Author: Marco

Marco G. Casteleijn grew up in a small town in the Netherland, and a good way to escape was to read. He has been writing short stories and poetry since high school and continues to do so. In addition, Marco has published several academic peer reviewed articles in respectable journals and is currently working on his lyric sequence “My Opaque Dreams" and a SciFi novel. Marco is currently working as a Senior Researcher at VTT in Finland where he resides with his heroic wife and three magical children.

Biotechnology and recyclability/circularity through Helsinki fashion week

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I had the lovely opportunity to discuss with Olivia Rubens, a positive knitwear women’s wear designer, super interesting topics such as sustainability and fashion. These topics within the realm of biotechnology, synthetic biology, the end-of-life of our clothes, and side-streams are at the core of our beliefs. We also discussed human & climate positive centered garments and microbes,  creating new circular systems and new materials from glucose with fungi, and enzymes that eat plastics.

(c) Olivia Rubens and Marco G. Casteleijn. 2020.

 

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Open letter to the Fashion Industry

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We are flooded with difficult choices every day. We are witnessing the fastest changes humanity has ever seen, and it is both wonderful and at times a bit scary. Yet happiness follows when we choose to do meaningful things.

We have the knowledge and capability to make almost anything, but what we choose to do now is more crucial than ever before.

In a world of decreasing resources, there really are no materials to waste. New ways to produce, manufacture, and consume are needed very, very soon.

There is a potential to make life sustainable for future generations and to create business opportunities for businesses today. Therefore, in order to combat climate change and save global resources, the production of materials needs to quickly move from virginal fossil raw materials to renewable or recyclable feedstocks.

From making proteins to the textile industry seems like a big leap, but surprisingly it is not. We are at a pivotal point to move away from non-sustainable materials to replace them with biobased materials. We are harnessing the power of microbes and fungi to create protein-based fibers, such as spider silk, or vegan bio-leather made from fungal mycelium. We are discovering new enzymes that can break down mixed textile materials, even plastics, into smaller molecules, which microbes use as food to make new materials. Biobased coatings, dyes, colors, biodegradable biobased plastics, and novel functionalities are being developed at an increasing rate.

More recently, “sustainable fashion” is an often-heard buzzword, but is the industry looking at real solutions to enter the much needed new models? For example, when creating clothing from leftover fabrics, are the textiles and garments designed for recyclability? What about the dyes and coatings used? How can we eliminate microplastic formation during washing completely? Can we produce buttons and zippers that are both sustainable to produce as well as easy to recycle? Can we make all clothing without the use of fields and animals? Should we?

Here the role of consumers also plays a key role. There will be a great need for personalized design in the future, a trend we can already observe in many industries. There is also a greater need for transparency. When AI and computational driven design of materials can design-test-build on the fly in-silico before the material is physically produced, novel materials will come to the market at an increasingly rapid pace. Before they do though, designers must ask from the material scientists: are there solutions to recycle these materials indefinitely. Is the material I use a virgin material or not? How long can people wear my clothes and how will it be recycled? How can I educate my customer and how can I play my part?

We can only get big answers if we ask big questions. Clearly we are at a remarkable point in our history to understand the big problems of our time, but if we do not solve our linear consumption behavior now, our amazing ways will soon find a planet who is no longer willing to sustain us.

~ Marco Casteleijn

Senior Scientist of VTT Technical Research Centre of Finland

Please join our “Waste to Wear” concept: http://wave.fi
or contact me: marco.casteleijn@vtt.fi for inquiries for biobased materials and enzymes for textile recycling.

 

Finding the best proteins

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There is a beautiful lesson to be learned from the Gossamer Condor[1]where the team of Paul MacCready succeeded where others have failed: to keep a, by man-powered aircraft in the air long enough to draw the infinity sign between two markers one half mile apart, starting and ending the course at least three meters above the ground. They did so on the 23rdof August, 1977.

Paul MacCready sought out to do “do more with less”, however the lesson I learned from this engineering achievement was: “fail fast to succeed faster”. Where other big companies, like Boeing failed, they succeeded. Paul MacCready’s team used cheap and simple materials and techniques so they could build 2 or 3 different configurations a day. They could sample what works and, more importantly, what does not work in a rapid way. Thus, when I start a lecture on making proteins, people look at first confused when I ask them if they have heard of the Kramer prize and its first winner.

So, what does it all have to with making and finding proteins? The process to produce recombinant proteins by use of living cells, currently the state-of-the art especially for the pharmaceutical industry[2], is a lengthy one. For some recombinantly produced antibodies there could be over 1000 different steps. At each of the steps the protein is exposed to different environments, which may interfere with its natural state.

In nature proteins are important, because within the cell and the body they provide structure, perform chemical reactions and pass on information. For example, collagen in connective tissue, α-amylase breaks down long-chain carbohydrates during digestion, or dopamine receptors in the brain. Within cells, many processes are put in place to make the right proteins at the right time, while folding them correctly into their native and active 3D-structures. However, by interfering with these natural processes and tricking cells to make more of the protein we would like to have, for example recombinantly produced human insulin[3], cells may not fold the protein of interest correctly. Misfolded proteins are not active, can induce adverse effects if administered as drugs, and if misfolded protein stick together they form insoluble complexes called aggregates, with no functionality and even greater adverse effects.

Therefore, when we start making small changes to proteins to see if they have an improved functionality compared to its original design, we need to test for many parameters, but functionality and protein stability are crucial. Before we make changes to our proteins, which may have the potential to become safe and functional drugs, we need to make sure our processes are designed well and that our means to evaluate the protein is sound. In the current landscape of protein preparation, the use of cells (i.e. biotechnology) is employed the most. However, one can argue that it takes long to make some protein with many different variations in cells: from weeks to months. How can we fail “faster to succeed faster” to evaluate more proteins?

One disruptive technology which has started to show its promise in an industrial setting[4]is the use of the cellular machinery that makes the proteins without its envelope. Proteins are made by other proteins, enzymes, who transcribe DNA into RNA and translate RNA into an amino acid sequence, a strand of the building blocks of a protein, that then folds into its 3D-structure. This technology is referred to as in-vitro transcription translation (IVTT) or cell free protein synthesis (CFPS)[5]. In short, a cell is cracked open like an egg, and its content is split in a soluble fraction and an in-soluble fraction. Part of the soluble fraction contains all the enzymes needed to make proteins, this part is isolated, often concentrated and then mixed with amino-acids, enzymes to provide energy, and other components needed to synthesize protein. RNA induces the process of protein synthesis, which is often transcribe from DNA within the mixture. Protein production in such an open system is short. Depending on the lysate used the expression can be between 1 – 16 hours, while the process is not designed to keep the cells alive and dividing at a set rate, but on protein synthesis.

Another powerful feature is that many different cell lysates can be used, for example rabbit reticulocyte, E. coli, mammalian cells, such as CHO or HeLa cells, L. tarentolae,tobacco plants, and many others.

The promise of recombinant protein preparation from GMO animals failed in the past; one main reason being the turn-over rate between preparation, purification and testing was very long. One can argue that the turn-over rates of cells are too long as well. In a market where the life-cycles of pharmaceutical products are getting shorter, while the overall volume is growing, more safe and well-designed protein-based drugs are needed. In this landscape CFPS has the potential to act as a valuable tool in the same way mylar foil was crucial for the Gossamer Condor’s success.

 

Marco G. Casteleijn © 2018

Publish first on BrighOwl blog: https://brightowl.pro/blogs/2018/06/finding-the-best-proteins/

[1]https://en.wikipedia.org/wiki/MacCready_Gossamer_Condor#cite_note-1

[2]Casteleijn M.G. and Richardson D. (2014) Engineering Cells and Proteins – creating pharmaceuticals. European Pharmaceutical Review, 19(4): 12-19

[3]https://www.dnalc.org/view/15255-Producing-human-insulin-using-recombinant-DNA-Walter-Gilbert.html

[4]https://en.wikipedia.org/wiki/Sutro_Biopharma

[5]Marco G. Casteleijn, Arto Urtti, Sanjay Sarkhel. 2013. Expression without boundaries: Cell-free protein synthesis in pharmaceutical research. Int J Pharmaceut, 440 (1): 39-47

 

Preparing recombinant protein: Tips to improve growth rate and yield

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I have been in the distinctive business of making proteins, looking at proteins, or finding proteins that have special properties. I mostly try to make them myself, and over the years I’ve had lots of success—and many failures. It’s part of research. We all know, from DNA we get transcribed RNA, which is translated into a strand of amino acids, which by forces of physics fold into a functional protein—sometimes with some help from chaperones. However, it does not always work. Even in a living organism with high adaptability, success isn’t guaranteed. Aggregation is a waste of energy for the organism, and now it needs time and energy to recycle the materials.

In industry, about 40 percent of all pharmaceutical proteins are made with recombinant protein production in bacterial strains, while the remainder is mostly by use of mammalian cells. In the lab, we use E. coli the most, simply because many tools are available—from cloning vectors to E. coli strains from DNA plasmid production, to cloning kits, simple transformation procedures, and plasmids designed for high yields of recombinant protein production. Many different media and fermentation procedures are designed by scientists and manufactures to increase yields of correctly folded protein. It all seems so simple, but it’s not.

Even a condensed, incomplete list of tools illustrates that we need to make many choices to find the right parameters. For now, I will skip most tools and focus only on protein yield. In the lab, we need just a little bit of protein, but as a bioprocess engineer, I always need to consider the choice of the expression system on the available means of the laboratory or production facility, modification needs of the protein produced, and compatibility of the gene control system with the bioprocess for production later on.

In terms of protein yield, either as yield per cell or total yield, it is important to remember that the product formation must be determined experimentally, and may either be growth associated or non-growth associated. The specific product rate formation, qp (kg kg h-1), is given by qp = qs Yp/s, where qs (kg kg-1 h-1) is the specific substrate rate and Yp/s (kg kg-1) is the yield coefficient. The specific product rate formation can also be expressed in terms of growth association or non-growth association by means of the Luedeking-Piret model: qp = αµ + β. Here, µ is the specific growth rate, β = 0 gives a complete growth association, and α = 0 gives a complete non-growth association. Protein product formation is mainly growth associated, at least in wild type cells. Low growth rate, for example in E. coli, may hinder product formation due to the maintenance cost of the cell, while fast product formations may hinder correct folding of proteins. These problems are related to protein production in living cells, but less so in cell-free expression systems.

Important parameters to consider are again plenty. Are we using a simple shake flask or do we have access to a fermenter? In either case, we still have to choose if we grow the cells in a batch phase or use a fed-batch culture, i.e. if we grow the cells following sigmoidal growth curve or a linear growth curve with a fixed number for µ. Batch phase in shake flasks is very common because it is cheap, however it has its own unique problems. For example, using LB medium would be a poor choice for long expressions. Cell densities are often low (OD600 < 6) due to acidification of the medium, while the pH in Terrific Broth (TB) can go up above a pH of 8.5, indicating high ammonia production due to utilization of amino acids as carbon source. The growth rate of E. coli is dramatically reduced under a pH of 5, and thus will not produce recombinant protein—it will start recycling it for its own maintenance. The utilization of amino acids as a carbon source will also lower the amount of recombinant protein.

The design of the shake flask (i.e. baffled or round), the size of the opening, and the choice of the cover are equally important, since E. coli can only grow and make recombinant protein if the oxygen transfer rate is as high as possible. Switching from an aluminium cover to very porous paper can increase the final biomass manyfold.

In conclusion, aside from the choice of DNA vector, E. coli expression strain, growth temperature, inducing agent and amount, and co-expression of chaperones, parameters to control the growth rate are equally important. Growth media, a good oxygen transfer, and a good buffering system have proven in my work to be important elements to obtain high yields of correctly folded protein.

(c) Marco Casteleijn. 2017

 

“Preparing recombinant protein: Tips to improve growth rate and yield” was published originally on the 23rd of May, 2017, in the ‘The Q’ . Republished with permission.

Embrace the other tribe

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eu-broken1

“I do solemnly swear (or affirm) that I will faithfully execute the office of President of the United States, and will to the best of my ability, preserve, protect and defend the Constitution of the United States.”

In our lifetime we have seen many upheavals in various countries around the world and many horrible atrocities committed against humanity. We are more and more connected to the flow of that news, yet more and more desensitised at the same time. Our sense of global humanity is an interesting construct, since we are hard-wired to live in smaller tribes and be empathic to familiar people. We can only really store a certain amount of people in our brain (1), beyond that realm we encounter “them” as another tribe.

While researching the tribe versus globalisation, I came across this opinion piece by  (2), and find truth in these lines regarding current events:

Important traumatic events have plunged people all over the globe into an identity crisis. Their response is tribalisation: going back to the tribe they know best.

and while this may give solace to ‘why is this happening?’, it does not give us a sense of security and stability.

This blog is mostly about writing and sharing our cooking project. Yet our collective identity crisis affects also me and our family. Our daughter flipping of the president elect during his press conference is her response to our insecurity about the future. I would like to stress here to be honest: firstly our family’s future, then the broader spread of humanity.

As a researcher I am depended on a favourable political landscape towards (my kind of) science to obtain funding. Without funding I will not be employed, since my salaries are depended on external funding. The EU has been a driving power in providing funding strategies beyond my current tribe in Finland. The political landscape in Finland to fund science has always been good, but resent years this is on the decline (3). This trend we also see in the USA, a frontrunner for scientific endeavours for many decades.

Now it seems we enter a new phase, something I am writing about in my very slowly progressing SciFi novel-thingy. At this rate it seems that it may even be fiction before I can get it published. A new world power is emerging, with another one waiting in the wings. Of course I am referring to China and India, the Dragon and the Elephant. Two countries that look at each other with suspicion, but with a clear understanding of the power of investments in new technologies and creating legislations that open a freedom of operation within their own tribes.

But a citizen of the World I am wary of the near future. The USA has through previous technical and scientific developments and acquisitions build up a wealth and power that influences the rest of the population with a literary unbalanced force. In the last election for example about 40% of the USA (which holds only 5% of the world population) decided to put a person in place with very limited views of the World. We can all argue about the values that that Tribe he represents and we can all argue about it as well, but for our global perspective, it is a step in the wrong direction. Scientific facts do not care about public opinion. Gravitational force is still here wether you deny it or not. Same about evolution, climate change, gender issues, GMOs, vaccinations, and the power of innovation.

Our future may be uncertain, I think our ancestors shared our concerns. They looked at the stars, they conversed with the bones of their ancestors, they invented religion to strengthen the bonds of the tribe in order to survive better. Will a collective connection directly to everybody else aid us? Do we need to enhance our brain (via science mind you) so that Dunbar’s number becomes infinite? Will an encounter with an off-Worldly tribe spark the birth of the Human Tribe? Many stories have been written about this.

So my take on it is that humanity is a collection of stories, and that the narrative of those stories is healing. Some people like poetry, others read horror stories, but at the end of the day we can tell each other stories about that one poem of inspiration or the scary bits of a horror story without forcing the other to read it. To find common ground, to revel in being human, to talk and listen and embrace the other tribe…

 

Sources:

(1) https://en.wikipedia.org/wiki/Dunbar’s_number

(2) https://euobserver.com/opinion/131413

(3) https://www.timeshighereducation.com/news/finland-funding-cuts-catastrophe-research

 

IMAGE CREDITS: CC / Flickr – Theophilos Papadopoulos

Kitchen lab – Episode #2 Yeah!

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Yes I am writing. Poetry, grants, and another grant (and even a third one), and a lonely short story. I will get to the book thingy soon. But I wanted to share our second episode of Kitchen lab.

Here our family will talk about how to make a tasty bbq also healthy. Please join us on our youtube channel and give it a thumbs up if you like it, comments are more than welcome. Of course I would love some science questions as well!

Kitchen lab update

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In our scientific endeavours at the University we serve three pillars:

  1. Research and pushing that little bubble of knowledge further into the unknown.
  2. Teach students.
  3. Serve society and open the dialogue with the public.

Carl Segan understood that very well and in the first rendition of the popular TV show “the COSMOS” and then the update by Neil Degrass Tyson brought some wonder to the people homes. Science, some teaching and entertainment.

Our little TV show Kitchlab is trying to do the same, and trust me I am not comparing me to the two well know scientist mentioned above, but hey we can all dream.

So after our first episode, soon our second, but just because it is so much fun here is our intro. Freshly pressed.

Our next episode is about how to do a tasty and health bbq. See you soon on our Youtube channel!

 

 

The Mondays

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IRES_RNAfold
“It looks like my story line is going… where?”

It is Monday. I am writing, yes the curse of November may be lifted yet. So what is going on? In the short fiction course I am taking we are required to (finally) write a short story. I am working on it. In the course I am a week behind, it has been busy, but these is writing going on.

This week we are finally looking at editing and I hope to get some insight into that, since it is still an elusive skill to me. The hyperlink story, brought to you by me and Ville where we deliver a story in 100 word chunks (or drabbles) via a challenged title has taken time, since each story is so packed with information or needs to deliver very precise. Also it is a hyperlink story, so each drabble connects to some others, while it may not be clear. This could be strutted easily if there was a plan prior. Yet we do not have. So structure is only applied while and after writing. Indexing took some time, then side drabbles run in parallel now to support other stories. It is becoming more and more fun, but if anybody would ever get something out of it I do not know.

So if you ever have time I invite you to read it if you want (you can find it all in The Cave (see link on top); some discussion or feed-back on hyperlink stories is something I am looking for.

Did you ever write in that format? Is it a rewarding forum? Or is a classic tale with a real beginning, middle and end better?