How do I classify different IISERs

DAAD final report Linnéa Jakob. RISE worldwide Research internship in India IISER Mohali, India I. General part p.

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2 DAAD final report Linnéa Jakob RISE worldwide Research internship in India IISER Mohali, India Contents I. General part p. 2-9 II. Technical part S I. General part 1. Preparations 1.1 Flight S Visa S Vaccinations S Money S Packing S In India 2.1 Living S eating S working S people and language S money S campus and weather S travel and excursions S security p. 9 1

3 1. Preparations 1.1 Flight For India you should first take care of the flight and then the visa. This is because the visa cannot be applied for earlier than two months in advance, but the flights still get more and more expensive over time. As soon as I knew the time, I booked my flight. Amazingly, the cheapest offer was ultimately what I found on the Lufthansa website itself, not via one of the comparison portals. In the end I paid almost 600 for the flight and went three months ago. I would also really consider whether to book your return flight immediately after completing the internship and whether you might not book from a different city than the outbound flight, as you would certainly like to travel in the end. For example, I booked my outbound flight to Chandigarh directly, but booked the return flight from Delhi a week after the internship ended. At that point in time I didn't have any specific travel plans, but it was clear to me that I really wanted to travel a little more and so Delhi seemed the more central location for my return flight. These "fork flights" can quickly become expensive via comparison portals, via the Lufthansa website, but, as described, it was consistently cheap because both flights were calculated separately. When changing at the airport in Delhi, it took me four hours of the six hours to get from the plane to the next gate. So if you need an international and a domestic flight afterwards, make sure that there is sufficient transfer time in between! 1.2 Visa First of all, you have to check whether your passport is still valid for six months from the date of the visa or whether you have a passport at all. If not, you have to apply for one first. You can start with the actual visa application two months before entry at the earliest. It is not the embassies who are responsible for issuing the visas, but service providers commissioned by them. In Hamburg and some other federal states, the IGCS (is responsible. You have to find out which service providers are responsible for your own state. You also have to fill out an online form on the following page: When applying, you have to decide whether you want a student visa or an entry visa ( = Entry Visa). I would say it doesn't matter which one you apply for, because ultimately the consulate decides which type of visa you issue. I applied for an entry visa, but was issued a student visa. I also have multiple entries "(x entries), which means that you can get out of the country and come back in again during the time of the visa. In the end, it would not have been necessary during my stay in India, but if there was a spontaneous opportunity to do a short trip abroad , it is good if the visa has x entries. There also seem to be no further difficulties in the application ng to cause so why not. It is advisable to take care of some visa documents in advance. In the international office of the university you have to ask for a letter, "Confirmation of Studies", which confirms that you have 2

4 studies at the university and that you will receive a DAAD scholarship in the corresponding period. In addition, they wanted to have the DAAD scholarship confirmation and a copy of the student ID, even if this was not listed in the consulate's list of documents. You need a letter of guarantee signed by your parents. A small text is sufficient here, however. I wrote "We hereby confirm that we will pay for our daughter ..., born ..., in the event of financial emergency in India for all costs of the stay and possible return flight costs.", With the address and signature of the parents. Furthermore, you have to ask your supervisor in India to write an invitation letter from the academic institution. On the phone I was told what should be in it: date of stay, passport number, own name and date of birth and the institution's letterhead. Others told me later that they were, for example, Also went without a passport number, so there doesn't seem to be any fixed criteria. They also asked me to provide a description of the scientific work I plan to do in India. Then I simply submitted the pdf file of the project description of the RISE-worldwide project. You must also submit copies of both parents' passports and a copy of the birth certificate. The required documents also included "Excerpt from the commercial register of the Indian company" and "Registration of the Indian organization issued by MHA", but neither of these is required for a state educational institution. The cost of the visa is 105.50 at the end of the course, but the exact amount depends on the type of visa. With the submission of some documents, the visa took me about three weeks. Although there is online tracking with an application number, it only works one week after the application, if the application has been passed on to the consulate by the IGCS. 1.3 Vaccinations You should seek advice from a tropical institute or doctor at an early stage. I was at the Globetrotter's tropical practice in Hamburg, which was easy, especially since you can come unannounced. Apart from the standard vaccinations, which you probably got as a child, I was advised to get vaccinated against rabies, typhoid, meningococcal meningitis and hepatitis A. I then did these four vaccinations. You should find out beforehand whether the health insurance company will pay for the vaccinations, because if they don't, it will be expensive and you have to think twice about each vaccination whether it is really necessary. I am at TK and they reimbursed me for the vaccinations, whereby I ended up with about 500 eko e ha e, ur u de pricelist Rah e zu e e. 1.4 Money If you don't already have one, you should get a credit card in good time. There are now numerous banks that offer free credit card accounts (coupled with a checking account) for students. I have an account with a credit card at INGDiba, which has proven itself in India. Before that I called there to say that I am going to India and the bank can then deposit with Visa that one is there. This is to prevent a security system from blocking the card because withdrawing money appears untrustworthy in India. To be on the safe side, I had a second credit card with me, from the DKB, but this bank refused to deposit with Visa that I was going to India. 3rd

5 Even so, this card worked fine. You cannot get rupee cash outside of India, but to get your first cash at the airport, you can bring a few euros with you and then have it exchanged. A little later, however, there is also the option of using an ATM (cash machine). You should definitely know all passwords and pins or write them down in encrypted form, it ultimately helped that I had everything ready at home so that my family could help me out with a missing "Verified-by-Visa" password, which I can later use for online bookings needed. 1.5 Packing The only items my professor recommended that I take with me were an adapter and an umbrella. Although I was there in the rainy season, I only used the umbrella twice because of the rain, but every day against the blazing midday sun, in which one had to go to lunch. It is common to use umbrellas as parasols and you will not attract attention if you protect yourself in this way, which I find highly recommended. After all, we are not used to the immense intensity of the Indian sunshine. The adapter is nice, but not absolutely necessary. Indian sockets have three holes, if you insert something into the upper one, a protection that closes the other two holes is pushed to the side. Then you can plug your European plug directly into the Indian socket. A thin ballpoint pen can easily be pushed into the upper hole. Because of this, I wouldn't plug in more than one adapter. When packing your clothes you have to consider that the workplace is in all likelihood in an air-conditioned room, although in India the air conditioning systems are actually set to very cool temperatures everywhere. So it can easily happen that you freeze inside, especially when you sit still at work for a long time. So also pack something warmer to cover with. In India you can of course go shopping for clothes and it is not expensive either. Therefore, overall, take less with you and then buy what seems appropriate to the circumstances. Whenever I was in Indian shops, I was amazed at what there was to buy. So there is really no need to be afraid of forgetting something, because ultimately you can get anything in India. 2.1 Housing In India it always seems to be the case at universities and colleges that the students all have to live and live in so-called "hostels". As a "summer project student", as my classification in India was mostly, I didn't have to live in a hostel. Before my arrival, my professor asked me whether I would like to move to the hostel (no air conditioning) or the guesthouse (air conditioning). Since I couldn't really imagine how I would cope with the heat and I was a bit afraid of the temperatures, I first decided to stay at the guesthouse when I was in Germany. The guesthouse was also very comfortable, but also quite expensive and also at the very end of the campus. I then quickly decided that I wanted to move to the hostel, which was mainly due to the fact that all the other students also lived there. The guesthouse was too isolated and lonely from the life of the other students. After two weeks 4

6 I was able to move into the hostel and in retrospect it was definitely the right decision to move. The hostel might not be comfortable by western standards, but it was perfectly adequate and I didn't spend that much time there anyway. There were single rooms (although some freshmen had to share rooms due to lack of space) with a small balcony where you could dry your laundry and shared bathroom for a hallway. The bathroom was also furnished to western standards, although there was only cold water when showering. However, since the rooms do not have air conditioning, it is a welcome cool-down, at least in summer. (For the winter, which also gets cool there, there is a boiler with hot water to fill, so that people fill the hot water in a bucket that they then take with them to wash). As I said, the hostel doesn't have air conditioning, but there is a ceiling fan in every room, which makes it tolerable. 2.2 Food As an Indian student you don't have to worry about food as you get three warm meals a day. I liked the food very much and it is one of those parts of a stay in India that are really very different from ours in Europe. For me, the "mess", i.e. the canteen or cafeteria, was located on the ground floor of my hostel. Breakfast, lunch and dinner were served there. For every day I paid a total of 105 rupees in cash for the meals, you pay a little less if you register in advance and pay via the account. Oh yes, it would also have to be said that all of the food in the mess was vegetarian. In general, the choice of vegetarian food in India is much larger. There are many vegetarians, even if their number is getting smaller and chicken is now considered to be an Indian national good, but even so, meat is not always eaten outside of the mess. Even at Burger King, half of the burgers are vegetarian. For vegetarians (I'm also a vegetarian in Germany) it is the perfect travel destination in this respect. The Indians were all very astonished that I was a vegetarian and my professor had even been seriously worried beforehand about how to feed me, since there is no meat here on campus. I think you are very accommodating to the Indians when you say that you would like to live mainly vegetarian one day and that meat consumption is not so important. 2.3 Working There is a lot of hard work in India. I was in a laboratory that worked on the interface between chemistry, biology and biophysics. In the laboratory there were seven PhD students and one master's student who were supervised by a professor. The professor was in his office all day and only came to the laboratory twice a day to look. So I then had more to do with the doctoral students. I worked with two doctoral students or supported their work. There was probably too little time for my own project. The doctoral students come to the laboratory around 9:30 in the morning, some even earlier and then actually stay there all day, until ten in the evening is normal. So they half live in the laboratory, but they also go to the hostel during the day to rest, but not too often. Saturdays were also worked and 5

7 sometimes ended a little earlier (maybe seven in the evening). It was not uncommon for experiments to take place at night, either because of the reaction times or because instruments were only free at night. Every now and then experiments were scheduled for Sundays and twice while I was there the "lab meeting" took place on Sundays because it suited the professor better. I didn't have fixed working hours, but I was told that it would be my decision how much I would like to work. Maybe I could have worked for fixed working hours and free Saturdays, but that would have isolated me from the lives of the other PhD students. For example, it was sometimes spontaneously decided to go to the mall in the evening or to take a walk on campus after dinner (the others then went back to the laboratory after the walk, I went to my room). But it must also be said that the professor in my laboratory was known to be ambitious and to get his students to work hard. It may be a little different in other laboratories. 2.4 People and language I was always treated very courteously and hospitably by all the people I met. They often suggest doing something together (although that doesn't necessarily mean something will come of it) and invite you to eat and taxi for almost anything. You have to make an effort if you want to pay. In any case, someone in the group pays for the meal and never each pays their share of the order. However, since the ordered dishes are usually shared anyway, this would not be that easy. For example, I was often with people in a café, where two coffees and a piece of brownie were ordered, the coffee is served halved in two cups by the waitress if requested and everyone shares the piece of cake, everyone gets a fork. I was absolutely the only non-Indian student on my campus. In larger cities, on the other hand, the campuses should be more international. Nevertheless, the excitement was mostly limited and I was rarely approached just like that by Indians who did not know me through anyone. Outside of campus, however, a lot of people looked around for me. One is quite noticeable, at least in a city like Chandigarh or Mohali (suburb), where there really weren't any non-Indian people to be seen. In Amritsar I was also approached by numerous people asking whether they or their children could take a picture with me. At first I was happy to go along with it, but at some point it starts to get on my nerves and slow me down. After I once told my Indian fellow travelers that it annoyed me a bit, I was stubbornly defended in Hindi from that moment on, so that I was a bit ashamed. The everyday language is generally Hindi. However, there are many different regional languages ​​in India that are more or less similar. In fact, each of these languages ​​has its own characters. Many people speak several languages, for example because they come from Kolkata (Bengali is spoken there) or from the region around my university (Punjabi is spoken there) or from the south, where there are many other languages. Also in the northern regions, e.g. Jammu and Kashmir, there are other languages. Most Indians speak Hindi 6

8 like a mother tongue, even if they come from a different region. English, on the other hand, is a foreign language for them too. Although it is of course very much a practice at the university that all science and teaching take place in English, there are strong differences in the quality of English between different people. Overall, I noticed that communication is a lot more difficult than speaking English with native speakers. This is due to the fact that what you have said often does not come across and that the other person's English is not perfect either. Not everyone in India can speak English by far. It is by no means the case that everyone somehow learned that a little bit in school, at least not these days, maybe yes, if the next generation prevails. Because of this, it can sometimes be a problem on the street not to know Hindi.I have seldom been out alone, but often things are only written in Hindi or you have to ask something. In general, there is much more demand in shops, which is also due to the fact that there are many more staff. You first have to get used to the fact that a security person is standing around on every corner and that a salesperson is guaranteed to be with you immediately when you look at something in a shop. The question of how much something costs is understood by everyone who works in shops. And with a little patience, someone who speaks some English will always be brought in from somewhere if you have communication problems. 2.5 Money When I was in India, the exchange rate between rupees and euros was roughly such that I converted by default with 70 rupees = 1 euro or 700 rupees = 10 euros. In India everything is actually cheaper than here. My accommodation in the hostel cost only 1000 rupees for a month (without meals). A lunch or dinner cost me 40 rupees in the Mess, a little more than 50 cents converted, I paid a total of 105 rupees every day for the three meals in the Mess. Eating outside is of course a bit more expensive, at least in restaurants, although you can still get a good meal and drink here for 300 rupees, i.e. a little under 5 euros. Street stalls can be much cheaper and often taste very good. In real shops, which are then often also chains, such as those found in the mall, you can pay by card. Other shops only accept cash and of course you can only pay for food in cash. So you should always walk around with enough cash. Overall, I got along well with the DAAD scholarship, both for the flight and for the running costs. 2.6 Campus and Weather My time at IISER Mohali was seven weeks from the beginning of August to mid-September. In northern India, this is not the hottest time, which is June and July, but the subsequent rainy season. Despite the name, it hardly ever rained when I was there. Most of the time the sun was shining or it was cloudy and damp, but without rain. Towards the end of my time, the sunshine became more abundant and the humidity decreased. The temperatures, however, always seemed to stay the same, around 30 degrees maximum. It was more pleasant in the early mornings and evenings, so you like to walk around outside. 7th

9 Several people told me that up until seven or eight years ago it would have rained a lot here in the rainy season and that it could perhaps be attributed to climate change. The IISER Mohali campus is very new as the institution was only founded in 2007. This is why not all of the buildings that are planned have been built by a long way. This leads to large open spaces on campus, which appear very spacious and green. Overall, IISER Mohali is not that exciting place. Apart from the academic buildings and the housing estates for professors and hostels for students, there was only the shopping area and the guesthouse, which is about a minute's walk from the Academic Block. The hostels, on the other hand, are only 5 minutes away, which is why I moved. On the way there is the "Shopping Area", which I think is a somewhat exaggerated name for two small shops and a milkshake bar. In addition, there is a small branch of the state-owned Kanara Bank. Although the shops are small, there is a surprising amount to buy in a confined space. Groceries of all kinds, consumer goods, sweets and, if you wish, you can also get something to buy a few days later. I could buy a towel, pillow and sheet there too. What can seem a bit strange is the fact that the campus is completely closed and therefore very, very isolated from the rest of the world. But it is also completely safe to walk around the campus. There is a fence around the campus and there is a security post at the only entrance where you have to explain why you want to drive in by car. There is a bus that runs every hour between the campus and the center of Mohali, it belongs to the institute and is free. There are a few shops in the center of Mohali and you can get a lot there. 2.7 Travel and excursions I went to India with the hope that I would be able to travel somewhere in the area almost every weekend. But it wasn't that easy to do. I didn't want to travel alone and there were no other interns at IISER Mohali, neither from RISE worldwide nor any other international students. So on the weekends I mainly did something with the students from the laboratory. They also worked on Saturdays, which was the reason for me not to fight for any free Saturdays, I wouldn't have had anyone to spend them with anyway. Sundays were regularly free, but this was not always kept and even the "lab meeting" was sometimes on Sundays. Of course, it was a good thing that I had no one else to travel on the weekend, because that's the only way I did private things with the Indian students and they took me to their weekend activities, mostly shopping or eating. I also took a little longer time off on two weekends so that I could travel with other Germans from RISE around the world in India who had found accommodation in other cities. We agreed to meet up on Facebook beforehand and so I went to Goa for one weekend and Dharamshala for one weekend. 8th

10 Even when I booked my flight, long before the internship, I decided to travel for another week afterwards. That's why I didn't book my return flight until a week after my internship had ended. It turned out that was spot on and I was very happy to end up having this week to travel. The planning for the trip was very spontaneous, but in the end we were four Germans from RISE worldwide who traveled together in the mountains in the north of India. It was a great week, even if I had to go back to Delhi earlier in order to have at least one more day there before my flight home. So I would definitely recommend planning additional travel time! 2.8 Security You can hear it from everyone who is told about your plans: Are you sure? Alone as a woman in India? Isn't that dangerous? And so on. I would recommend: just do it anyway. In my entire time nothing has happened to me and I have always felt safe, often even safer than in Germany. The perceived security (and I guess also the actual security) is so high because there are so many people everywhere in India. Even if someone were to attack me now, there would be so many others who would definitely help me! Or rather: nobody would dare to do something bad in such a large audience anyway. In any case, those were always my thoughts and nothing ever happened. It goes without saying that you shouldn't be careless and stick to the usual tips, such as not walking around alone in dark alleys at night, and after all, exactly the same behavior is advisable in Germany. Another thing to be prepared for is that the well-intentioned advice on security in India itself will not get any better, it will get worse. Everyone from professors to students to taxi drivers advise you: don't walk around alone anywhere! It's totally dangerous! Just don't let yourself be intimidated and let yourself be robbed of your freedom, that's what I did at the beginning and never left the campus alone. Towards the end I traveled around more and more and found more and more that the warnings were exaggerated. 9

11 Report on my DAAD Rise worldwide Internship in Dr. Samrat Mukhopadhyays Laboratory at IISER Mohali, India Advisor: Dr. Samrat Mukhopadhyay Time of internship: Intern: Linnea Jakob Place: IISER Mohali, India Content 1. My project 2. Introduction Aggregates, amyloids and fibrils Prion proteins Sup35 Yeast and the Ade1 marker Yeast transformation 3. Methods used Sup 35 mutants Expression of Sup35 NM Purification Fluorescence spectroscopy AFM 4. Conclusion 5. References 1. My project I have spent nearly two months at IISER Mohali (Indian Institute for Science Education and Research), which is a university-like public institution in India with a strong focus on research. There I was working in the "Amyloid biology laboratory" of Dr. Samrat Mukhopadhyay, which was part of biological as well as chemical sciences departments. The research mainly focuses on looking at protein aggregation using an array of biophysical tools like fluorescence spectroscopy, AFM (Atomic Force Microscopy), CD (Circular dichroism), Raman spectroscopy. During my tenure I was working on Sup35 protein which is a translation termination factor in yeast which is also known as yeast prion protein. My work mainly aimed at purification and investigation of the aggregation kinetics of Sup35 and to understand the molecular basis of amyloid fibrils of Sup35. This report will be about the backgrounds of this research and also about the work that I have carried out at IISER Mohali. 10

12 2. Introduction aggregates, amyloids and fibrils Amyloids are protein aggregates with characteristic cross-beta structure [1]. The formation of amyloids is not restricted to a specific group of proteins, but seems to be a general property of proteins under certain conditions [2]. Amyloid deposits in the body can cause several diseases, examples for disease-causing proteins that form amyloids are beta-amyloid and tau in Alzheimer's disease, alpha-synuclein in Parkinson's disease or prion protein in Creutzfeldt-Jakob disease [3]. But there is another class of amyloids, where the amyloid structure is used by an organism to maintain important functions in cells which are known as functional amyloids. Prion proteins Prion proteins (PrP) are a group of proteins that can switch between two conformations, the cellular (PrP C) and the scrapie form (PrP SC) [4]. The conformational transition from α-helical to β-sheet rich prion is known to be associated with several neurodegenerative diseases which are collectively known as spongiform encephalopathies. PrP SC can self-template by promoting the conversion of PrP C into more and more scrapie form. Another property exhibited by prion is polymorphism / strain phenomenon that is the same polypeptide giving rise to structurally and morphologically distinct species. The "yeast prion protein" Sup35 is not related to the mammalian prion protein; the reason why it is then called yeast prion protein is that it shares most of the features of mammalian prion protein such as self-propagation, strain phenomenon, and species barrier [5]. Due to ease of genetic manipulation yeast is widely used as a model to prion propagation and strains. Sup35 a yeast prion-like protein Sup35 is a protein in yeast involved in translation termination, by recognizing stop codons [6]. Sup35 contains three domains, the N-, M-, and C domain. The N domain is essential for aggregation, while the M domain maintains the solubility of non-prion form and the C domain is involved in the translation termination function of Sup35 [8]. NM region is an IDP (intrinsically disordered protein), that means in its native structure it does not contain any secondary structural elements, so there is no defined structure [7]. The yeast colonies formed out of cells containing normal, soluble Sup35 are called [psi -], while yeast containing the other, aggregated Sup35, is called [PSI +] [1]. In [PSI +] cells the concentration 11

13 of soluble Sup35 is very low, and aggregated form of Sup35 will not be able to recognize stop codons, so that there will be a read-through giving rise to new phenotypes [6]. Ade1 marker is used to visualize these strains. Fibrils formed at 4 C and at 37 C were found for example to have slightly different effects on the cells after transformation [9]. 4 C fibrils leads to aggregation of nearly all of the soluble Sup35, leading to white colonies. Fibrils formed at 37 C lead to formation of pink colonies, because this fibril type leaves more soluble Sup35 in the cell. The structural difference between the fibrils formed at 4 C and at 37 C leads to these phenotypic characters. [PSI +] and [psi -] yeast colonies and the Ade1 marker In order to distinguish the yeast [PSI +] and [psi -] strains, a marker was used, which leads to coloring of the yeast cells in white ([PSI + ]) or red ([psi -]) [10]. This marker is based on the adenine synthetic pathway. For synthesis of adenine different enzymatic steps are neccessary, and in Ade1 mutation one of these enzymes is not functional [10]. The mutation is incorporating a new stop codon within the gene that encodes the enzyme, so that this translation is stopped too early. As this enzyme is now not functional any more, it leads to yeast cells not being able to synthesize their own adenine properly. Instead, an intermediate metabolic product (phosphoribosylaminoimidazole, AIR) is accumulating. This intermediate polymerizes in higher concentrations. These polymers have a red color, so they lead to visibly red colored yeast colonies [10]. Now why is this a marker for identifying the conformation of Sup35? Because Sup35 is a translation termination factor. And when most of Sup35 is forming aggregates, there is only very few functional Sup35 left in the soluble form. The lack of enough Sup35 leads to some stop codons being not recognized, leads to read-through. In the case of the mutated Ade1 there will be a read through leading to the synthesis of Ade1 enzyme [10]. Therefore there will not be any accumulation of red pigment and the colonies appear white. Depending on the fibril strength and availability of soluble Sup35 there is also an intermediate possibility, which leads to formation of varying colors ranging from white to red [9]. This happens because of different variants of fibrils formed by prion Sup35. For instance, when fibrils at 37 C are transformed into yeast it gives pink colonies because these fibrils are not so strong thus less propagation leaving some soluble molecules of Sup35. 12

14 Yeast transformation In order to show that the Sup35 prion protein can infect [psi -] transformation experiment was done. Transformation in this case means to make a [PSI +] strain out of a [psi -] strain by inserting Sup35 in prion conformation into the [psi -] strain [11]. The prion Sup35 was added in the form of aggregates, which were converted into smaller parts before. To make the cells competent for taking up these large particles, the cell wall was removed by an enzyme, lyticase. These results in the formation of spheroplasts, which have nearly no cell wall left [11]. These are treated with different buffer ingredients, like PEG and DTT, which both enhance the efficiency of transformation. Expression of Sup35 NM To produce large amounts of the Sup35 protein, it was expressed in E. coli cell culture. The E. coli strain used for the work was BL21 (DE3) pLysS. BL21 (DE3) is the strain name, and plyss is a plasmid [12]. The T7 promoter, which is placed in front of the Sup 35 gene on the transformation vector, is used as a switch for the expression. In its chromosomal DNA the strain contains the gene for the T7 RNA polymerase, which is under control of a lac promoter [12]. Now this lac promoter can be switched on by the addition of isopropyl-1-thio-β-d-galactopyranoside (IPTG), which is a strong inductor. The plyss plasmide contains a resistance gene against the antibiotic chloramphenicol, so that chloramphenicol can be added to the culture to prevent any other bacterial growth [12]. Ampicillin is also added to prevent the growth of E. coli cells which are not containing the expression vector, because the resistance gene for ampicillin is encoded on the vector carrying the Sup35 gene. Another way to prevent contamination with other bacteria or any other components was setting up a primary and a secondary culture. The E. coli cultures were grown on Luria broth (LB) medium. For preparation of 100 ml LB medium 2.5 g dry LB mixture was dissolved in water in a flask, closed by a cotton plug. Before use the LB medium was sterilized by autoclaving and the cell culture work was done under sterile conditions. The concentration of ampicillin in the primary and secondary culture was 100 µg / ml, the concentration of chloramphenicol was 35 µg / ml in the primary culture, in the secondary culture 17.5 µg / ml. The recombinant E. coli strain with the vector containing Sup35 wildtype or tryptophane mutants were prepared some time ago and were stored in a glycerol stock at -80 C. To start a culture, a 13

15 small amoung of this glycerol stock was used to inoculate the primary culture. The primary culture was grown over night at 37 C and 200 rpm. For the primary culture, 10 ml LB medium were used, for the secondary culture 1 L. The growth of the secondary culture was started by adding a 10 ml aliquot of primary culture to the 1 L (1%). Beginning from three hours after start of the secondary culture, the OD was checked in the biophotometer. If the OD was in between, the culture was induced by the addition of 400 µl 1 M IPTG to the 1 L secondary culture, which gave a final concentration of 400 µm in the culture. Now the culture was left for incubation for one hour and then it was centrifuged for 30 min at 4 C and 4000 rpm. The centrifugation leads to formation of a cell pellet and a supernatant without cells. The cell pellets were resolved in lysis buffer (containing 10 mm Tris and 1 mm EDTA) and stored at -80 C. Purification Tryptophane mutants Naturally Sup35 does not contain any tryptophane. Several mutants have been made by incorporating single tryptophanes in different amino acid positions throughout the protein and it was shown that these mutants do not have any effect on the structural properties of Sup35. As tryptophane is an intrinsic fluorophore it can be used for fluorescence spectroscopy experiments and this gives detailed information about the exposure to water of residues in certain positions while aggregation. These mutants as well as wild type Sup35 had to be purified first. Ni-NTA column The solid phase in this adsorption chromatographic method is made out of agarose beads with bound nitrilotriacetic acid (NTA), which is a negatively charged moiety and which immobilizes the positively charged Ni 2+ ions [13].The affinity of His-tagged proteins to Ni 2+ ions is very high and so Sup35, which was expressed with an incorporated His-tag out of six histidines, binds to the resin. Through different washes the other proteins and macromolecules can be removed, after that an elution is done to separate the Sup35 from the Ni 2+. Elution has to be done by a molecule which binds to Ni 2+ with an even stronger affinity and is present in large amounts, we used 250 mm imidazole for this purpose. Cell pellet stored at -80 C was put into boiling water for 20 minutes. Then this was centrifuged for 30 min at 4 C, rpm. The Sup35 protein was now in the supernatant, which was transferred to 14

16 a new centrifugation tube. Then a 100% saturated ammonium sulfate solution was added and the protein was precipitated in 50% ammonium sulfate. The solution was stored at 4 C for two hours for precipitation. Then it was centrifuged again for 20 min at 4 C, rpm. This time the Sup35 protein was in the pellet and the supernatant was discarded. The pellet was then dissoved in 8 M guanidinium chloride (GdnCl) buffer (8 M guanidinium chloride, 20 mm phosphate buffer, ph 8) and this was left to incubate at room temperature over night. Then the protein solution was centrifuged for 30 min at rpm, 4 C. The protein in the supernatant was then purified by Ni-NTA column. After washing the column with water Ni-NTA was equilibrated with 20 ml of 8 M GdnCl. Then the protein solution and resin were incubated under rotation for one hour to make the His-tagged protein bind to the Ni 2+ ions. After filling the resin in a column and collecting the flow through, different washs followed. First 10 ml 8 M GdnCl buffer, then 10 ml 10 mm imidazole buffer (8 M urea, 20 mm phosphate buffer, ph 8), then 20 ml 1 M NaCl buffer (8 M urea, 20 mm phsophate buffer, ph 8), then 10 ml of 10 mm imidazole. Elution was done with 20 ml of 250 mm imidazole (8 M urea, 20 mm phosphate buffer, ph 8). To check how much protein and how much contamination with nucleic acids is there, absorbance at 260 nm and 280 nm was measured using biophotometer. Q Sepharose A Q sepharose column works through anion exchange chromatography. There are anion exchangers as well as cation exchangers. An anion exchanger is positively charged, so that anions can bind, according to the strength of the ionic bond. Q sepharose resin is made out of agarose beads with bound quarternary ammonium [14]. This quarternary ammonium is positively charged and therefore an anion exchanger. Proteins, which are negatively charged, bind to Q sepharose. Nucleic acid is even more negatively charged and binds the Q sepharose even stronger. This different velocities in passing causes through the column and that leads to the separation of protein and nucleic acid. To help the molecules to elute after binding the quarternary ammonium groups, an increasing gradient of NaCl was used. As the concentration of NaCl increases, Cl-ions bind to quarternary ammonium with very high affinity making the protein to elute. Q sepharose column was done to further purify the protein from DNA contamination. The Q sepharose column was equilibrated with 1 M NaCl buffer (8 M urea, 20 mm phosphate buffer, ph 8). After adding the protein solution to the Q sepharose resin and collecting the flow through, an adapter for the FPLC was attached. The FPLC system was used to provide a steadily increasing 15

17 NaCl gradient. As Buffer A 8 M Urea (8 M Urea, 20 mm phosphate buffer, ph 8) was used, as Buffer B 8 M Urea with 1 M NaCl (8 M Urea, 1 M NaCl, 20 mm phosphate buffer, ph 8). The gradient was set with a target of 50% after 60 ml, the pressure maximum was set as 0.1 MPa and the flow rate was set as 1.5 ml / min. When the UV absorption started to rise, fractions of 2 ml were collected. Normally the protein would start eluting at around 100 mm of NaCl. Q sepharose column chromatography monitored by FPLC is shown in figure 1. Fig. 1: Q sepharose purification run with FPLC system. On the x-axis, purification progress is shown as volume (ml). Blue: UV absorption (280 nm), red: conductance. First peak at A 280 is the unbound protein, second one is the protein of interest and the third peak corresponds to nucleic acid. Later the gradient was stopped and the concentration of buffer B (including NaCl) was set to 100%, which leads to the strong rise of conductance. Purity was checked by running the fractions on SDS-PAGE, see figure 2. Fig. 2: 12% SDS-PAGE fractions of Q sepharose purified Sup35. 16

18 Fluorescence spectroscopy Fluroescence spectroscopy was used for measuring the kinetics of the fibril formation. To detect amyloids, amyloid markers thioflavin T (ThT) was used. After binding to amyloids fluorescence quantum yield of this fluorophore increases [15]. Thus amyloid fibril formation is monitored by fluorescence emission intensity of ThT. Figure 3 shows an example of an amyloid formation kinetics of a mutant of Sup35, monitored with ThT fluorescence. The aggregation of Sup35 had lag-time followed by exponential phase and then a saturation phase. The lag-time was around 20 minutes. The sigmoidal growth equation was used to fit the kinectics, see Figure 3. Looking at the lag time is an important part of the kinetics of amyloid formation. The lag time can give information about the nucleation process occurring in the beginning of the fibril growth. Together with other data it is useful for studying the process of amyloid formation, which is the aim of the research in this lab. Fig. 3: ThT fluorescence monitored Sup35 mutant (triplicate) Atomic Force Microscopy (AFM) To gather information about the fibril morphology AFM was used. AFM of Sup35 fibrils were imaged in tapping mode. The AFM images revealed that the height of the fibrils is 4-5 nm. 17

19 4. Conclusion During my tenure I could learn protein expression and purification using Ni-NTA and Q-sepharose column chromatography. Also I got to know about techniques like fluorescence spectroscopy and AFM. Using fluorescence spectroscopy aggregation kinectics Sup35 was monitored. Morphology of the fibrils were discerned by utilizing AFM. 5. References 1. Nilsson, Melanie R., Techniques to study amyloid fibril formation in vitro, Methods, 2004, Volume 34, Fändrich, M., On the structural definition of amyloid fibrils and other polypeptide aggregates, Cellular and Molecular Life Sciences, 2007, Volume 64, Pulawski, Wojciech et al., Ubiquitous Amyloids, Applied Biochemistry and Biotechnology, 2012, Volume 166, Prusiner, Stanley B., Prions, PNAS, 1998, Volume 95, Narayanan S. et al., Yeast prion- protein, sup35, fibril formation proceeds by addition and substraction of oligomers, Chembiochem, 2006, Volume 7, Pezza, John A. et al., Amyloid-associated activity contributes to the severity and toxicity of a prion phenotype, Nature communications 5, 2014 , Article number Ferreon, AC et al., Single-molecule fluorescence studies of intrinsically disordered proteins, Methods in enzymology, 2010, Volume 472, Salnikova, Aleksandra B. et al., Nonsense Suppression in Yeast Cells Overproducing Sup35 (erf3) Is Caused by Its Non-heritable A. myloids, The journal of biological chemistry, 2004, Volume 280, Ter-Avanesyan, Michael et al., Unraveling prion structures and biological functions, Genome Biology, 2006, Volume 6, Dept. of Cell Biology & Physiology, Washington University School of Medicine, King, Chih-Yen et al., Transformation of yeast by infectious prion particles, Methods, 2006, Volume 39, BioDynamics Laboratory Inc., Product information on Competent Cell BL21 (DE3) pLysS, Qiagen, Ni-NTA Agarose, GE Healthcare Life Sciences, Q Sepharose Fast Flow, ctid = 21228 & storeid = 11251 & langid = Wolfe, Leslie S. et al., Protein-induced photophysical changes to the amyloid indicator dye thioflavin T, PNAS, 2010 , Volume 107,