 So today, yeah, I'm going to talk about some insect odent receptor work we're doing and I will say that this is quite a new topic for me and also I think for structural work in general. So there's definitely a lot we still I won't be able to answer and I'm really open to have feedback and suggestions on this work and I think already a lot of the talks today have been really helpful. So just who I am. I'm Cassie. I'm a postdoc who just started Lund University and the new Max Planck Center for next generation insect chemical ecology, which is an absolute mouthful of a name. And yeah, I'll just give you a little introduction as to why we're interested in this work and then move on to what I've been doing so far. So, in sector affection, like, why do we care a lot of people today have talked about sort of medicinal targets and things like that and I think that stuff like what we work on tends to get a little overlooked. And in sector factions really important. As I said in the abstract, I'm going to talk about moths and mosquitoes for two, they're important for two different reasons moths are pests usually of agriculture and we're really interested in controlling them. And mosquitoes are of course pests of humans. So we're interested in them from medicinal kind of human health standpoint and all factions really important as a sense for insects and in fact it's basically the major sense that is used in most insects and already we can use chemical ecology approaches to manage and control insect populations. As a kind of colloquial example, I'm sure many people spray deep onto themselves to avoid getting bitten by mosquitoes. But there are lots of field applications for this work already and we hope to to understand in sector faction to develop better methods in this area and to kind of exploit the chemical ecology of these insects against them. So a little introduction to the system and the proteins we're going to talk about today in the peripheral insect or factory system which happens in the antenna so I'm not thinking about what's going on in like the brain or anything. We have two main groups of proteins these odent binding proteins which are not membrane bound proteins so not of interest to us today. But what is interesting is the odent receptors. And these are heteromeric complexes so they form a complex with something called the odent receptor co receptor or orco. And orco is highly conserved across all insects but you actually only have like one orco per species, whereas you have then a range of odent receptors which have different specificities for different ligands. So it's a really interesting system. There's a tetramer so with the orco subunits, and each subunit is a seven transmembrane domain protein. I think initially they were thought to have a really similar structure to gpcrs, but they actually have an inverted topology to what gpcrs do. So, they're kind of unique, and there is of course limited structural data for these types of proteins. So there's a huge amount of functional studies that show that these receptors vary so much in their function in their specificity for example so you have some receptors which only respond to one ligand, and that's the highly specific. And you have some which are more broadly tuned to a range of ligands so there really must be some differences between them but we don't really understand these. In terms of structural work so far I can't claim to say that I've had any part in any of it but I will give you a rundown of what there is. The first structure was published three years ago now 2018 by Vanessa Reuters lab group who are lab group from America, and they published this cryoem derived structure which was produced in HEC 293 cells. So the HEC cells are used quite commonly for functional assays of these proteins. I can say a little bit about why we choose not to use these for our work in a minute, but this was published as a structure of orco so this is just the coreceptor in a homeomeric complex. Very recently the same lab group has gone on to just published this as a preprint so it's not fully published yet, but you can go check it out if you're interested of a odent receptor but this is again in a homeomeric complex so they chose this odent receptor from an ancient research which I think, and don't quote me on this maybe predates the evolution of the coreceptor of orco so they have this homeomeric complex of an odent receptor which has a binding pocket, and their intention was to look at the binding pocket and how this opens, what is an ion channel that obviously causes a neurological kind of response. So we identified a binding pocket and some of the functionality of this protein, and this is a huge step in the right direction for our work, but unfortunately OR5 is not a specifically tuned receptor. They found that it was quite broadly tuned and they didn't find that much specificity in the pocket. So, we still have a lot of unanswered questions, and they still also haven't done an odent receptor, but they found a coreceptor complex which is how it is in insects themselves so we really are interested in seeing how this functions, because I think it's not quite the same if you have four binding pockets, and you're showing a mechanism of opening versus one binding pocket that should activate the opening. So there's still a lot of work to be done. So our aim was to try and do some structural work with these proteins, but in yeast. Well, a lot of people have talked today about why it's a great system for membrane structure, kind of determinate membrane protein structure determination. We also people have mentioned hexels and I think hexels are really a valid way of doing this for cryo EM. One of the reasons we were interested in not doing. Hexels was one we wanted to create a high throughput system that produces a lot of protein and because we might do crystallography as well we don't know yet. And also because there is actually some inconsistency sometimes with the hexels in terms of functional studies that we do. And you can get some kind of weird results and all consistent with other functional assays so although we don't really know about the functionality in yeast we at least have a fresh start to see how it goes. So I kind of just put on here like an overview of what our process was going to be, but of course this is a lot of work condensed into a very simple diagram. Right now I'm at this point so I decided this project in this year really so at the point where we've transformed some yeast and we're seeing what happens. So in terms of the choice of receptors there are so many to choose from and there this is a list here of the ones we went with. We tended to choose highly specific receptors apart from one on there which has an unusual functionality which I won't talk about too much today but it has some two lig and cell response to. And we also have collaborators and Swedish agricultural university who are interested in mosquito receptors so I can't really tell you detail of why they chose these ones because in this case I'm following instruction rather than coming up with the idea myself. But we decided to go for about 12 just because this seems like a reasonable number to start with, but really this, the reasons why these are on this list there are so many I could have picked and at some point I just have to pick 12 basically so you know that read too much into this. We initially did the cloning. A few people talked about some of the design elements to this today. And we did a multiple site gateway cloning to produce a plasmid which has a ZSN resistance marker. It has our odent receptor co receptor gene and has an EFP fusion odent receptor gene. The reason being that we thought that with the odent receptor it tends to be seen that you don't get functional expression of this without the orco in place the orco has some fun. It has a role to play in expressing that. So we figured that if we saw GFP that the OR would be expressing and therefore the orco as well. Obviously that's an assumption and we will have to check later on that that that's the way it works. They also have constant constitutive promoters, and we didn't, you know, intend to direct them anywhere to express in the yeast because we just wanted to see if we could express them in yeast at all really to begin with. It was a bit of, it's, it's one of those projects where you go in and you have no expectations that anything will work but you've just got to see what happens. So we generated the this these plasmids and in the E. coli and then did the linearization to transform them into yeast. We also generated our transformments with zeosim. After that I've done some additional selection with high zeosim concentrations and also checking fluorescence because it being constitutive we can see expression in just the colonies that we initially get. And of course there will be a lot more optimization steps that will come after this. So far we have four or all the complexes that I believe are successfully cloned and I've got many more to keep trying. How am I checking that it worked PCR sequencing fluorescence, kind of all the standard things but just to let you know that this is what we're doing. Right now I am continuing with checking these and transforming and performing optimization, but I wanted to share we did some initial confocal microscopy pictures of one of the initial transform colonies so after no optimization. And I think we see that there is GFP expressing but as you can see I think it's not in that many of the yeast cells. So this is certainly something we want to try and optimize and this might involve things like fact sorting or, you know, optimizing based on the fluorescence. But I think it looks like the GFP is expressing which was a huge, hugely positive sign for me. And just the kind of close up I guess of that one I am no expert on these kinds of things and certainly this was not an optimized confocal kind of experiment we really just the confocal guide not really even worked with you so much so we were just seeing if we could get any pictures really. But I think it does look as if it is expressing towards the membrane, which I think is again a positive sign because we didn't really target anything or really know what's going to happen. So it's kind of a lot of preliminary work so far. But I have a lot, a lot of time, a lot of things to do going forward so of course I need to optimize the growth of these strains that we've generated by monitoring for example biomass or the fluorescence. I mean there are two approaches how can I grow the most and how can I grow the most that's expressing the most. So I'm hoping to sort the cell lines using facts so that we can get rid of those use that aren't expressing. And of course, there's a protein purification and detergent screening steps. And the big question as well for me is that we need to determine that this is a functional protein, and this will certainly be something that's going to need a lot of thought and assay design to figure out a way of doing this. Just say thanks for listening. I'm happy to answer any questions or you can always email me afterwards if you can't think of something now. Just to say thank you to my supervisors Krista and Matt's to Bao Zhang who I think is here and he helped so much with the cloning and help design everything and show me how to do everything. Thank you so much. Thank you to Guppy and Hannah who showed me how to work with yeast. I don't think even mentioned that we're using pickier yeast with X 33 strain if anyone is interested. And of course, Ola who is the microscope person because I can't say the word who helped you with the confocal to begin with and I think will help me going forward to generate some better confocal images. So that's me. Thank you very much. Thank you. You are co expressing the receptor with the this orco code receptor or you call it. Do you have you considered like putting you only have the gfp tag on the receptor. Yeah, have you considered like putting a visual tag on orco just so you can see that you have both. I wanted to think about this but we wanted to reduce the amount of kind of tagging we did just because if this turned out to work we hope that that would help us then, you know, messy further down the line and save us some work. I think it's, it's like there is a chance that the orco will express on its own without the OR, but there is very little chance that the OR will express without the orco so I think the idea of putting it on the OR was that if we see this gfp expression, we have both expressing because this is more likely. Like I said that's that's based on work in other systems so I don't know that that will be the case and certainly that's something we have to look at. Yeah, so speaking about other system would we have a question from Arianna about you if you considered insect cells. Yeah, and we did basically this was the decision between yeast and insect cells, because I think the insect cells are a valuable option for this. It just came down to the fact that we had expertise in the yeast stuff on hand and that we felt that we could do maybe higher throughput assays this way. But the idea is like if this doesn't work we go to insect cells next you know so it's definitely a consideration. And then we have a question about from Martin Caffrey if humans also have orco. Humans do not have orco so human odor receptors are actually RGPCRs I believe they're completely different in mammals from insects we don't have we don't do odor infection in the same way as insects and yeah we don't have orco it's insect specific.