold a second slide at an angle 2. While maintaining contact with the bottom slide, pull the top slide back to contact the drop which will spread by capillary action 3. Maintain firm contact with bottom slide and push the top slide in one motion to produce the smear Neutrophil WBC  Most abundant white blood cell (40-70%)  Acts as a phagocyte  Regarded as first responders- arrive at site of infection rapidly and isolate the foreign agent ASAP  12-15 µm across  Have a distinctive multi-lobed nucleus Eosinophil WBC  2-3% of WBCs  Filled with acidophilic (attracted to acidic structured) granules  Granules are released by degranulation during an allergic response, asthma, or as an anti-parasitic- to cause harm to parasite but can also cause harm to host tissue e.g. allergic response also causes harm to patient  12-17 µm  Bi-lobed nucleus Monocyte  2-8% of WBCs

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Mono-lobed nucleus (kidney bean shaped) Able to differentiate into macrophages (phagocytes)- able to engulf and digest foreign material Generally stored in the spleen 15-18 µm

Small lymphocyte  20-45% of WBC  Only found in higher vertebrates (vertebrates that have a jaw (gnathostomata))  Differentiate into T and B cells- key to the adaptive immune response  7-10 µm  Very large nucleus

Locust leg kick Experiment apparatus  Stimulator- Produces electrical stimulation that triggers the kick  Yellow dial- control voltage  Green dial- frequency (1 Hz= lowest setting: one stimulus per second)- goes to 100 Hz  Need to make sure voltage high enough to stimulate but not too high to fry the leg so need to calibrate that at the start of the experiment      

Transducer Contains a hook which is what the locust leg will kick This hook is only sensitive in one direction Cable coming out translates analog information into digital signal Box picks up data and feeds through to laptop App on laptop- dataview

When not calibrated- if set up and locust leg kicks, data would be produced but nothing to compare it to so must calibrate it to give context To calibrate  Apply known forces in the form of weights with different grams (5.02g, 10.02g, 16.99g, 25g) made of plasticine  Traces that come from weights create a figure used to translate information coming from locust leg  Hook produces an output every time it is touched- produces peaks, size of peak proportional to how hard you press down on it  Need to set dial so that peaks coming from hooks are visible on the screen  Hang playdough weights on hook- wobbly line shown to match swinging of playdoughwant to have it as stationary as possible  The weight is hanging on so applying force and then lift it up by cupping with hand and lifting- want transition from weight being on hook and not being on hook to be smooth  Data shows cliff in force- want a sharp cliff Need to get two pieces of information from cliff traces 1. voltage before cliff 2. voltage after cliff Take average from section before cliff and after cliff

NEED TO PREPARE AN EXCEL SPREADSHEET WITH W (GRAM), F (NEWTON), V (DIFF), V (UPPER) AND V (LOWER) AS TEXT IN ADJACENT CELLS IN THE TOP ROW There is no voltage difference between input and output when there is no weight on the hook WE WANT FORCE NOT GRAMS CONVERT FROM GRAMS TO NEWTONS MULTIPLY GRAM VALUE BY 0.00981 

Making a figure with voltage on y axis and weight expressed as force in newtons along the x axis

If there are problems with this initial stage, the data points on the linear trendline will be all over the place and there will be a small R 2 value- we want the R2 value to be as close to 1 as possible Compare data coming from locust leg to this standard curve Experiment  Place plasticine on the bed and make a trough for thigh of locust leg to go into  There is a small and large nerve that triggers the muscle in locust leg- the large one is big enough that as long as the electrode as into the cuticle but not all the way through the leg, should be able to stimulate it well  Thigh rests on bed, tibia down the side, ankle kicking into hook Locusts drop their leg when predated Need to use plasticine to hold leg in place as locust kick with so much force Push electrode underneath leg- not through the plasticine Set to 1Hz with voltage turned all the way down and start turning up- leg starts kicking once per second Set voltage a bit higher to ensure the leg wont stop kicking...