GFP Practical Report

GFP Practical Report GFP is very useful as a reporter protein. After its discovery in 1962 its practical applications were put into use 30 years later by adding the coding DNA of GFP before the stop codon of other proteins. This allows for an easily detectable marker of the proteins presence without needing additional cofactors or causing any harm to the organism. The spectral characteristics of GFP can be changed by making mutations to the protein. In this investigation a Y66W mutation was made to wildtype GFP in order to produce a shorter excitation and emission wavelength. The mutation was made using QuikChange site directed mutagenesis. The protein was then cloned into BL21(DE3) pLysS for expression. The cells were then lysed and applied to a Ni-NTA column. This fractionated the lysate in order to analyse these fractions using SDS-PAGE, fluorescence and Bradford assays. It was found that the Y66W mutation was successfully added but due to another mutation in the stop codon additional amino acids were added to the C terminus of the protein. It was also found that purification was partially successful as GFP was eluted in the correct fraction. This is supported by the Bradford and fluorescence assays. The green fluorescent protein (GFP) is a 238 amino acid protein with a molecular mass of 26,870 Da. It was first isolated from the jellyfish species Aequorea Victoria by Osamu Shimomura in 1962 (1). GFP is expressed in small photoorgans that are situated in the umbrella of the jellyfish. Douglas Prasher first realised the potential of GFP as a reporter protein (2). As proteins are smaller than the resolving power of electron microscopes, Prasher thought the GFP gene could be added into the gene for haemoglobin before the stop codon. This would allow the protein of interest to maintain all of its functions but would have the GFP protein at its C terminal end. This means that detection of GFP fluorescence would also indicate the presence of haemoglobin. Furthermore, GFP does not require additional cofactors or substrates to fluoresce. This means that it works extremely well as a non-invasive method of detection of protein expression. GFP is also non-toxic so it is able to be used in vi vo without causing damage or harm to the organism. Crystallisation studies (3) have shown that GFP has a barrel structure with the chromophore buried in the centre. This chromophore is comprised of 3 amino acids (Ser 65-Tyr 66-Gly 67) that undergo a series of spontaneous cyclisation reactions to create the active chromophore. Wild type GFP has a major excitation peak at 395 nm and a minor one at 475 nm with an emission peak at 509 nm. In vivo GFP is coupled to the protein aequorin which induces a blue glow when it interacts with Ca2+ ions and breaks down luciferin. This light is able to excite GFP and cause fluorescence. In vitro this is not the case, however GFP fluorescence can be easily induced by irradiating GFP with UV light. As with all proteins, GFP can be mutated. By mutating key residues, such as residues in the chromophore, it is possible to change the characteristics of GFPs fluorescence. The first of many mutations was the S65T mutation (4). This mutation improved the characteristics of the protein including increased photostability, fluorescence and a shift of the major excitation peak. This investigation is based on the engineering of GFP to create a mutant of GFP with a shorter excitation and emission wavelength by inducing the Y66W mutation. The aims of this investigation were as follows. To carry out site directed mutagenesis of GFPuv to clone into pET28c and transform the products into XL-1 super competent cells. Extraction of the plasmid after incubation overnight to check the purity and concentration of DNA. Preparation and transformation of BL21(DE3) cells. Lyse these cells and fractionate the lysate to purify his tagged GFP using a Ni-NTA column. Finally, detection of purified GFP by SDS- PAGE, Bradford assay and fluorescence. The workflow of the investigation can be found in figure 1 in appendix 1. A more detailed protocol can be found in the BIOC2302 semester 2 practical manual on pages 6-15 with rationale for all experiments. In site directed mutagenesis I 31 ÃŽ ¼l of water was added to the PCR reaction to give a total reaction volume of 50 ÃŽ ¼l. In site directed mutagenesis II a supplied culture of cells was used in the experiment rather than cells from the transformation colonies in site directed mutagenesis I. His tagged GFP was used instead of the mutant in the protein purification experiment in order for easier administration as the process is the same. Site directed mutagenesis I Before the wet lab work began it was first necessary to design primers for QuikChange to induce the Y66W mutation into the wild type GFP. These can be seen as figure 2 in appendix 2. These were created using the QuikChange primer design tool on the Agilent website. The site directed mutagenesis was carried out using the primers supplied to induce the correct mutation. The products of this were cloned into the pET28c plasmid and the XL-1 super competent cells. The cells were plated as per the BIOC2302 practical manual and left to incubate overnight. Site directed mutagenesis II Upon checking the plates in the next session it was found that no transformed colonies had grown so a new culture was supplied. The undigested plasmid control grew approximately 50 colonies The culture of BL21(DE3)pLysS cells was set up and the OD600 were recorded. They can be seen in table 1 in appendix 3. Within 50 minutes the culture had reached an OD600 of 0.483 meaning the cells were at the correct density for lysis. The cells were prepared as per the BIOC2302 practical manual and the recombinant plasmid was extracted. The concentration measured was 121.7 ng/ÃŽ ¼l and the A260/A280 was 1.86 using nanodrop. Therefore, the ethanol precipitation was not carried out. To prepare for sequencing 4.11 ÃŽ ¼l of this solution was diluted, with 5.98 ÃŽ ¼l EB buffer, to the correct concentration. This was then sent to be sequenced, the results of which can be seen in appendix 4. The primer has been highlighted in green and is surrounded by a box with the mutated codon in red. A deletion also occurred in the stop codon of the mutant as highlighted by the second box with deleted bases highlighted in blue. Protein purification The plates were inspected in the next session. It was found that the 200 ÃŽ ¼l transformation plate grew 3 colonies and the 50 ÃŽ ¼l transformation plate grew none. Transformation efficiency can be calculated for the 200 ÃŽ ¼l plate as 37 transformants/ÃŽ ¼g of DNA. The cells were weighed and found to be 0.539 g so 2 ml BugbusterTM used. After lysis and fractionation the SDS-PAGE samples of each fraction were prepared and loaded onto the gel. The Bradford assay was carried out while the gel ran.   The BSA standards were calculated and the contents of each standard well can be seen in table 2 in appendix 3. The fractions were then diluted into their wells and the contents can be seen in table 3 in appendix 3. The plate was filled according to the map in figure 2 in appendix 5. The plate was ran and the absorbances for the BSA standards were taken from the plate readout and inputted into table 4 in appendix 5. From here a calibration graph was set up using GraphPad Prism and can be seen as graph 1 in appendix 6. This graph shows that the data points for the standards do not fall near the line of best fit. The absorbance results from the plate readout for all of the fractions were imputed into table 5 in appendix 7. The equation of the line from graph 1 was then used to calculate the concentration of protein in each of the fractions. All of these values were also inputted into table 5. With the Bradford assay complete the SDS-PAGE gel was disassembled, stained and a picture was taken. A map of the gel can be seen as figure 3 in appendix 7 and the picture of the gel can be seen as figure 4 in appendix 8. By looking at the picture it can be seen that in lanes 2, 3, 4 and 9 there are dark bands spanning the entire lane. In 5, 6 and 8 there is faint banding across the well. In well 7 there is a distinct small band in between the 25 kDa and 37 kDa molecular markers. Lane 8 shows no bands at all. Finally the fluorescence assay was carried out as per the map of the microtiter plate in figure 5 in appendix 7. The results from the plate readout were inputted into table 5. From here a graph comparing the log of protein concentration compared to fluorescence of each fraction was plotted and can be found as graph 2 in appendix 6. This shows elution 1 with the highest fluorescence and the unbound x10 had the lowest. However, when comparing protein concentration the unbound fraction had the highest and wash 2 had none. Percentage fluorescence was also calculated and inputted into table 6 in appendix 9. The first aim of this experiment was to transform the site directed mutagenesis products into XL-1 super competent cells. The correct primers were used in order to induce the Y66W mutation into the parental DNA. However, no colonies that were meant to take up the mutated plasmid grew but the undigested control grew around 50 colonies. This means the cells did not take up the plasmid because otherwise they would have grown on the plate. This could be due to a mistake made in making the PCR reaction mixture or the DNA may have become damaged at some point in the experiment. Additionally, the suppliers of the XL-1 super competent cells advice to avoid large changes in temperature. This was unavoidable in this experiment and may have contributed to the cells not taking up the plasmid. In the future more care should be taken while plating and preparing the cells. Also preparation of any reaction mixtures should be checked very closely in order to ensure the correct reactants are added in the correct amounts. In site directed mutagenesis II the cell culture was lysed when the OD600 was 0.483. That is because E.coli cells are most likely to be made competent when they enter early log phase. This corresponds with an OD600 of 0.4-0.5. The DNA concentration extracted in this experiment was found to be 121.7 ng/ÃŽ ¼l and an A260/A280 of 1.86. This means that the DNA is good quality as the desirable range for A260/A280 is 1.7-2.0 and the concentration was much higher that what was required. However, in future experiments to test for reliability multiple results should be taken. Furthermore, the data could have been confirmed by using the spectrophotometric method alongside using nanodrop. The sequencing results in appendix 4 confirmed the successful incorporation of the Y66W mutation into GFP, creating the CFP mutant. However, the second mutation at the stop codon deleted 2 bases including the first base of the stop codon. This means that when the protein is expressed the ribosome will not stop and instead will continue to add amino acids onto the C terminus of the mutant until it reaches a new stop codon. There 144 bases between the original stop codon and the next in frame stop codon meaning 48 additional amino acids will be added to the C terminus. This codon can be seen highlighted in purple below the original stop codon. These additional amino acids could affect the folding or could increase the likelihood of aggregation of the mutant protein. In the protein purification experiment the 200 ÃŽ ¼l transformation plate grew 3 colonies and the 50 ÃŽ ¼l transformation plate grew none. The transformation efficiency on the 200 ÃŽ ¼l plate was 37 transformants/ÃŽ ¼g of DNA. The reason why this is so low could be due to a number of factors such as the plating technique or the cells may not have been left to chill on ice for the optimum amount of time. However, the negative control did not grow any colonies, confirming that all of the bacteria on the transformation plate were transformed. Again, more steps should be taken if this was to be carried out again to ensure that proper plating and prepping protocol is followed. The Bradford assay shows that in wash 2 there was no protein in the well. This means that any protein found in elutions 1 and 2 should all be His tagged GFP that bound the Ni-NTA column. This can be confirmed by the fluorescence results as the elution 1 fraction contained the majority of the total fluorescence with 44.13% of the total. However, all other fractions also produce some fluorescence. This could be due to GFP contamination in the other fractions. This could have occurred due to the resin being saturated, preventing further binding to the column. It could also be due to aggregation of the protein obscuring the His tag and preventing binding. Furthermore, the plots on the calibration graph do not fall on the line of best fit. This means that the equation of the line is not accurate and protein concentrations calculated using it are also inaccurate. Therefore, there could be more protein in each fraction than was calculated. This could account for the fluorescence in the wash and unbound fractions. The Bradford assay is quite limiting. This is due to the fact the assay only measures protein concentration rather than GFP concentration. This means that it is unsure whether the protein concentration measured in elution 1 and 2 is all GFP or it is contaminant protein. The same can be said for the other fractions, its unsure whether the protein concentration measured has been contaminated by GFP. In the future this assay should be carried out again to try and reduce contamination. The calibration graph should also be repeated until all of the data points fall on the line of best fit. Otherwise none of the calculated protein concentrations are accurate. Finally, the SDS-PAGE results shows banding in wash 1, 2 and elution 1 and 2. This suggests that there is contaminating protein in all of these fractions. Elution 1 shows a clear band at approximately the 26-27 kDa mark as it is present just above the 25 kDa marker and is well below the 37 kDa marker. This suggest the band in elution 1 is GFP as it is the appropriate size and is in the expected fraction. Another source of error could be due to the amount of pressure applied to the pipette. This will vary from person to person and will affect the volume of the solution being pipetted. As such small volumes were being used and there was a lot of solutions to be pipetted it is very possible a mistake was made. This mistake would have a big effect on the concentration and therefore could have a big effect on the absorbance values. These errors can be avoided in future by using the appropriate pipette for the volumes being used. Further reduction in errors can come from correct technique and by doing replicates and averaging values. There could be some error in the microtiter itself. There may have been markings or scratches on the plate that werent seen at the time. This could affect how the light passed through the reader and therefore affect the absorbance values. In conclusion, the aims of this investigation were to induce the Y66W mutation into wild type GFP using QuikChange site directed mutagenesis. Furthermore, the protein was to be expressed in competent BL21(DE3) pLysS cells. Finally wildtype GFP was to be purified using a Ni-NTA column and the fractions analysed with SDS-PAGE, fluorescence and Bradford assays. The investigation successfully introduced the Y66W mutation into wildtype GFP. However, the stop codon was also mutated adding an extra 48 amino acids on the C terminal of the protein.   A band indicating the presence of GFP was found at the 26.9 kDa mark in elution 1, indicating it was bound to the column and was eluted. However, all factions were contaminated with other protein. References      Ã‚   1. Shimomura, O., Johnson, F., and Saiga, Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous. s.l. : J. Cell. Comp. Physiol. 59:223-39, 1962. 2. Prasher, D., Eckenrode, V., Ward, W., Prendergast, F., and Cormier, M. Primary structure of the Aequorea Victoria green-fluorescent protein. s.l. : Gene 111 (2)229-33, 1992. 3. Ormo, M., Cubitt, A., Kallio, K., Gross, L., Tsien, R., and Remington. S. Crystal structure of the Aequorea Victoria green fluorescent protein. s.l. : Science 273:1392-5, 1996. 4. Heim R, Cubitt AB, Tsien RY. Improved green fluroescence. s.l. : Nature. 373 (6516): 663-4., 1995.

Vampire Academy Chapter 12

TWELVE SLEEP CAME RELUCTANTLY THAT NIGHT and I tossed and turned for a long time before finally going under. An hour or so later, I sat up in bed, trying to relax and sort out the emotions coming to me. Lissa. Scared and upset. Unstable. The night's events suddenly came rushing back to me as I went through what could be bothering her. The queen humiliating her. Mia. Maybe even Christian – he could have found her for all I knew. Yet†¦none of those was the problem right now. Buried within her, there was something else. Something terribly wrong. I climbed out of bed, dressed hastily, and considered my options. I had a third-floor room now – way too high to climb down from, particularly since I had no Ms. Karp to patch me up this time. I would never be able to sneak out of the main hall. That only left going through the â€Å"appropriate† channels. â€Å"Where do you think you're going?† One of the matrons who supervised my hall looked up from her chair. She sat stationed at the end of the hall, near the stairs going down. During the day, that stairwell had loose supervision. At night, we might as well have been in jail. I crossed my arms. â€Å"I need to see Dim – Guardian Belikov.† â€Å"It's late.† â€Å"It's an emergency.† She looked me up and down. â€Å"You seem okay to me.† â€Å"You're going to be in so much trouble tomorrow when everyone finds out you stopped me from reporting what I know.† â€Å"Tell me.† â€Å"It's private guardian stuff.† I gave her as hard a stare as I could manage. It must have worked, because she finally stood up and pulled out a cell phone. She called someone – Dimitri, I hoped – but murmured too low for me to hear. We waited several minutes, and then the door leading to the stairs opened. Dimitri appeared, fully dressed and alert, though I felt pretty sure we'd pulled him out of bed. He took one look at me. â€Å"Lissa.† I nodded. Without another word, he turned around and started back down the stairs. I followed. We walked across the quad in silence, toward the imposing Moroi dorm. It was â€Å"night† for the vampires, which meant it was daytime for the rest of the world. Mid-afternoon sun shone with a cold, golden light on us. The human genes in me welcomed it and always sort of regretted how Moroi light sensitivity forced us to live in darkness most of the time. Lissa's hall matron gaped when we appeared, but Dimitri was too intimidating to oppose. â€Å"She's in the bathroom,† I told them. When the matron started to follow me inside, I wouldn't let her. â€Å"She's too upset. Let me talk to her alone first.† Dimitri considered. â€Å"Yes. Give them a minute.† I pushed the door open. â€Å"Liss?† A soft sound, like a sob, came from within. I walked down five stalls and found the only one closed. I knocked softly. â€Å"Let me in,† I said, hoping I sounded calm and strong. I heard a sniffle, and a few moments later, the door unlatched. I wasn't prepared for what I saw. Lissa stood before me†¦ †¦covered in blood. Horrified, I squelched a scream and almost called for help. Looking more closely, I saw that a lot of the blood wasn't actually coming from her. It was smeared on her, like it had been on her hands and she'd rubbed her face. She sank to the floor, and I followed, kneeling before her. â€Å"Are you okay?† I whispered. â€Å"What happened?† She only shook her head, but I saw her face crumple as more tears spilled from her eyes. I took her hands. â€Å"Come on. Let's get you cleaned – â€Å" I stopped. She was bleeding after all. Perfect lines crossed her wrists, not near any crucial veins, but enough to leave wet, red tracks across her skin. She hadn't hit her veins when she did this; death hadn't been her goal. She met my eyes. â€Å"I'm sorry†¦I didn't mean†¦Please don't let them know†¦Ã¢â‚¬  she sobbed. â€Å"When I saw it, I freaked out.† She nodded toward her wrists. â€Å"This just happened before I could stop. I was upset†¦Ã¢â‚¬  â€Å"It's okay,† I said automatically, wondering what â€Å"it† was. â€Å"Come on.† I heard a knock on the door. â€Å"Rose?† â€Å"Just a sec,† I called back. I took her to the sink and rinsed the blood off her wrists. Grabbing the first-aid kit, I hastily put some Band-Aids on the cuts. The bleeding had already slowed. â€Å"We're coming in,† the matron called. I jerked off my hoodie sweatshirt and quickly handed it to Lissa. She had just pulled it on when Dimitri and the matron entered. He raced to our sides in an instant, and I realized that in hiding Lissa's wrists, I'd forgotten the blood on her face. â€Å"It's not mine,† she said quickly, seeing his expression. â€Å"It†¦it's the rabbit†¦Ã¢â‚¬  Dimitri assessed her, and I hoped he wouldn't look at her wrists. When he seemed satisfied she had no gaping wounds, he asked, â€Å"What rabbit?† I was wondering the same thing. With shaking hands, she pointed at the trash can. â€Å"I cleaned it up. So Natalie wouldn't see.† Dimitri and I both walked over and peered into the can. I pulled myself away immediately, swallowing back my stomach's need to throw up. I don't know how Lissa knew it was a rabbit. All I could see was blood. Blood and blood-soaked paper towels. Globs of gore I couldn't identify. The smell was horrible. Dimitri shifted closer to Lissa, bending down until they were at eye level. â€Å"Tell me what happened.† He handed her several tissues. â€Å"I came back about an hour ago. And it was there. Right there in the middle of the floor. Torn apart. It was like it had†¦exploded.† She sniffed. â€Å"I didn't want Natalie to find it, didn't want to scare her†¦so I-I cleaned it up. Then I just couldn't†¦I couldn't go back†¦Ã¢â‚¬  She began to cry, and her shoulders shook. I could figure out the rest, the part she didn't tell Dimitri. She'd found the rabbit, cleaned up, and freaked out. Then she'd cut herself, but it was the weird way she coped with things that upset her. â€Å"No one should be able to get into those rooms!† exclaimed the matron. â€Å"How is this happening?† â€Å"Do you know who did it?† Dimitri's voice was gentle. Lissa reached into her pajama pocket and pulled out a crumpled piece of paper. It had so much blood soaked into it, I could barely read it as he held it and smoothed it out. I know what you are. You won't survive being here. I'll make sure of it. Leave now. It's the only way you might live through this. The matron's shock transformed into something more determined, and she headed for the door. â€Å"I'm getting Ellen.† It took me a second to remember that was Kirova's first name. â€Å"Tell her we'll be at the clinic,† said Dimitri. When she left, he turned to Lissa. â€Å"You should lie down.† When she didn't move, I linked my arm through hers. â€Å"Come on, Liss. Let's get you out of here.† Slowly, she put one foot in front of the other and let us lead her to the Academy's medical clinic. It was normally staffed by a couple of doctors, but at this time of night, only a nurse stayed on duty. She offered to wake one of the doctors, but Dimitri declined. â€Å"She just needs to rest.† Lissa had no sooner stretched out on a narrow bed than Kirova and a few others showed up and started questioning her. I thrust myself in front of them, blocking her. â€Å"Leave her alone! Can't you see she doesn't want to talk about it? Let her get some sleep first!† â€Å"Miss Hathaway,† declared Kirova, â€Å"you're out of line as usual. I don't even know what you're doing here.† Dimitri asked if he could speak with her privately and led her into the hall. I heard angry whispers from her, calm and firm ones from him. When they returned, she said stiffly, â€Å"You may stay with her for a little while. We'll have janitors do further cleaning and investigation in the bathroom and your room, Miss Dragomir, and then discuss the situation in detail in the morning.† â€Å"Don't wake Natalie,† whispered Lissa. â€Å"I don't want to scare her. I cleaned up everything in the room anyway.† Kirova looked doubtful. The group retreated but not before the nurse asked if Lissa wanted anything to eat or drink. She declined. Once we were alone, I lay down beside her and put my arm around her. â€Å"I won't let them find out,† I told her, sensing her worry about her wrists. â€Å"But I wish you'd told me before I left the reception. You'd said you'd always come to me first.† â€Å"I wasn't going to do it then,† she said, her eyes staring blankly off. â€Å"I swear, I wasn't going to. I mean, I was upset†¦but I thought†¦I thought I could handle it. I was trying so hard†¦really, Rose. I was. But then I got back to my room, and I saw it, and I†¦just lost it. It was like the last straw, you know? And I knew I had to clean it up. Had to clean it up before they saw, before they found out, but there was so much blood†¦and afterward, after it was done, it was too much, and I felt like I was going to†¦I don't know†¦explode, and it was just too much, I had to let it out, you know? I had to – â€Å" I interrupted her hysteria. â€Å"It's okay, I understand.† That was a lie. I didn't get her cutting at all. She'd done it sporadically, ever since the accident, and it scared me each time. She'd try to explain it to me, how she didn't want to die – she just needed to get it out somehow. She felt so much emotionally, she would say that a physical outlet – physical pain – was the only way to make the internal pain go away. It was the only way she could control it. â€Å"Why is this happening?† she cried into her pillow. â€Å"Why am I a freak?† â€Å"You aren't a freak.† â€Å"No one else has this happen to them. No one else does magic like I can.† â€Å"Did you try to do magic?† No answer. â€Å"Liss? Did you try to heal the rabbit?† â€Å"I reached out, just to see if I could maybe fix it, but there was just too much blood†¦I couldn't.† The more she uses it, the worse it'll get. Stop her, Rose. Lissa was right. Moroi magic could conjure fire and water, move rocks and other pieces of earth. But no one could heal or bring animals back from the dead. No one except Ms. Karp. Stop her before they notice, before they notice and take her away too. Get her out of here. I hated carrying this secret, mostly because I didn't know what to do about it. I didn't like feeling powerless. I needed to protect her from this – and from herself. And yet, at the same time, I needed to protect her from them, too. â€Å"We should go,† I said abruptly. â€Å"We're going to leave.† â€Å"Rose – â€Å" â€Å"It's happening again. And it's worse. Worse than last time.† â€Å"You're afraid of the note.† â€Å"I'm not afraid of any note. But this place isn't safe.† I suddenly longed for Portland again. It might be dirtier and more crowded than the rugged Montana landscape, but at least you knew what to expect – not like here. Here at the Academy, past and present warred with each other. It might have its beautiful old walls and gardens, but inside, modern things were creeping in. People didn't know how to handle that. It was just like the Moroi themselves. Their archaic royal families still held the power on the surface, but people were growing discontent. Dhampirs who wanted more to their lives. Moroi like Christian who wanted to fight the Strigoi. The royals still clung to their traditions, still touted their power over everyone else, just as the Academy's elaborate iron gates put on a show of tradition and invincibility. And, oh, the lies and secrets. They ran through the halls and hid in the corners. Someone here hated Lissa, someone who was probably smiling right to her face and pretending to be her friend. I couldn't let them destroy her. â€Å"You need to get some sleep,† I told her. â€Å"I can't sleep.† â€Å"Yes, you can. I'm right here. You won't be alone.† Anxiety and fear and other troubled emotions coursed through her. But in the end, her body's needs won out. After a while, I saw her eyes close. Her breathing became even, and the bond grew quiet. I watched her sleep, too keyed up with adrenaline to allow myself any rest. I think maybe an hour had passed when the nurse returned and told me I had to leave. â€Å"I can't go,† I said. â€Å"I promised her she wouldn't be alone.† The nurse was tall, even for a Moroi, with kind brown eyes. â€Å"She won't be. I'll stay with her.† I regarded her skeptically. â€Å"I promise.† Back in my room, I had my own crash. The fear and excitement had worn me out too, and for an instant, I wished I could have a normal life and a normal best friend. Immediately, I cast that thought out. No one was normal, not really. And I'd never have a better friend than Lissa†¦but man, it was so hard sometimes. I slept heavily until morning. I went to my first class tentatively, nervous that word about last night had gotten around. As it turned out, people were talking about last night, but their attention was still focused on the queen and the reception. They knew nothing about the rabbit. As hard as it was to believe, I'd nearly forgotten about that other stuff. Still, it suddenly seemed like a small thing compared to someone causing a bloody explosion in Lissa's room. Yet, as the day went on, I noticed something weird. People stopped looking at Lissa so much. The started looking at me. Whatever. Ignoring them, I hunted around and found Lissa finishing up with a feeder. That funny feeling I always got came over me as I watched her mouth work against the feeder's neck, drinking his blood. A trickle of it ran down his throat, standing out against his pale skin. Feeders, though human, were nearly as pale as Moroi from all the blood loss. He didn't seem to notice; he was long gone on the high of the bite. Drowning in jealousy, I decided I needed therapy. â€Å"You okay?† I asked her later, on our way to class. She wore long sleeves, purposefully obscuring her wrists. â€Å"Yeah†¦I still can't stop thinking about that rabbit†¦It was so horrible. I keep seeing it in my head. And then what I did.† She squeezed her eyes shut, just for a moment, and then opened them again. â€Å"People are talking about us.† â€Å"I know. Ignore them.† â€Å"I hate it,† she said angrily. A surge of darkness shot up into her and through the bond. It made me cringe. My best friend was lighthearted and kind. She didn't have feelings like that. â€Å"I hate all the gossip. It's so stupid. How can they all be so shallow?† â€Å"Ignore them,† I repeated soothingly. â€Å"You were smart not to hang out with them anymore.† Ignoring them grew harder and harder, though. The whispers and looks increased. In animal behavior, it became so bad, I couldn't even concentrate on my now-favorite subject. Ms. Meissner had started talking about evolution and survival of the fittest and how animals sought mates with good genes. It fascinated me, but even she had a hard time staying on task, since she had to keep yelling at people to quiet down and pay attention. â€Å"Something's going on,† I told Lissa between classes. â€Å"I don't know what, but they're all over something new.† â€Å"Something else? Other than the queen hating me? What more could there be?† â€Å"Wish I knew.† Things finally came to a head in our last class of the day, Slavic art. It started when a guy I barely knew made a very explicit and nearly obscene suggestion to me while we all worked on individual projects. I replied in kind, letting him know exactly what he could do with his request. He only laughed. â€Å"Come on, Rose. I bleed for you.† Loud giggles ensued, and Mia cut us a taunting look. â€Å"Wait, it's Rose who does the bleeding, right?† More laughter. Understanding slapped me in the face. I jerked Lissa away. â€Å"They know.† â€Å"Know what?† â€Å"About us. About how you†¦you know, how I fed you while we were gone.† She gaped. â€Å"How?† â€Å"How do you think? Your ? ®friend' Christian.† â€Å"No,† she said adamantly. â€Å"He wouldn't have.† â€Å"Who else knew?† Faith in Christian flashed in her eyes and in our bond. But she didn't know what I knew. She didn't know how I'd bitched him out last night, how I'd made him think she hated him. The guy was unstable. Spreading our biggest secret – well, one of them – would be an adequate revenge. Maybe he'd killed the rabbit, too. After all, it had died only a couple hours after I'd told him off. Not waiting around to hear her protests, I stalked off to the other side of the room where Christian was working by himself, as usual. Lissa followed in my wake. Not caring if people saw us, I leaned across the table toward him, putting my face inches from his. â€Å"I'm going to kill you.† His eyes darted to Lissa, the faintest glimmer of longing in them, and then a scowl spread over his face. â€Å"Why? Is it like guardian extra credit?† â€Å"Stop with the attitude,† I warned, pitching my voice low. â€Å"You told. You told how Lissa had to feed off me.† â€Å"Tell her,† said Lissa desperately. â€Å"Tell her she's wrong.† Christian dragged his eyes from me to her, and as they regarded each other, I felt such a powerful wave of attraction, it was a wonder it didn't knock me over. Her heart was in her eyes. It was obvious to me he felt the same way about her, but she couldn't see it, particularly since he was still glaring at her. â€Å"You can stop it, you know,† he said. â€Å"You don't have to pretend anymore.† Lissa's giddy attraction vanished, replaced by hurt and shock over his tone. â€Å"I†¦what? Pretend what?†¦Ã¢â‚¬  â€Å"You know what. Just stop. Stop with the act.† Lissa stared at him, her eyes wide and wounded. She had no clue I'd gone off on him last night. She had no clue that he believed she hated him. â€Å"Get over feeling sorry for yourself, and tell us what's going on,† I snapped at him. â€Å"Did you or didn't you tell them?† He fixed me with a defiant look. â€Å"No. I didn't.† â€Å"I don't believe you.† â€Å"I do,† said Lissa. â€Å"I know it's impossible to believe a freak like me could keep his mouth shut – especially since neither of you can – but I have better things to do than spread stupid rumors. You want someone to blame? Blame your golden boy over there.† I followed his gaze to where Jesse was laughing about something with that idiot Ralf. â€Å"Jesse doesn't know,† said Lissa defiantly. Christian's eyes were glued to me. â€Å"He does, though. Doesn't he, Rose? He knows.† My stomach sank out of me. Yes. Jesse did know. He'd figured it out that night in the lounge. â€Å"I didn't think†¦I didn't think he'd tell. He was too afraid of Dimitri.† â€Å"You told him?† exclaimed Lissa. â€Å"No, he guessed.† I was starting to feel sick. â€Å"He apparently did more than guess,† muttered Christian. I turned on him. â€Å"What's that supposed to mean?† â€Å"Oh. You don't know.† â€Å"I swear to God, Christian, I'm going to break your neck after class.† â€Å"Man, you really are unstable.† He said it almost happily, but his next words were more serious. He still wore that sneer, still glowed with anger, but when he spoke, I could hear the faintest uneasiness in his voice. â€Å"He sort of elaborated on what was in your note. Got into a little more detail.† â€Å"Oh, I get it. He said we had sex.† I didn't need to mince words. Christian nodded. So. Jesse was trying to boost his own reputation. Okay. That I could deal with. Not like my reputation was that stellar to begin with. Everyone already believed I had sex all the time. â€Å"And uh, Ralf too. That you and he – â€Å" Ralf? No amount of alcohol or any illegal substance would make me touch him. â€Å"I – what? That I had sex with Ralf too?† Christian nodded. â€Å"That asshole! I'm going to – â€Å" â€Å"There's more.† â€Å"How? Did I sleep with the basketball team?† â€Å"He said – they both said – you let them†¦well, you let them drink your blood.† That stopped even me. Drinking blood during sex. The dirtiest of the dirty. Sleazy. Beyond being easy or a slut. A gazillion times worse than Lissa drinking from me for survival. Blood-whore territory. â€Å"That's crazy!† Lissa cried. â€Å"Rose would never – Rose?† But I wasn't listening anymore. I was in my own world, a world that took me across the classroom to where Jesse and Ralf sat. They both looked up, faces half smug and half†¦nervous, if I had to guess. Not unexpected, since they were both lying through their teeth. The entire class came to a standstill. Apparently they'd been expecting some type of showdown. My unstable reputation in action. â€Å"What the hell do you think you're doing?† I asked in a low, dangerous voice. Jesse's nervous look turned to one of terror. He might have been taller than me, but we both knew who would win if I turned violent. Ralf, however, gave me a cocky smile. â€Å"We didn't do anything you didn't want us to do.† His smiled turned cruel. â€Å"And don't even think about laying a hand on us. You start a fight, and Kirova'll kick you out to go live with the other blood whores.† The rest of the students were holding their breaths, waiting to see what we'd do. I don't know how Mr. Nagy could have been oblivious to the drama occurring in his class. I wanted to punch both of them, hit them so hard that it'd make Dimitri's brawl with Jesse look like a pat on the back. I wanted to wipe that smirk off Ralf's face. But asshole or not, he was right. If I touched them, Kirova would expel me in the blink of an eye. And if I got kicked out, Lissa would be alone. Taking a deep breath, I made one of the hardest decisions of my life. I walked away. The rest of the day was miserable. In backing down from the fight, I opened myself up to mockery from everyone else. The rumors and whispers grew louder. People stared at me openly. People laughed. Lissa kept trying to talk to me, to console me, but I ignored even her. I went through the rest of my classes like a zombie, and then I headed off to practice with Dimitri as fast I could. He gave me a puzzled look but didn't ask any questions. Alone in my room later on, I cried for the first time in years. Once I got that out of my system, I was about to put on my pajamas when I heard a knock at my door. Dimitri. He studied my face and then glanced away, obviously aware I'd been crying. I could tell, too, that the rumors had finally reached him. He knew. â€Å"Are you okay?† â€Å"It doesn't matter if I am, remember?† I looked up at him. â€Å"Is Lissa okay? This'll be hard on her.† A funny look crossed his face. I think it astonished him that I'd still be worried about her at a time like this. He beckoned me to follow and led me out to a back stairwell, one that usually stayed locked to students. But it was open tonight, and he gestured me outside. â€Å"Five minutes,† he warned. More curious than ever, I stepped outside. Lissa stood there. I should have sensed she was close, but my own out-of-control feelings had obscured hers. Without a word, she put her arms around me and held me for several moments. I had to hold back more tears. When we broke apart, she looked at me with calm, level eyes. â€Å"I'm sorry,† she said. â€Å"Not your fault. It'll pass.† She clearly doubted that. So did I. â€Å"It is my fault,† she said. â€Å"She did it to get back at me.† â€Å"She?† â€Å"Mia. Jesse and Ralf aren't smart enough to think of something like that on their own. You said it yourself: Jesse was too scared of Dimitri to talk much about what happened. And why wait until now? It happened a while ago. If he'd wanted to spread stuff around, he would have done it back then. Mia's doing this as retaliation for you talking about her parents. I don't know how she managed it, but she's the one who got them to say those things.† In my gut, I realized Lissa was right. Jesse and Ralf were the tools; Mia had been the mastermind. â€Å"Nothing to be done now,† I sighed. â€Å"Rose – â€Å" â€Å"Forget it, Liss. It's done, okay?† She studied me quietly for a few seconds. â€Å"I haven't seen you cry in a long time.† â€Å"I wasn't crying.† A feeling of heartache and sympathy beat through to me from the bond. â€Å"She can't do this to you,† she argued. I laughed bitterly, half surprised at my own hopelessness. â€Å"She already did. She said she'd get back at me, that I wouldn't be able to protect you. She did it. When I go back to classes†¦Ã¢â‚¬  A sickening feeling settled in my stomach. I thought about the friends and respect I'd managed to eke out, despite our low profile. That would be gone. You couldn't come back from something like this. Not among the Moroi. Once a blood whore, always a blood whore. What made it worse was that some dark, secret part of me did like being bitten. â€Å"You shouldn't have to keep protecting me,† she said. I laughed. â€Å"That's my job. I'm going to be your guardian.† â€Å"I know, but I meant like this. You shouldn't suffer because of me. You shouldn't always have to look after me. And yet you always do. You got me out of here. You took care of everything when we were on our own. Even since coming back†¦you've always been the one who does all the work. Every time I break down – like last night – you're always there. Me, I'm weak. I'm not like you.† I shook my head. â€Å"That doesn't matter. It's what I do. I don't mind.† â€Å"Yeah, but look what happened. I'm the one she really has a grudge against – even though I still don't know why. Whatever. It's going to stop. I'm going to protect you from now on.† There was a determination in her expression, a wonderful confidence radiating off of her that reminded me of the Lissa I'd known before the accident. At the same time, I could feel something else in her – something darker, a sense of deeply buried anger. I'd seen this side of her before too, and I didn't like it. I didn't want her tapping into it. I just wanted her to be safe. â€Å"Lissa, you can't protect me.† â€Å"I can,† she said fiercely. â€Å"There's one thing Mia wants more than to destroy you and me. She wants to be accepted. She wants to hang out with the royals and feel like she's one of them. I can take that away from her.† She smiled. â€Å"I can turn them against her.† â€Å"How?† â€Å"By telling them.† Her eyes flashed. My mind was moving too slowly tonight. It took me a while to catch on. â€Å"Liss – no. You can't use compulsion. Not around here.† â€Å"I might as well get some use out of these stupid powers.† The more she uses it, the worse it'll get. Stop her, Rose. Stop her before they notice, before they notice and take her away too. Get her out of here. â€Å"Liss, if you get caught – â€Å" Dimitri stuck his head out. â€Å"You've got to get back inside, Rose, before someone finds you.† I shot a panicked look at Lissa, but she was already retreating. â€Å"I'll take care of everything this time, Rose. Everything.†

Analysis of the Standard Enthalpy of Combustion for Alcohols Essay

Aim: To investigate the standard enthalpy change of combustion for 5 consecutive alcohols in the alcohol homologous series, methanol, ethanol, propan-1-ol, butan-1-ol and pentan-1-ol, by using a calorimetric method to calculate the heat gained by the 100cm3 water in the experiment, and thus the heat lost by the alcohol lamp at standard temperature and pressure (298 K and 101.3 kPa). Background Knowledge: Alcohols are organic compounds containing Oxygen, Hydrogen and Carbon. The alcohols are a homologous series containing the functional –OH group. As we move down the homologous series of alcohols, the number of Carbon atoms increase. Each alcohol molecule differs by –CH2; a single Carbon atom and two Hydrogen atoms. Combustion is the oxidation of carbon compounds by oxygen in air to form CO2 and H2O. Combustion produces heat as well as carbon dioxide and water. The enthalpy change of combustion is the enthalpy change that occurs when 1 mole of a fuel is burned completely in oxygen. When alcohol undergoes complete combustion it produces carbon dioxide and water as products, and energy is released. The standard enthalpy of combustion of an alcohol (à ¢H °comb) is the enthalpy change when one mole of an alcohol completely reacts with oxygen under standard thermodynamic conditions (temperature of 25 °C and pressure of 101.3 kPa). The standard enthalpy change of combustion of alcohols varies depending on their molecular size. The greater the number of carbons, the higher the standard enthalpy of combustion, as there is the presence of more bonds. The larger the alcohol molecule, the more bonds will be broken and formed, and therefore more heat will be produced. Using experiments, the standard enthalpy of combustion of an alcohol can be found, buy first finding the heat released during the reaction using the equation Heat=mass of water Ãâ€"specific heat capacity of water Ãâ€"rise in temperature of water Note: The specific heat capacity of water is 4.18 Jg-1 °C-1. and then finding the number of moles of alcohol burnt, and dividing the heat by this number. Equipment: 1. 250 cm3 Conical flask 2. 100 cm3  ± 0.08 cm3 pipette 3. Loggerpro thermometer 4. 5 x different consecutive alcohol spirit burners (eg. methanol, ethanol, propanol, butanol and pentanol) 5. Stand 6. 2 x clamps 7. Scales 8. 1500 cm3 distilled water 9. Heat proof mat 10. Matches Method: 1. Connect the temperature sensor to the datalogger. Connect the datalogger to the computer. Ensure the datalogging software is loaded and set to record the temperature of the sensor. Set the sampling rate to 1 sample per second for 210 seconds. 2. Using the pipette, pipette 100 cm3 distilled water into the conical flask. 3. Set up the stand, and clamp the conical flask 25 cm from the table. Also clamp the temperature probe 30 cm from the table, so that it is submerged in the distilled water but not in contact with the conical flask walls. 4. Weigh the alcohol lamp (including its cap) using the scales and record the mass. 5. Place alcohol lamp directly under the conical flask on a heat proof mat. 6. Click ‘collect’ on datalogger to start recording the temperature. After 30 seconds, light the alcohol lamp. 7. When the datalogger reaches 210 seconds immediately extinguish the flame by replacing the cap. ‘Store the latest run’ in loggerpro. 8. Re-weigh the alcohol lamp (including cap) as soon as possible after extinguishing the lamp. 9. Repeat steps 2 – 8 with the same alcohol to obtain trail 2, and trial 3 results. 10. Repeat steps 2 – 9 for 4 other consecutive alcohols. 11. Calculate the average change in mass of each alcohol and calculate the change in temperature of water for each trial. 12. Calculate energy absorbed by this using q=mcà ¢T then calculate à ¢H °comb=qn 13. Plot the graph of à ¢H °combversus number of carbons in alcohol. Apparatus: temperature probe datalogger device 5 cm 25 cm alcohol lamp loggerpro collector on computer heatproof mat 100 cm3 distilled water conical flask clamp clamp Variables: 1. Independent The alcohol used to heat water will be changed, however all alcohols will be primary. The range of alcohols will be 5 consecutive alcohols from the homologous series; methanol, ethanol, propan-1-ol, butan-1-ol, pentan-1-ol. 1. Dependent The change in temperature of the 100cm3 distilled water when heated by an alcohol lamp. 1. Measure the initial temperature and final temperature using loggerpro. The change in temperature can be calculated by: ΔT=T(final)-T(initial) 1. Controlled Finding the à ¢H using à ¢H °comb=qn Controlled Variables How is it controlled? Effect on experiment if uncontrolled Type of liquid Using only distilled water for all trials throughout the experiment. Different liquids could result in a difference in the strength of attractive forces between particles, meaning a different specific heat capacity which would affect the calculation of energy gain to water using the equation q=mcà ¢T, and thus an incorrect enthalpy change value. Volume of liquid used Measure 100cm3 of distilled water by using 100 cm3  ± 0.08 cm3 graduated pipette for each trial. If the volume was not exactly 100 cm3 it would directly affect the mass of the water which will affect the q=mcà ¢T value and thus the à ¢H value. Material glassware Use the same brand and materials of a conical flask for all trials. Different materials have different conductivity and may absorb more heat from the alcohol lamp, affecting the overall heat absorbed by the distilled water. Using the same material and brand of conical flask ensures that this is the same for each experiment. Temperature of surroundings For standard enthalpy of combustion, the temperature must be 25 °C however in a classroom this is hard to control, so for each experiment the temperature will stay constant at 19 °C. If the surrounding temperature was to be changing, the distilled water could be losing more, or gaining more heat energy from the surroundings, directly affecting the temperature change and therefore, q=mcà ¢T and the à ¢H value. Distance between the conical flask and alcohol lamp A clamp will be set at a distance of 25 cm from the table, and this the flask will sit at the same height each trial. If the distance changes, the heat lost to the surroundings varies and the heat that reaches the bottom of the calorimeter also varies. This will lead to a difference in rise in temperature of water (à ¢T), and therefore an incorrect calculation for q=mcà ¢T and à ¢H value. Pressure of surroundings For standard enthalpy of combustion the pressure must be 1 atm, however in a classroom this is hard to obtain, so all experiments will be done in a room with the same pressure. Might influence the vapour pressure point, which will affect the q=mcà ¢T value, and thus the à ¢H. Duration of heating The water will be headed for 180 seconds. This ensures that all experiments have the same time to heat the water which directly effects the change in temperature and thus the q=mcà ¢T calculation and the à ¢H value. References: http://gandhijkt.org/blog/wp-content/uploads/2011/03/chemistry-sample-lab-report.pdf http://www.ausetute.com.au/heatcomb.html http://www.s-cool.co.uk/a-level/chemistry/chemical-energetics/revise-it/enthalpy-changes

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