 Thank you very much. Also for the organizers of this nice workshop, I will present the Baccala virus insect cell system and how I'm using that also for the expression. You can use it for the expression of the memory coding. So the Baccala virus insect cell system was basically developed already 40 years ago, so it's quite old. And it's a quite versatile system, which can be used for protein production. You can produce virus-like particles. And also coming to the question of if you see an increase in other expression tools, definitely see that Baccala is growing a lot with the emerging of cryo-M, where you need less sample amounts. So a lot of people are using Baccala system now. And the heterologous memory and protein expression is definitely difficult. So insect cells are one of the expression tools to overcome protein expression problems, especially if you think of reconciliation or think of the memory composition, which can be quite different, for instance, in insects, in yeast or bacterial hosts. So the Baccala virus system that we're currently using mostly is derived from the Baccala virus of the Grapha-Californica. It's a virus of around 140 kilopayers. And what we are using to express our proteins with the system is the very late polyhedron promoter, normally, or the P-10 promoter. These proteins, the polyhedron or P-10, are normally expressed in the very late stage of the virus cycle, and are not really needed for the virus in a laboratory environment. But are needed for the virus to spread in the native environment. So one can use this promoters to express high amounts of proteins in the very late stage of the infection cycle. The Baccala virus is also easy to modify. You can add easily several kilopayers of brain genes into its genome. And you can also modify the Baccala virus to instinctively leading proteases. You can add also proteins that may enhance glycopilation or protein stability. You can add chaperones, for instance. So coming to the systems that are mostly in use nowadays, there are basically two systems, which is the first Baccala derived system. So you have a Baccala which basically is a modified virus genome, which is able to be replicated in E. coli cells. So the Baccala virus has been modified with some E. coli genes, for instance, antibiotic resistance and the Lexa gene, where you can integrate your genes of interest, thereby disrupt the Lexa gene, and be able to do a blue-white screening to see if your gene of interest had been integrated into the Baccala virus. And then you can transfect this and generate virus from it. The other system that has been developed is a system that uses thermologous recombination in the cell. So what you use is you use an inactive form of the Baccala virus DNA and transfect it together with a transfer vector, which carries your gene of interest. And thereby, you're restoring an essential gene, restoring the function of the Baccala virus in the cell and also introducing your gene of interest. These two systems have pros and cons, especially on how expensive they are in use. So the multi-baccala system or the Baccala system, which uses the Baccala derived Baccala virus, is quite cheap. You can easily modify it in the lab because it can be amplified in E. coli, but it has been shown that Baccala virus derived from these Baccala meats are often less stable compared if you use the homologous recombination. There you are, you're limited on the supply of the virus DNA, which you then co-transfect together with your transfer vector. But of course, this system is very easy to use in high throughput screenings if you're screening a lot of protein or expression. So I learned the back-mit derived system, and I'm going to also present you that you can do not only was-to-one protein expression, but you can do multiple protein expression. This is some work which has been done in the Berger's lab. Developed a multi-baccala system where you use several donor and acceptor vectors to generate a large expression cassette, which you can integrate into the Baccala virus genome, and thereby expressing multi-protein complexes. This can, of course, lead to different expression levels, and this can be also modified by expressing, for instance, one expression construct which all the protein consists with a linker that can be then cleaved by a type cleavage site, for instance, in the cell. There are different cell lines available, and the most commonly used one is SF9, which is derived from the MOT, and you also have SF21 cells, which also derived from the MOT larvae, and they also seem to yield some higher protein levels in expression. The reason why you're using SF9 cells is they are most robust, keeping them in culture, and they also generate a very good virus type, especially in the beginning. And there are also several cell lines available, for instance, on like dilation, protein stability, or enhanced expression, which one can test. Another expression cell line is high 5 that you can use. And high 5 cells normally express higher amounts of proteins, but this also very much depends on your interest how much protein you can express, and I would definitely recommend to test several cell lines, and normally you start with the SF9 cells and then go into the other cells and see if you can boost your expression levels. The maintenance of insect cells is rather easy, I would say, compared to other cell lines. So you need, of course, to work sterile. You need a 27 degree incubators, and you don't need CO2 atmosphere, as for instance, for the million cell lines. You can grow them in adhering cultures on suspension cultures, which you would probably do then for the expression of proteins, and there's also, you can use normal take-off last, roller-bottles. The healthy concentration also depending very much on the cell line, but generally speaking, it's around half a million to two million cells, and then also depending on which media you're using. So there are so many companies providing media for insect cell expression, and they will also give you recommendations on which cell density can be used with their media and how to maintain your insect cell cultures in their media. So definitely, if you have any questions or have troubles, have a look into the user manual. There are different additives that you can use, for instance, serum, FPS, you can add lipids, antibiotics, and other sort of actions. I generally like to keep my expressions as simple as possible in the beginning, and then see if one can boost, for instance, expressions. For instance, with the membrane proteins, you can add lipids. FPS have been shown to stabilize viruses, but often it also lowers the yields that you get in protein expressions. And antibiotics you can use, but it's no need, but it may help you to overcome bacterial contaminations. So now we'll shortly present to you the back-to-back system, how I also learned it, or the multi-back system, which starts basically with cloning your gene of interest into a transfer vector, which can be then shuttled together with other donor vectors to a larger multi-protein expression construct, or directly shuttled into the baculovirus genome in E. coli cells. You will do a back-to-back selection in bacteria, do blood-to-blood white screening, and then isolate the back-to-back. So this figure I'm showing here is from one of Imoberdas' publications on his multi-back system, and normally you do the transaction then in adherence cell culture, where you generate low virus titers. So after a few days, you should be able to see some signs of infection. If you have something like a YP gene integrated into your virus, you will be able to quantify this and also see if your cells have been infected and produce, for instance, YP. The virus titer you will generate in the beginning here will be very low. So you need to amplify the virus further, which can be done then in Shakerflask. So you will harvest the medium, basically, where the virus has been secreted in and then infect Shakerflask with your virus. And this normally also takes a few days, depending on your virus strength, and you will constantly measure densities to check for the proliferation rest. And normally this will then also give you the virus one generation, which will be able to give you already nice protein expressions in most of the cases. So one thing that you might want to think about is the virus titer. So you definitely need a strong virus to infect a lot of cells. You can quantify this by different methods. I personally have to admit that I don't really like these methods because especially we're thinking about protein expression here. So one thing that you want to consider definitely is checking your protein expression on an SDS trail or using the YP signal to see where the protein expression is maximized. And another thing is that you're constantly measuring cell densities. You should check for the proliferation rest. So infective cells will not proliferate anymore, which corresponds then to an activity of around 100%. Which also corresponds here to the multiplicity of infection of three or greater, where you're at around 95% of infection. As you can see, so this is in an optimal world, this will take around two weeks, but let me tell you, it often takes much longer for this. So there are several systems that try to overcome this. For instance, this system where you use a DNA EI complex that you can use for a transaction and you can directly go and shake a plastic and this will give you a virus that is already strong, often strong enough to directly infect larger cell cultures. The virus normally is pretty stable already at four degree. So you need to avoid light exposure. You definitely want to store some virus at minus 80, which keeps it stable for longer. And another method to keep your virus stable is in insect cells. So you can use baccalaureate infected insect cells that can be harvested quite early after infection where they only started to produce a lot of virus particles without protein expression. And this you can freeze and use also for infection of larger cultures later. And this gives a really nice reproducible expression of proteins. So coming to the optimization, especially also thanks to Roslin, she mentioned a lot of these things in the beginning of her talk. So something like synthetic genes, you should definitely consider infect cell expression is very expensive, maybe not as expensive as mammalian cell culture, but maximizing your protein amounts is definitely advisable. You need to think about the membrane composition, especially for the membrane proteins. You have levels of, you have some levels of cholesterol, although they are low, you have high phosphorydure inositol, but no phosphorydure serine. So you need to consider that. So if you know something about the protein, at least something like this, you can add this, this has been also shown for GPCRs. And especially GPCRs have been studied a lot concerning the expression of membrane protein, of expression in insect cells. So if you're looking for advisors, you can definitely go and look for GPCR expressions in insect cells. There are a lot of data on that. You might want to consider co-expression for instance with the multi-buck system, chaperones, binding partners. And also what I also experienced is that the Paul H promoter or the P10 promoter can be different and expression your protein levels. So if you have established expression, at some point, you can also try to use different cell lines for instance high-file cells or SF9 cells, or you can use for the bacular virus modifications. This is very similar to modifications in SF9 cells, this introduction of probably glycosylases and ways to glycosylate your protein, which gives you a more native mammalian life translation pattern. And something that you want to avoid, especially in expressions often is the serum because it has been shown to lower your protein expressions. And with this, I'm more or less at the end and I would like to thank my current group at Orhus University and Bjorn Pedersen's lab where I'm trying to express SLCs in insect cells. I'd like to thank Maria, she's taking care of the insect cell lab that I'm using. My old lab in Berlin, where I used insect cells to express large scaffold associated proteins, membrane associated scaffold proteins. And I was also lucky to have a short stay in the Mojagas lab funded by the Biostructics program during that time to learn the expression of multi-protein complex in insect cells. That's done and I'm happy to answer questions. Thank you so much for your presentation. It was very nice. We have some questions here. The first one was from Rajiv. He asked, how can we take care of the stoichiometry while co-expressing multiple protein? So the stoichiometry is definitely a problem. That's because, yeah, protein levels are just differently expressed. You can use different promoters to try or just the same promoter to polite. Or with the system, this link system where you basically co-express all the proteins as one construct and then use something that could have a key which, but of course this cannot be done for all proteins because this might hinder folding and so on, so, yeah. Thank you. We have another one from Chris State. He asked, why are there recombinant buccal viruses made using the BACMIT system? Less stable than if made by recombination in insect cells. What is the difference in how? So this is not exactly known, but it's presumably because of the introduction of the foreign genes from E. coli that you introduced into the buccal virus. So buccal virus also are constantly trying to eliminate gene fragments that are not needed. So you also need to consider this if you're expressing proteins and have several virus generations, at some point you might lose your gene of interest that you want to express and need to go back to probably a frozen cell stock or something like this. But yeah, it's not exactly known, but presumably the introduction of foreign genes. Thank you. Thank you so much. Do you see media batch to batch variation and does that affect special levels in quality? This has definitely been seen. We also saw that also in my old lab that can be batch differences. There are nowadays ways to overcome this because often these media use some magies to lay it or animal forces, but there are now available some chemically defined medias which should give you a more reproducible expression because they are chemically defined. They should be always the same. But of course, also when using FPS, as in addition, there can be also batch variations. You can talk to your supplier if you definitely see that a certain batch is worse than the one before. Thank you. Thank you so much. Thank you so much for your presentation and for your answers.