Engineering Life for Long Term Space Missions

Stories can inspire people to imagine and create the future. In fact, a lot of our predictions about the technology of the future come from science fiction stories. Take for example the mission to the moon in the novel “From the Earth to the Moon” published in 1865. Jules Verne tells the story of how the US sends 3 men to the moon, in a spaceship named Columbiad, launched in a rocket that weighs 9,000 kg at the cost of 12.1 bn dollars. 104 years later, in 1969 the US in fact sent 3 men to the moon in a spaceship named Columbia, weighing 11,000 kg, at the cost of 14.4 bn dollars. The list of science fiction inspiring today’s technology is long and although not all come true (sorry, still no lightsabers…), many of them are really accurate.

Stories can inspire people to imagine and create the future

One of the most popular subjects for science fiction novels are missions to and colonization of Mars. As a human species we have been setting our eyes on Mars for a long time. Many projects are currently under development. For example, Mars Direct is a project with the goal of starting a human colony on Mars within the next decade (1). In the same manner, NASA aims to have technologies to land humans on Mars by 2030 (2). In the private sector, Elon Musk, the CEO of Space Exploration Technologies Corporation (SpaceX) has targeted 2026 for a Mars landing with plans to establish permanent colonies(3). Maybe  one day the first generation of humans will contemplate a blue sunset on Mars, but before that could happen we have a long way to go. Because, the farther you go into space, the less you can depend on Earth.

Continue reading Engineering Life for Long Term Space Missions

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Meet the Fellow: ADAM MOL

Today we hear from Adam Mol, who is working in Beatrix Suess’ lab at TU Darmstadt, Germany!

What is your background in science (and otherwise)? What led you to decide to do a PhD?
I completed my studies in the field of Biotechnology at the University of Silesia, Katowice, Poland. My master project was carried out in the Department of Genetics where I was working in plant genetics. This research was related to drought resistance in cereal crops. My work there allows me expand my knowledge about molecular biology and piqued my interest of Science. This experience gave me a great opportunity to enter into real scientific life for what I would like to thank my advisors Prof. Mirosław Małuszynski and Dr. Agata Daszkowska-Golec.

Next as a graduate student I have been continuing my scientific carrier as a member of Vilardell’s lab at the Molecular Biology Institute of Barcelona (IBMB), Barcelona, Spain. I have been investigating research related to alternative splicing in human cells. My work at IBMB was very important step in my scientific carrier which gives me opportunity to improve both my theoretical and experimental knowledge about biomedicine.

And now I am convinced that I would like to continue my scientific interests as a PhD student.

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What does your lab do? What is a brief description of the project you work on?
Currently I do my doctoral studies at Synthetic RNA Biology group headed by Prof. Beatrix Suess at Technical University of Darmstadt, Darmstadt, Germany. A main focus of our research is the development of active aptamers. The group has very good background in in vitro selection of aptamers as well good establish in vivo screening system. Also we try to find application of aptamers as synthetic riboswitches. Second focus in our group is related to disease. We work with natural regulatory RNA: siRNA in bacteria, microRNA in inflammations as well with alternative splicing in hypoxia.

Continue reading Meet the Fellow: ADAM MOL

Antibiotic Resistance and Metabolism

This week, we hear from Leonie about the relationship between a very important global health problem – antibiotic resistance – and research on metabolism.

According to the Centre for Disease Control and Prevention at least 23,000 people die each year in the United States due to infections from antibiotic resistant bacteria. But the problem is not limited to the US and occurs in all parts of the world. The World Health Organization calls antibiotic resistance a major global health threat. Therefore, limiting or reversing antibiotic resistance evolution as well as the development of new antibiotics is crucial to guarantee treatment of bacterial infections also in future.

But hey – why are we talking about antibiotic resistant on our metaRNA blog?

One strategy to deal with the emerging health threat is to understand the resistance mechanisms in greater detail.  87% of the metabolites in a bacterial cell change their abundance when exposed to antibiotics. Some changes seem to be dependent on the antibiotic class whereas others appear to be general metabolic changes caused by antibiotics.

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Taken from “How to Fight Back Against Antibiotic Resistance” by Gautam Dantas and Morten Sommer in  American Scientist.

So far metabolomics hasn’t played a big role in the antibiotic resistance research field, even though there is evidence that not only metabolic profiles change during antibiotic treatment but also resistance-conferring mutations directly affect the bacterial metabolism. For example the amino acid metabolism or lipopolysaccharide synthesis can be affected by the development of antibiotic resistance. Other mutations were shown to affect the choice or ability to process different carbon or nitrogen sources.

The analysis of resistant mutations in regard of metabolic fluxes offers consequently a great potential to understand resistance mechanisms and what exactly happens in resistant cells. A detailed understanding of these processes might help to open up avenues of potential new treatment strategies or the development of new drugs. Another link between resistance and metabolism are reversible phenotypic adaptations of bacteria to antibiotics that are not manifested in the genome. These adaptations can result in temporarily highly resistant bacteria, called persisters. This phenotypic adaptation is also an important piece to understand the puzzle of antibiotic resistance and is consequently an interesting target of research.

Meet the Fellow: LAURA LLEDO

Laura is our Meet the Fellow this week! She works with robotized SELEX.

I am collaborating with the Life & Medical Sciences (LIMES) Institute for my PhD. This Institute is part of Bonn University, in Germany. The LIMES Institute is an internationally oriented center for biomedical research. The main scientific focus of the institute is to explore the regulation of lipid metabolism and the immune system in health and disease, and decipher the signaling processes that take place both within and on biomembranes. I am working in Günter Mayer’s lab, focused on aptamer selection by Systematic Evolution of Ligands by Exponential Enrichment (SELEX link to post). We are many international scientists with different backgrounds in our working group. Our work is connected, helping each other as a team so we can reach our particular goals leading to the final selection of great aptamers.

Aptamers are short and stable nucleotide molecules that bind with high specificity and affinity to a certain target. They have plenty useful applications like diagnosis, drug delivery, therapeutic and bio-imaging. The SELEX procedure is very time consuming as each selection cycle consists of multiple steps and to select a good aptamer, 5 to 15 selection cycles are necessary.

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Continue reading Meet the Fellow: LAURA LLEDO

On SELEX and Sausages

This week, we hear from Adrien who expands our knowledge of aptamers by explaining an important technique, called SELEX, which is used to create specific aptamers in the lab.

Recently, we published a post about the wonderful qualities of aptamers, but we left you with important questions, like how are they formed? How can we identify one single sequence in a pool which contains millions of billions of different sequences? The answer to that question in one word is: SELEX, meaning: Systematic Evolution of Ligand by EXponential enrichment (Yeah, I know, it is more than one word…). This technique is a screening method that has never stopped evolving for the last two decades. The original paper on SELEX is from Larry Gold and Craig Tuerk, written in 1990, where they isolated a high affinity nucleic acid ligand that interacted with the T4 DNA polymerase. Nowadays, there are tens of different SELEX techniques which are derived from this original one and each technique has a different application.

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Fig 1. Different SELEX techniques. From RNA Aptamer Evolution: Two decades of SELEction. G. Aquino-Jarquin & J. Toscano-Garibay. Int. J. Mol. Sci. 2011.

Here is a list of the different evolutions in SELEX techniques 20 years after the original publication. The evolution in the technique can be Setting the ground, Improving the libraries, Entering in the cell environment, Regulation and detection and Updating SELEX with modern Technologies

Continue reading On SELEX and Sausages

Meet the Fellow: IGNAZIO GERACI

Today, we meet Ignazio Geraci, a Fellow who designs riboswitches for a living!

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What does your lab do? What is a brief description of the project you work on?

If you would ask my PI, we basically are tool-makers. Need to thump? We make an hammer. Need to look closer? We make the loupe. Need to detect real-time intracellular metabolite fluctuation in single cells? Well my friend, our answer is aptamers.

Aptamers are a special class of nucleic acid molecules that can blabla… You can google what they are (or look at this recent post!). For us, these tiny little fellas are like highly-specialized workmen. They do one job, but they do it flawlessly. And of course they do, they have been specifically selected for it!

The process of selecting these molecule, called SELEX, enables us to identify specific aptamers starting from 1015 different sequences. If you were wondering how big this number is… it would cover the distance between the Earth and the Sun. In micrometers. Understanding that, you can get a glimpse of how many sequences, and possible diverse structures, we are working with. What I will be trying to do in my PhD is to implement aptamer section in order to sort for other attributes in addition to target specificity. It will be demanding, it will be challenging, but I am positive I will get the best out of it, also due to the enthusiastic research group I am part of. In any case, wish me luck!

Continue reading Meet the Fellow: IGNAZIO GERACI

If you don’t like the game, change the rules: An insight into directed evolution

This week, we hear again from Eduardo on how we can use the phenomenon of evolution to our advantage (in a lab setting!) to create specific cellular behaviours that we are interested in.

Evolution is a widely accepted scientific fact. It posits that the reason we are here today, the reason the living world is the way it is, is evolution. All life on Earth started off at the same point, with certain molecules that got more and more complex until the molecules essential for life came to be (For more on these first molecules and their impact nowadays, check our previous article on riboswitches). These molecules eventually got together and assembled into the first cell. And from there, they just kept going, mutating and diversifying.

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Fig 1. Tree of Life, showing current biodiversity and how it originated from the same starting species. Taken from http://www.evogeneao.com.

There are a few inherent characteristics to the process of evolution, such as:
A)   Randomness: as mutations don’t have a particular focus of their own, and just become selected depending on the conditions in which they arise
B)   Large timescales: The timescale needed for these mutations to actually make a functional difference in the population of organisms is quite long.

Continue reading If you don’t like the game, change the rules: An insight into directed evolution

Meet the Fellows: LEONIE JAHN

Let’s meet Leonie Jahn, who works in Dr Morten Sommer’s group on metabolism and antibiotic resistance!

 

The group I am doing my PhD in is quite diverse in its research. We try to contribute to small step solutions for global problems such as climate change or antibiotic resistant bacteria. We do so by finding sustainable alternatives for the production of certain chemicals or by studying the evolutionary mechanisms leading to antibiotic resistance.

 

I love the diversity of activities that come along with research

 

My project falls in the field of antibiotic resistant bacteria. I had no doubt to start a PhD since I love the diversity of activities that come along with research: the critical thinking that result in a lot of unsolved questions, the creative part of designing experiments to answer those questions, doing practical work in the laboratory (sometimes mindless but meditative, sometimes frustrating but challenging and often fun), analyzing data, interpreting and visualizing results as well as communicating them in presentations or publications. In addition, I like to work in an international environment, where it is normal to talk to people from almost all continents every day. In my free time I like reading, documentaries or podcasts to all sorts of topics (psychology, physics, philosophy, politics, literature, art, history etc), yoga and spending a lot of time with family and friends.

ANCIENT WISDOM: What are riboswitches?

Riboswitches are at the heart of many RNA-based technologies and they are the link between SELEX-based aptamer development and applications inside the cell. Today, three Fellows of our network answer questions about riboswitches: Sara tells us where they came from, Adrien explains how we can use them, and Eduardo forecasts the future of riboswitch design.

 

Why did riboswitches develop? How do they fit into the story of evolution?

SARA: “Adaptation to surroundings is essential for all living organisms, from the smallest bacteria to us. There are many ways a cell can sense the environment, most of which include complex pathways that coordinate like the best orchestra to allow accurate and efficient responses.

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“The term “RNA world” refers to the hypothesis that self-replicating ribonucleic acid molecules (RNA) are the precursors of all current life forms on Earth. Imagine, for a moment, a living cell with no proteins, no lipids…and no DNA (deoxyribonucleic acid). How could it sense the environment and, more importantly, adapt to it? The answer may lie in the RNA molecule.

“There are special RNA molecules able to act as direct sensors of both external and internal changes, with no need of proteins or cofactors! These molecules, so-called riboswitches (RIBOnucleic acid SWITCH) are present in the vast majority of bacteria species where they serve as sensors for  vast range of ligands (amino acids, anions, metals, purines, cofactors…).

“Some riboswitch classes, particularly those that sense molecules like TPP (thiamine pyrophosphate; a eukaryote-specific riboswitch), AdoCbl (adenosylcobalamin), and FMN (flavin mononucleotide) are exceptionally widespread (see figure, Barrick and Breaker 2007). These riboswitch ligands are thought to be molecular relics from an RNA World. Furthermore, the most widespread riboswitch classes have some of the most complex aptamer structures. These information-rich structures are unlikely to have emerged during late evolution. Rather, widespread complex riboswitches most likely have an early and perhaps RNA World origin.

“The main function of riboswitches is to modulate gene expression (and thereby adapt to environmental changes) by ligand-induced conformational changes using a wide range of means, in many cases organism-specific and even species-specific.“

 

How can the complexity and variation of natural riboswitches be used in synthetic systems?

ADRIEN: “Based on the principles of riboswitch regulation, a versatile set of riboswitches have been engineered. By combining RNA-based sensing domains with regulatory domains, a riboswitches can serve as a synthetic RNA regulator with multiple properties. Many tools have been developed exploiting these riboswitch properties for systems with conditional gene expression.   

“Most mechanisms of engineered riboswitches used in bacteria involve either the Shine-Dalgarno sequence (a conserved nucleotide sequence where the ribosome can bind and produce the protein encoded in the gene upstream), or the terminator (a sequence which have a specific folding pattern which physically stops the RNA polymerase).

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(A), example of regulation of gene expression with the Shine-Dalgarno (SD) sequence. In absence of ligand (orange pentagon), the aptamer part of the riboswitch (blue) is not folded. This leaves the SD sequence (green) accessible for the small ribosomal subunit (30S) and the translation occurs.

(B) Regulation of transcription termination. The aptamer domain (blue) is fused to a spacer (grey) and followed by a sequence complementary to the 3’ end of the aptamer (red). In absence of ligand, the complementary sequence binds to the aptamer and forms a terminator hairpin, which stops the RNA polymerase (RNAP). Upon ligand binding, the aptamer is formed but the terminator hairpin is not, allowing the transcription and subsequent translation of the gene. Both of these riboswitches were constructed using the synthetic theophylline aptamer. (Groher & Suess, Bioch.and Bioph. acta 2014)

“Riboswitches are also used in some higher organisms like yeast, the eukaryotic “model organism”. There are many ways to regulate gene expression in yeast with riboswitches. Some examples include control of initiating translation (the process by which proteins are created) or ribosomal shunting through allosterically controlled ribozymes, which is another mechanism for translation initiation. My (and also Adam´s) favourite method of regulation is control by splicing. To make a long story short, splicing is the step which eliminates non-coding sections of an RNA transcript (introns), of the pre-mRNA, transforming the pre-mRNA into mRNA. If the intron is not eliminated, the ribosome will not produce the right protein. This is further explained in the figure below.

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“In order to regulate gene expression by splicing, some riboswitches are positioned within introns. To remove the intron, the spliceosome need to recognize specific sequences like the 5’ splice site (5’SS), the Branch point (BP) and the 3’ splice site (3’SS). If the aptamer part folds (blue in the figure below), in presence of its ligand (orange pentagon in the figure below) hiding these specific sequences, the spliceosome (U1 in the figure below) will not remove the intron and the gene will not be expressed.”

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What is the future of riboswitch-based technology? Is it easy to build and use them?

Eduardo:Based on our continuously growing knowledge on RNA biology, the development of artificial riboswitches has become a reality. However, there are several problems to take into account when doing so. The design of artificial riboswitches is based, most of the time, on its sequence similarities to known riboswitches and their predicted physicochemical characteristics. The sequence is responsible for the spatial structure the riboswitch will take, thus affecting the way it binds to its ligand, and what happens after that. That is why, apart from sequence, physicochemical properties must be taken into account when designing artificial riboswitches.

“A riboswitch structure is useless if it cannot overcome a certain energy threshold when binding its ligand (which can be any small proteins to sRNA). By overcoming this energy threshold, it is allowed  to change its conformation and then regulate gene expression. There are even cases in which the objectively best – that is as modeled by chemistry data – energy values needed for the riboswitch to change its conformation may not actually be the ones that are selected. So it is important to keep that in mind, and to refine our searching and developmental methodologies.

“Based on all these facts, riboswitches have shown that they are a very important tool for synthetic biology research, and a very exciting field to work in. You will hear more about them and our work in future posts.”