Richard Jacobs:  Hello. This is Richard Jacobs with the Future Tech and Future Tech Health podcasts. I have Christopher Reinkemeier: Reinkemeier, the Ph.D. student at Johannes Gutenberg-University Mainz. The Institute for molecular biology and the European molecular biology laboratory. So Christopher thank you for coming. How are you doing?

Christopher Reinkemeier:  I’m doing fine. How about you?

Richard Jacobs:  Good, good. Yeah. I found you because I saw you are part of a group that is creating a synthetic organelle that will be inside of living cells if I have it right. Can you tell me a little bit about what’s recently happened?

Christopher Reinkemeier:  Yes. So, we recently published synthetic organelle. So that synthetic organelle, it can do a specific translation of one mRNA to produce specifically modified protein and these specifically modified protein carriers than a non-canonical amino acid, as a specific side. So, it has already previously been established a technique of genetic code expansion where you record, for example, the Stop Code on, in your protein coding sequence to put a non-canonical amino acid. And we now developed an organelle based on, phase separation and, micro driven motor proteins to greater organelle, which only recruits one mRNA and then only translates this mRNA with an expanded genetic code. So that only just specific mRNA carries the modification.

Richard Jacobs:  Well, most organelles in the cell do a crazy amount of things like let’s say arrived the zone, but yours is ultra-specific where it only creates one thing. Is that right?

Christopher Reinkemeier:  Yeah, that’s right. So, our organelle only creates this one protein basically. So, to do this one concept where you rely heavily on is the concept of phase separation, which has, in recent years have gotten a lot of intention in cell biology because there are a lot of membrane-less organelles which organize things. So, maybe all the traditional organelles you think of like Mitochondria or the nucleus and their own membrane encapsulated. That’s, proteins can also form membrane-less organelles, which you can imagine like when you mix oil and water, they won’t mix and you will have oil droplets in water or vinegar when you make dressing for a salad or something like that. So this is also a principle which cells use because then in this oil droplet in the water, the oil is very highly concentrated. It also happens for proteins and so what we do is we concentrate two components to make the expanded genetic code, which is the tRNA synthetase to tRMA and to mRNA for prudent of choice into a concentrated phase so that really only this mRNA gets to sneak on alkaline amino acids by tRNA.

Richard Jacobs:  Okay. Let’s go over a little bit of basic just for listeners in case. So, organelles are components inside of a cell that does things like the ribosome, I guess creates proteins. You have the nucleus, you have mitochondria, etc. So you’re trying to create a, essentially a synthetic one that has just one purpose as proof of concepts. And then later on, and I’m sure you’ll expand if this works. I do have a question. I’ve thought about this, you know, if you think of the body, the skin is like a membrane that keeps the insides in and outsides out. Then you have different parts of the body, like the, you know, I guess the peritoneum keeps the organs in the stomach area and then organs themselves have linings and cells to keep things in and out. And then cells have linings, the membranes that keep stuff in and out. What about Organelles? What is the surface of an Organelle look like? Does it have a membrane? Like how does it keep a separate self, inside of the Cell Cytoplasm? And how did you guys create a boundary that keeps your synthetic organelle in his own place, inside of the cell without, you know, being dissolved or without interacting with the rest of the cell?

Christopher Reinkemeier:  So, inside the cells, there are basically two different kinds of organelles. One, again, like a cell encapsulated in the membrane, so just the more classical organelles, like Mitochondria, has a membrane or the Golgi apparatus, which modifies proteins. They all have a membrane and they have things concentrated inside of the membrane, but they always need to transport machinery. Then there are other organelles in cells like, for example, the Nucleolus or some of the stress granules, which are called membrane less organelles because they form inside of the cell by interaction of proteins and RNA so they basically without being enclosed by a membrane, they’re formed by inter-molecular interactions between RNAs and proteins for example. And they locally concentrate specific components and they dynamically exchange with the surrounding cytoplasm because they are not surrounded by a membrane. So basically everything which is in the cytoplasm has potential access to this, but two different affinities between proteins and armies determine how concentrated components are inside of these membrane-less organelles. And this is exactly what we now use to create this synthetic organelle.

Richard Jacobs:  In the non-membrane organelles, how do they stay fixed in the cell or do they? Or do they migrate throughout the cell-like, when I think of the cell nucleus, I think of they just sitting in the center, but does it migrate?

Christopher Reinkemeier:  So, this nucleus is a membrane-bound organelle but there’s a structure inside of the nucleus which is the Nucleolus and this membrane-less. And these specific structures are very dynamic. So in general, a lot of these, membrane-less organelles they can, for example, in response to stress and they can form, grow and then also dissolve again. So they are very dynamic and, I think when you compare it, for example, to the traditional membrane-bound, they’re even more dynamic than these ones. So they move a lot in cells and they grow and can shrink.

Richard Jacobs:  Okay. They migrate, they grow, they shrink, etc. Gotcha. So the synthetic organelle, that you made, you told me a little bit about what it does. That was pretty technical, but what is the point? Where are you trying to do? What mechanism or pathway within the cell or what role or function does this organelle serve?

Christopher Reinkemeier:  So, the purpose of our organelle is to put really specific modifications into proteins. So this technology of generic code expansion can be used, for example, to put reactive amino acids into a protein at a specific site. And then you can afterward then react your protein with, for example, what we often do it with a fluorescent dye so that you can label your protein and use this for microscopy, for example, for super-resolution microscopy or because this technique has been around for already 120 years, there are a lot of different amino acids, which can be put into a protein. And with this you can, for example, get photo control of a protein and you can just then activate a protein when you’re shining a light on it or something like that. Or put specific, poor sensitive modifications to decide like free common applications of this technology. But what we can now do is we can make sure that this only happens for, one of the mRNAs in the cell and that only one protein gets modified. So, when you don’t want to label your protein, for example, for microscopy, you can be sure that only the protein you want to study is actually labeled with the fluorescent dye afterward.

Richard Jacobs:  But why do this inside a cell? Why not just make like a cell size organelle that can do this and just put it in the body itself. Why do it inside of the cell?

Christopher Reinkemeier:  So, the point why we do this inside of a cell is so that we can study the protein in its natural environment. So worth the technique before you can already easily purify a protein and then, have the protein you modify. But this isn’t just purified and you can’t do In Vitro experiments with it. But if we really want to understand a protein in its natural context, what it’s actually doing, it’s always good when you can do this inside the cell or inside an organism. So, therefore, it’s better when you can do this incorporation to eukaryotic cells, If you want to study eukaryotic protein,

Richard Jacobs:  Well this wouldn’t be a solution, this wouldn’t be a therapy where, if somehow gets a synthetic organelle into all the cells of the body or all the cells of a specific tissue. Or is that one of the goals? Just not now.

Christopher Reinkemeier:  Well, I think that’s a bit very much preliminary requests. Because what we are doing is at a very much of the beginning of what you can do with this technology for the Janko expansion. So we only use it to expand this for generic or to use it as a serological tool. I guess that would be still if you thinking about medical applications, that would be still a lot of more development be necessary to do that.

Richard Jacobs:  Okay. So, you’ve run this experiment and has worked and what have you observed from running it and so?

Christopher Reinkemeier:  So what we observed is when we actually combined these two components, one is the phase separating component and one is microtubule motor protein, which moves along microtubules that we actually are able to, inside cell, form these big synthetic organelles and that these synthetic organelles can still perform the very complex task of translating a protein. And this, also with the expansionary quote so that you basically then have cellular housekeeping proteins are translated with the normal genetic code of the 20 million assets and only your protein of interest is basically translated with a second genetic code which has then non-canonical amino acid on top and can then be specifically modified. And this, to our surprise, works actually surprisingly well. So we get between around like 10 fold selectivity in our organelle, always membrane-less and can still exchange with the surrounding cytoplasm and we still get relatively high efficiency and selectivity for the protein we want to produce. And this works for a bunch of different proteins.

Richard Jacobs:  And how do you get this stuff into the cell? You have to inject it or you found a way for the cell to naturally engulf it and take it in?

Christopher Reinkemeier:  So, we work with tissue culture cell lines and we just use a transient transfection with a plasma DNA and then the cells produce to proteins which formed the organelle itself. And also that’s all the other steps. So with transient transfection, you just put it with your classmate and then you put a chemical reagent on this and then it’s just taking up by the cells.

Richard Jacobs:  Interesting. So, it’s taken up by the cells, you observe it goes through the cell-membrane, migrate to somewhere inside the cell and then start working?

Christopher Reinkemeier:  Yeah. So because we just put a plasma DNA, it’s then transcribed and translated by to cell and the cell makes all the proteins which we need to form the organelles. So this, in molecular biology is a relatively standard tool to do these transient transfections where you just, basically express a protein inside of mammalian cells using a plasmid for this.

Richard Jacobs:  Okay. So, again what have you observed by observing this functionality inside the cell? Anything different from what you expected or is there really no difference?

Christopher Reinkemeier:  Well in terms of because our experiences are usually relatively short term, so we didn’t see something like toxicity or unhealthy effects on the cells so far. For this, you would need probably like the longest stable expression of this. But this is difficult to achieve. So, in our short term of the experiment, the cells look healthy and we are able to do this selective expression of the protein we want to express.

Richard Jacobs:  Okay. So what would be the next stage in these experiments? Now that you’ve done this, you know, where do you go from here?

Christopher Reinkemeier:  So, the next stage is to actually thoroughly optimize this. So this is not the first step where we showed that we can get the selective expression, but what we use so far is, for example, we use, big phase separating proteins which are also naturally occurring in cells. And they involve probably when you express them for long term, they might not be that healthy because they are also involved in some new general rate of disease like ALS for example. So what we try now is to minimize our phase operating systems so that this just really develops into a really healthy like, organelle structure in the cell so that ideally at some point, you can just use it as a, add on organelle, which is completely healthy for the cell and you can just use this organelle to express whatever protein you want with a second genetic code so that you get special proteins produced in your cell.

Richard Jacobs:  Okay. Has this given you an understanding of how the other organelles inside the cell work or any insights into how, you know, DNA is transcribed or how proteins are made or what are some ancillary learnings that you’ve gotten that maybe you didn’t anticipate?

Christopher Reinkemeier:  I think definitely for the phase separation field this is one of the first papers which showed us that you can actually design an organelle which can do a really complex task. So when you imagine translation, this is for your cells, one of the most complex things they have to do. So you need hundreds of different molecules to work together to actually produce a protein which includes different tRNAs, derived with Jones, a bunch of translation factors and if you can synthetically put this into an organelle, which then does something more with this translation machinery. It was actually for us, quite surprising because when you think of this, we just put five components into the cell. We get this organelle and then, we can actually really recruit all the other components to make a fully functional protein bio-molecule okay and so honestly this organelle, we now use this for clinical expansion, but conceptually, the principle that you can in membrane-less compartment, concentrate factors can also be used for lot of other stuff, which you can think of like where you maybe want to treat on as differently or you want to perform asymmetric reactions more efficiently that can all potentially benefit from designing a synthetic organ where you can then have the synthetic like reaction reactor in your cell to perform certain tasks you’re interested in more efficiently.

Richard Jacobs:  Okay. Very interesting. So, what’s ahead for the next six months or a year with the work or is it going to be years before you’re really at a point where you think you understand what’s going on inside the cell a lot more?

Christopher Reinkemeier:  I guess for this, at the moment, as I already mentioned, we are surprised that this work with simple components actually works out well. So, we are obviously working on improving this fervor to get really the most healthy system we can think of and also, which would obviously be ultimately fast and very good as when we were at this can get a really good, cell language can dust this all the time and not only when you transient transfect to make this stable. But this is a term at the moment still turns out to be not working that well. So this is something we need to work on. So that we can have the cell language, can do this orthogonal translation permanently.

Richard Jacobs:  Oh, so your goal would be to have a permanent structure inside of, okay. Well, I’m assuming what your goals. What’s your big ambitious goal for doing this? What do you hope that would happen? What would be ideal?

Christopher Reinkemeier:  So what I would hope what would happen would be that this organelle which we have at the moment can be long-term stable in the cell. Constantly this organized translating organelle so that you have one space into cell where you can basically operate a second genetic code, which is distinct from the normal genetic code because your cell can still perform everything with the normal genetic code, which it needs to survive and to be happy and you have just added an organelle, where you can then, produce special proteins with special functionalities so that you can modify your protein however you want to.

Richard Jacobs:  Okay. Very good. So what’s the best way for people to learn more and to get in touch with the questions or you know, to request papers or submit papers, etc.? How do they interact with you?

Christopher Reinkemeier:  So the best way to reach us is the web-page of my group, the group which I’m in switches at the Lemke Labs. So you can Google Lemke Labs in Heidelberg or go over to the website of the institute, which is EMBL Heidelberg or embl.de and you can go over research, you can then find the Lemke Lab and that’s the best way to get in touch with us and also find our recent publications to this.

Richard Jacobs:  Very good. Well, I appreciate you coming on the podcast. Thanks for talking about this. It’s been crazy avant-garde stuff. Very, very interesting.

Christopher Reinkemeier:  Thanks a lot. You’re very interesting.