Team:Marburg/Human Practices

Human Practices

Accessible Science

We have a moral duty to remove the barriers to participation, and to invest sufficient funding and expertise to unlock the vast potential of people with disabilities...
-- Stephen Hawking

Although equality is a universal human right, prejudice and perceived disabilities too often exclude people with special needs from many opportunities. Especially active participation in Natural Science is much disrupted.

We decided to challenge this status quo. To have a lasting impact, and due to the complexity of this topic, we had to focus our efforts on the needs of one group of disabled people in particular: Visually impaired people.

According to the World Health Organization (WHO), 253 million people worldwide live with vision impairments. 217 million have a moderate to severe visual impairment and 36 million are blind. In Germany, 1.2 million blind people lived in 2002. Of these, 600 live in Marburg and 150 of them are students of the Philipps University.

That is a third of all blind students in Germany!

Marburg has taken many steps to make the city more livable for blind people. You can find talking bus stops and elevators, shopping aids in nearly every supermarket and special rooms in the library of the Philipps University. One of the reasons why Marburg has become this hub for blind people is the BLISTA. It is a nationwide competence center for the blind and visually impaired. It established the first high school for visually impaired students worldwide. Nowadays, this Carl-Strehl-School is the only high school in Europe preparing blind students for higher education. Despite these ideal conditions, visually impaired students are only rarely found in fields like biology or chemistry. We intended to show that anyone believing blind people incapable of doing well in these fields is mistaken.

Therefore, the crucial role in our Human Practices project fell towards public engagement and close cooperation with the BLISTA (engl.: German Institute of the Blind) to facilitate equality and accessibility in Science. In the following paragraphs, you can see for yourself.

Self-experiment - Dinner in the Dark

Since Marburg is known as the “capital for blind people,” the bar Caveau is offering a dinner in the dark named “Finstaverne” (engl.: Sinistavern). Here you get the opportunity to experience one evening in total darkness, enjoying some food and drinks with your friends or colleagues. Compared to other such evenings the only difference is the complete absence of visual impressions. The iGEM Marburg 2018 team used this opportunity to get a feel for how everyday situations, like having a dinner, in the absence of visual impressions is affecting us.

In small groups, a waiter guided us into a room without any light sources. The first challenge was to find your place on the table without pushing anyone or anything. Because we all often rely exclusively on our eyes, the first moments without them were unfamiliar to us. Once we were sitting, we started to wonder, how big the room could be, how many people were there and which team members were sitting on the tables next to us. All these questions we tried to answer by acoustic and haptic clues alone.

Then it was time to order food and drinks. Therefore, most of us had to ask more than once what food selection was available. Some of us decided to order something that could be easier to eat without seeing it rather than soup or spaghetti. They thought eating a sandwich could be more manageable. We realized that eating any kind of food was the key challenge of this evening for most members of our team. The ones who ordered sandwiches were wondering how they lost all the toppings while eating.
After several hours, it was time to leave. Here comes the last challenge of this evening. How to pay without knowing how much money you have in your hands? Some Members were well prepared and assorted the money bills in their wallet, beforehand. Most of us did not think about that before entering the dark room and had to guess if they had picked the right bills and coins. Fortunately, the waiters were very kind and trustworthy. Being more experienced than us they were kind enough to help us with the payment.

Team iGEM Marburg late at night in front of the
Team iGEM Marburg late at night after our Dinner in the Dark we head home we new impressions

We had a lot of fun and enjoyed our dinner on that evening. At the same time, it was impressive how you experience basic things like your food and drinks differently, if your only judge is what you feel, smell or taste. Your perception of a bar changes if you cannot see all the people around you and are limited to hear a lot of voices and conversations mixing from different directions.

It was difficult to adapt to this new situation but we all managed to eat, drink and to pay. However, this experience gave us a small insight into challenges for the visually impaired. Although we know that this experience is not equivalent to real everyday situations for people with visual impairment, it still gave us an idea of the challenges some people have overcome day by day.

Interviews: Studying Science with Visual Impairment
Interview with the Dean of Biology Astrid Brandis-Heep

Our hometown Marburg is known as “the capital of blind people” but visually impaired students can only rarely be found in fields like biology or chemistry. What could be the reasons for this? We used the opportunity to talk to Dr. Brandis-Heep, the dean of students in the department of biology.

iGEM Marburg 2018: "Are biology students with visual impairment in our department?"

Dr. Brandis Heep: “From time to time there are students with visual impairment in the department of biology. The practical work in the lab is more challenging for them”

iGEM Marburg 2018: “Are there special lab spaces for students with special needs? How do they manage to do the practical work?”

Dr. Brandis Heep: “If students need special support they can get help from assistants and besides that all our lab courses are done in groups of two or more students. The big advantage is that students can help each other. So both sides learn from each other. This would not be possible if students have to pass the courses alone.”

iGEM Marburg 2018: ”Are there special safety instructions for students with visual impairment?”

Dr. Brandis Heep: “They should be able to follow the safety instructions given to all students who work in the lab. Sometimes special instructions are necessary depending on the grade of visual impairment.”

iGEM Marburg 2018: “Are students with handicap allowed to provide a replacement if they are not able to complete a course?”

Dr. Brandis Heep: “In our department, usually every student is free in choosing the modules he or she wants to take. Students with a handicap will choose lectures and courses in the way they are able to take part. This means not all practical courses may be possible. Sometimes they need an assistant to do the practical work and so they do not need to provide replacements. They always need intensive advices before choosing a module.”

iGEM Marburg 2018: ”Are there special features that must be taken into account during the application procedure for students with visual impairment?"

Dr. Brandis Heep: “The application procedure is the same but in our experience the students get in touch with us before they start as a student at the faculty of biology. We talk about their disability and the possibilities we can offer. This gives us the opportunity for individual arrangements. For example, the lecturer can give them the PowerPoint slides, so they can follow the lecture on their laptops.”

The fact, that some students with visual impairment successfully completed their biology studies has strengthen our motivation to bring science closer to pupils of the Blista School. In a conversation with a student of the Blista School, he told us that he sometimes thinks about breaking up the high school diploma because he thought there was no way for him to study biology. He told us that he is very interested in science and especially in microbiology. However, after we told him about our conversation with Dr. Brandis-Heep and he had worked with us in the lab for one day, he gained new courage, that graduating and finding a job in this field is possible for him. It made us very happy that we were able to show people new ways to realize their dream.
We are very thankful that Dr. Brandis-Heep gave us the opportunity to ask these questions and supported us with our human practices project.

Interview with our PI Gert Bange

For several years, our PI Prof. Dr. Gert Bange mentors the iGEM Teams of Marburg. In Marburg, he is known as one of the pioneers in including students with visual impairment in scientific research. We used the opportunity to talk to him and could benefit from his experience in cooperation with visually impaired students. He told us, that he had visual impaired students working on their own project in his department. We had many questions to ask about daily lab situations.
One of our questions was, if this student was focused on doing bioinformatics or if he also did practical work in the lab. Prof. Dr. Gert Bange said that he was performing practical experiments like everyone else in his lab. We told him that some people are precarious if visual impaired students are able to work in a laboratory where they are confronted with many chemicals and different sources of dangers.Our PI answered us that of course, it is important to label some bottles and equipment in braille or with other haptic marks. In addition, it is necessary to have a well-structured and organized workspace with clearly defined places for the equipment. According to him, it did not cost much effort to prepare a special workspace for a visual impaired student because anyway lab spaces should be well organized.
He never had the feeling, that visual impairment was a barrier in daily lab work for his student. He confirmed that his students was working independent, efficient and was reliable. Prof. Dr. Gert Bange strongly believes that no matter if, someone has a handicap, everyone should get the same opportunity to show his or her expertise and knowledge.

The complete scientific community would benefit of all those talents, if we start encouraging people with disabilities to study STEM-fields. In order to do so we would like to lay the foundation stone for a scientific world without hindrances for visually impaired people with our barrier-free Wiki.
We thank Prof. Dr. Bange for sharing his impressions and experience with us and supporting our ideas.

Visit of chemistry lesson in the Carl-Strehl-School for the Visually Impaired
Students are working in groups with their customized laptops.
In the context of our human practices project, we had the opportunity to visit the chemistry lessons of a 12th grade from the Blista School for pupils with visual impairment. About 15 students were taught about the nomenclature of alkanes. The teacher catered for students with different needs using various methods. One of these methods attracted our attention when we entered the classroom. Every student possesses his or her own laptop. Asking the students, we figured out, that the school is providing laptops for each student.
Dr Tobias Mahnke explaining the content of the lesson in one-on-one conversation.
The teachers upload worksheets on their intranet and using their laptops, the students are able to use these worksheets by scaling them up or using a screen reader. Additionally, he printed the worksheets in normal print with a big font size and in braille depending on their individual handicap. Another haptic method is the usage of magnets or models in different shapes, to give the students an idea of atomic bonds and orbitals. Additionally, auditory methods were used to distinguish between different chemicals in tubes.

After the lesson, we had some time to talk to the teacher and he confronted us with a very interesting question: “How do you explain to a blind student how a flame looks and behaves like?”

This is a very difficult task because they, of course, can neither touch the flame nor hear it. Tobias Mahnke showed us how they solved this problem. With a kind of heat formable piece of paper, he is able to emulate the shape of a flame and now the students can feel where the hottest position is.

We were impressed by the different methods of teaching students with visual impairment and could learn many techniques to prepare their visit to our lab. In general, we thought back to our chemistry lessons in school and wished that our teachers also used a variety of methods and not the only visualization by sketches. It would have been a lot easier to imagine and understand the configuration of molecules and other complex topics.

It would be a big win for everyone if there could be more communication and exchange between schools teaching students with special needs and regular schools because both could benefit from each other and learn a lot, as we did during our human practices project.

Our team member Memduha listening in on how the visually impaired students try to solve a chemistry problem with their magnetic boards
Two of the pupil who attended the class as we visited the Carl-Strehl-Schule. One of the two also took part in our Open Laboratory Days.

Excerpt: Study Perspective for the Visually Impaired.

Governments throughout the world can no longer overlook the hundreds of millions of people with disabilities who are denied access to health, rehabilitation, support, education and employment, and never get the chance to shine.

— Stephen Hawking —

With the knowledge from our Open Laboratory Days we knew that most experiments could be adapted to remove barriers, the next logical step for us was to find out why there weren't more disabled students at our departments. What was the university politics regarding equal access to education and how were they implemented? Our university has an office of disability issues which is also responsible for blind and partially sighted students. That was the first address for us to get answers. Contacting them was easy and already in the return e-mail, it turned out that they were eager to hear about our project and the planned cooperation with the Carl-Strehl-School.

We met with them in a relaxed setting and got to ask as well as informing them that, actually, we were neither the first iGEM Team in Marburg, nor the first one to collaborate with the School for the blind and visually impaired.

The main service they provide for students is to know regulations and, even more importantly, the people in the different departments responsible for their implementation. Consequently, it is their job to provide disabled students contacts and guidance while being not directly involved. But they had a lot of useful information for us. For example, about state-funded aids for students who need assistance or additional time during exams. They also told us that the Philipps-University is the center of higher education in Germany with the highest quota of visually impaired students. We found out that the people responsible for how visually impaired students were dealt with in actuality were the same that also coordinated the whole education program in the departments.

Thinking back to our own time as newcomers at the university, knowing whom to contact is a valuable thing. Since we now knew the contact details interesting to the students, we put them together in one comprehensive list, which we provided them with. To further encourage the pupils to choose their path without fear that they stood no chance, we arranged interviews with visually impaired researchers in the middle of a Natural Science course, or that have successfully obtained a degree in their respective field. We had a bioinformatic student present, that was eager to answer questions of interested pupils.

We hope those measures will prove helpful in diminishing their concerns, since such personal reports are a bit more compelling than just our word for it. Some of us bonded with our aspiring researchers and we became personal contacts for future questions and study subjects. We hope that our efforts will start their respective science careers and that we remain a source they can rely on. Our initiative lay the groundwork for a lasting, sustainable interaction of future iGEM Teams with new generations of BLISTA pupils.

Integrated Human Practices

We acquired information from all three pillars that built the foundation of research - Society, Academia, and Industry - to step up our game and bring our iGEM project to the next level. We believe that our efforts and achievement reflect the interdisciplinary nature of our project and benefit the real-world applicability and future viability of Vibrio natriegens and the iGEM competition results.

Society - Accessible Webdesign

“The power of the Web is in its universality. Access by everyone regardless of disability is an essential aspect.”

Tim Berners-Lee (inventor of HTML and founder of the World Wide Web)
Logo for Webaccessibility

Accessibility in science does not end at providing a barrier-free lab. When getting into contact with Victor Kratz one of the topics we discussed was accessibility in the digital space. In the past years globalization and digitalization have shaped the world as we know it. Globalization is accelerated by digitalization and the internet is more important than ever before. The web has the ability to work for all people regardless of language, location, gender, age, income or ability. It removes barriers in communication and interaction that many people with disabilities face in the physical world. Yet, badly designed websites and applications can create barriers which lead to the exclusion of possible users. At this point in time e.g. a lot people with visual impairment need to adjust web pages to make them accessible to them. Barrier-free web design provides accessibility to all people regardless of ability or interface. In our conversation with Victor Kratz he mentioned that a lot of websites still are not suited to be accessed via a screen reader.

As scientist, we use the web a lot basically everyday nowadays. We’re having access to scientific publication, are doing bioinformatic analysis and can order e.g. oligos through online shops from science companies. It generally plays a huge role. Because of this, it is crucial to provide access to all those applications to everyone regardless of their abilities. A huge part of iGEM is the presentation of our work. We hold presentations, make posters and design a wiki. Habitually, we should always ask ourselves this one question: Is everyone able to access the data I’m presenting? We decided to design our wiki in an accessible manner to tackle this question. Through this, we aim to make the results of the iGEM competition accessible to everyone. And by this taking a step towards a more inclusive scientific community.

But how do you design an accessible wiki? We did some research and put together a guide for you!

Accessible Wiki - The guide
  • Navigation

    Many users rely on a keyboard for navigation. All elements of your wiki should be accessible when using only a keyboard as the navigation device. This means that you can focus on every relevant elements using the Tab key.
  • Colour and Contrast

    A high contrast between background and e.g. text should be used. This is especially important for buttons and symbols since they can’t be modified by the user. Additionally, red and green should not be used as contrasting colours since people with colour blindness can’t distinguish between those two colours. Additionally, for symbols and buttons, use a combination of colour, shape and text instead of only using colour to distinguish
    Figure showing an example graphic on how to use contrast.
  • Scalability

    Font sizes, distances,areas etc. should be set relatively so that they can be adjusted. This can be achieved by using % or em as units instead of pt or px.
    body {font-size: 14px}
    body {font-size: 14pt}
    body {font-size: 14%}
    body {font-size: 14em}
    Distances should not be created using transparent pictures. Instead, distances should be formatted using HTML or CSS.
  • Headers

    Headings should be defined as headings instead of defining them as . Heading should be descriptive. Instead of “Welcome”, your heading should contain important search keys.


  • Lists

    List should be formatted as lists instead of using wordwraps and hyphens.



  • Text

    Color or font style should not be used as the sole distinctive feature. Instead use e.g. bold styles to let it stand apart.
    This is a text in which this word is important.
    This is a text in which this word is important.
    This is a text in which this word is important.
    Animated text can also cause problems and should be described by an alternative text in the source code. (See Figures and Videos)
  • Frames

    Information that belongs together like navigation and content should always be shown in one frame. Otherwise, users have to switch between those frames. Frames should be named with titles like “navigation” or “content” for better orientation.
  • Figures, Videos and Audio

    Don’t only convey information using images, audio or videos. To better accommodate people using a screen reader, it is possible to add an alternative text to the source code of figures. By this, the screen reader reads the alternative tags instead of reading the filename you uploaded. This is crucial for obtaining information about the graphic.

    If graphics do not convey any information and their sole purpose is to accessorize the website you should still put a blank alt tag in the source code. By this the screen reader skips this graphic. Without any alt tag, it would read the filename.

    It is possible to check for accessibility of your wiki. In the settings of most browsers, graphics can be turned off. Image Maps should not be used, as they are only accessible using a computer mouse.

    This is an example picture of a cell.
  • Tables

    Tables should be arranged, so that they can be read row for row from left to right. Additionally, a description containing a summary that is not shown but can be read using a screen reader is helpful for understanding the content.
  • Links

    Links to other sites should be in form of a descriptive text instead of non-descriptive links, such as “click here”. If a graphic is used as a link symbol, the alternative text in the source code should point to the information the link leads to. External links should be labeled.

Academia - How to design a perfect cloning chassis for synthetic biology

In this years iGEM project our goal was to accelerate synthetic biology. As one of its most abundant methods, the process of cloning was targeted for improvement by our strain engineering subgroup. Therefore, engineering Vibrio natriegens, the fastest growing organism, as a cloning host became one of our main objectives. When we started to design our cloning strain, we asked ourselves, what changes need to be achieved to fulfill the demands for a next-generation cloning chassis.

For example, what genetic properties are desirable for other iGEM Teams, research groups, and the synbio industry to consider establishing a new strain in their laboratory?

Figure 1: Meeting with Prof Dr Zurbriggen. Our Skype conversation with Prof Dr Zurbriggen.
This question led us to contact an external expert for academic cloning applications, Prof. Dr. Matias Zurbriggen (Institute for synthetic biology, Heinrich Heine University Duesseldorf), to discuss our first design ideas for our Vibriclone strain.

At first, he mentioned general concerns about V. natriegens. He would be worried about the mutation rate for the genome as well as on introduced plasmids due to the fast growth of the organism. Its size might cause limitations for plasmid transformation, and recombination or deletion on cloned plasmids might prove to be an issue.

Afterwards, he suggested the integration of common genetic tools, such as the ability for blue-white screening, into our strain design. This would shorten the adaption phase in laboratories with an established cloning standard and make switching to V. natriegens more desirable. The transition towards V. natriegens would then become more accessible for everyone in the future regardless of their preferred cloning method.

After this inspiring discussion, we went back to the sketch board to implement all of his valuable feedback for the updated version of our Vibriclone 2.0 strain. We especially focused on the integration of the lac operon to allow alpha complementation for blue-white screening (for further reading about alpha complementation, click here). Furthermore, taking all the general concerns about V. natriegens to heart, we integrated more input by testing the mutation rate for the genome and plasmids. We demonstrated that both mutation rates are not increased in comparison to E. coli, although this fear is understandable due to the fast doubling time. We consider it crucial integrate feedback of a large variety of people to succeed in our final goal to fully establish V. natriegens as a cloning chassis and replacing E.coli, Stepping outside of the box and considering various points of views helps us to facilitate an easy adaption of our Vibriclone strain for diverse laboratories with different areas of research.

Industry - Protein Expression with Crystals First
Figure 1: Meeting with Serghei Glinca.
Members of our team sitting together with Sergej Glinca talking about our wonderfull possibilities.

Crystals First is a start-up from Marburg that made it their mission to accelerate and improve the quality of drug design processes. We had the pleasure of featuring the CEO of Crystals First, Serghei Glinca, as one of our speakers for the German iGEM Meetup. There he sensitized us to the importance of protein expression in the context of drug discovery, which lead us to design our protein expression strain. After that, they invited us to present our project to the pharmacy department of the Philipps University Marburg. This resulted in not only valuable feedback but also in the opportunity to test our project with a real-world problem.

As a proof of concept for our expression strain VibriXpress (click here for desciption) Dr. Stefan Merkl from the company Crystals First recommended the overexpression and purification of human matriptase. Matriptase is a membrane-spanning serine peptidase and a valuable drug target for medical research, because it promotes cancer (Uhland, 2006). By degradation of extracellular matrix proteins, it allows cells to migrate and also activates growth factors. To find new drugs against matriptase it is crucial to get high amounts of that protein. Unfortunately, in E. coli production of matriptase leads to the formation of occlusion bodies and by refolding of proteins the amount of correctly folded matriptase is limited to 1-5%. Since V. natriegens comes from salt marshes with frequently changing environments, it succumbs a lot of stress, making the availability of chaperons and other protein folding helper essential. We expected that V. natriegens might have a better chance to produce high amounts of correctly folded matriptase than E. coli.

Therefore, our plan was to transform a matriptase-bearing plasmid into V. natriegens, overexpress the protein and finally purify it. For this plan we were equipped by Crystals First with two plasmids, one plasmid harboring the wild type matriptase controlled by a T7 promoter and one plasmid with a C122S mutation of matriptase, which is assumed to promote correct folding. This plasmid has an ampicillin resistance gene and is controlled by a T5 promoter, that can be read by housekeeping polymerases. Due to the pending construction of our expression strain VibriXpress, we only considered the second plasmid as suitable target. Since V. natriegens cannot be addressed by ampicilline we adapted to the derivative carbenicillin instead. Our experiments with pMMB-tfox showed that carbenicillin can be used to select ampicillin-resistant cells (click here for results).

However, despite various attempts, successful transformation of the matriptase plasmid into V. natriegens was not feasible. None of our several transformations via electroporation led to any colonies. For troubleshooting, we conducted a control experiment with another plasmid bearing an ampicillin resistance gene and got colonies on our plates. Our explanation for these results is that different ampicillin resistance genes have different substrate specificities and the gene from the matriptase plasmid probably is specific for ampicillin but not for carbenicillin. Therefore, we propose to clone the matriptase gene into another backbone, for instance with kanamycin or chloramphenicol resistance and then repeat the experiment to investigate V. natriegens for potential enhanced protein yield.

Industry - Investigation on Up-Scalability for Vibrigens

To take a closer look at the industrial promises V. natriegens might pose we contacted individuals heavily involved or representing with one industrial branch. We reached out to biotechnologist, Robert Giessmann, from the research group of Prof Dr. Peter Neubauer who possess a long-standing expertise in making laboratory procedures applicable to the needs of industrial production. They pose the valuable interface to enable scale-up of academic successes.

As a first step to tinker V. natriegens to that purpose, he suggested tracking how the conditions in the media changes during V. natriegens growth. Especially important are the differences in dissolved oxygen and pH levels over time. He mentioned that these factors heavily impact growth, OD as well as organism and product behavior. Furthermore, he wanted us to consider that the choice of media, and a controlled distribution and can facilitate how well V. natriegens performs in a bioreactor. We received his generous offer to conduct suitable experiments for his advice in their specialized laboratory in Berlin since our home University does not offer the same technical equipment.

Figure 1: Sensor-equipped shaking flask.
Shaking flasks with imbedded chemosensors.

Cultures were prepared in a small sensor-equipped shaking flask. Their two imbedded chemosensors on the surface react to the shifts of pH and oxygen. Results are interpreted by a connected software and deliver time-resolved changes in media condition. The interaction is wireless via Bluetooth ensuring that the cultures are constantly shaking with 250 rpm in an incubator at 37°C. This technical set-up avoids interruption of the growth process and provides us with more accurate and detailed data.

Firstly, we took a detailed look at the normal growth media for V. natriegens, LB supplemented with v2 salts, in various combinations with a baffled and unbaffled flask. Secondly, we underwent various steps towards the utilization of different media including the EnPresso medium system with gradual and continuous glucose supply. Interestingly, and in contrary to the known behavior of V. natriegens under anaerobic condition, the pH levels showed a steep increase with the rapid consumption of dissolved oxygen. The pH became increasingly alkaline until the value went out of the measurement range of the employed chemosensors.

Figure 2: Normal growth conditions
Observed changes in dissolved oxygen and pH levels in normal growth medium without baffled flasks.

Concerns arose that the chemosensor might be incompatible with the secreted products of the organism. However, after normalization of oxygen in the test tube, the pH downregulated itself diminishing such fears.
Considering the continued although slower growth of V.natriegens under alkaline conditions, its resilience across a broad pH range might render the organism suitable to develop pH resistant strain. Such an engineered organism would be useable for metabolic engineering processes in laboratories unable to constantly regulate the pH-levels in medium. We deduced that the high pH-level might stem from the production of ammonia and other amine products.

To countermeasure the depletion of oxygen we investigated the effect of a baffled flask and the in vitro controlled medium conditions created by of EnPresso B tabs. We also switched to this medium to investigate the eligibility of V. natriegens for even higher growth density which is crucial for higher product yield, especially for proteins as targets in the pharma industry.

The results in figure XY clearly show that we could efficiently stabilize the growth conditions for V. natriegens with EnPresso and baffled flasks are sufficient enough to maintain the needed high oxygen levels.
More importantly, the optical density increased to 25 which is an outstanding sixty percent boost compared to our measured top OD with 14 further proving the potential scale-up possibilities when adapting V. natriegens as a production host. The increased cell densities of V. natriegens in combination with EnPresso support suggestions we obtained from Karsten Shürrle (DECHEMA) to consider the emphasize of yield over time when profitably running a white bioreactor.

Figure 3: growth in baffled flasks:Observed changes in dissolved oxygen and pH levels in normal growth medium with baffled flasks.
Figure 4: Growth in Enpresso medium Observed changes in dissolved oxygen and pH levels in EnPresso growth medium without baffled flasks.

We implemented our new found knowledge on the high oxygen dependency of V. natriegens from our collaboration in Berlin to increase general cell density in our test samples with newly constructed plasmids back at home. Thereby, we could obtain more genetic material in a shorter time to enabling us to speed up distribution and characterization of our Marburg collection.

Aside from this small perk for everyday labwork, we gained know-how that in addition to our results in metabolic engineering group will lead to further steps towards the real-world applicability of V. natriegens in the industry.

Industry - Industrial application for Vibrigens

To expand on our knowledge from our collaboration with the TU Berlin we got into contact with Senior Fermentation Expert, Prof Dr. Oskar Zelder, from BASF to answer the question: What expectation a new organism has to fulfill to become a suitable industrial chassis.

We discussed in length how diverse the demands and challenges for such an organism can be and need to be highly specialized for the task at hand. There a major differences rather one is aiming for a small molecule production, like our 3-hydroxypropionic acid (3-HPA) platform chemical, or if the main purpose is protein expression and secretion. He informed us that there is no such thing as “all size fits all”.

Figure 1: Online Meeting with Prof. Dr. Oscar Zelder
We got invited to discuss our Vibrigens project with this BASF fermantation expert.

However, target-specific discussion for 3-HPA led to general design question concerning bioreactor set-up since not only the production of a target molecule need to be considered but also its isolation and purification. In the case of an acidic molecule like 3-HPA an acidic medium and in consequence a bacterium which is resistant against low pH-levels would be most suitable. This would unlock distillation as a cheap and efficient purification method.

More immediate for our project, however, is the following aspect:
In industrial biotechnology, everything is a little bigger. Fermenter can easily have a volume of over ten cubic meters. This is much bigger than the maximal volume that can be realized with glass, so stainless steel has become the most common material choice. Stainless steel is much more resistant to corrosion than ordinary steel, but it is not impervious. Chloride ions, for instance, can expedite deterioration. Our favorite microorganism, V. natriegens requires an almost three-fold higher sodium concentration than the current standard, E. coli. And sodium is mostly added as sodium chloride, table salt. So we came to realize by Prof Dr Zelder’s feedback that this high chloride content of current media formulations may hold V. natriegens back for in industrial applications.

So he gave us the valuable input to investigate ways to reduce the chloride content in our medium by providing alternative chloride-free sodium sources. One such easy method, he suggested, would be to plate out a thick biofilm of a V. natriegens culture on agar plates mainly devoid of chloride and wait for colonies to adapt to the growth condition over time.

Taking this input, we went back to the laboratory to find suitable sodium salts to compete with sodiumchlorid (NaCl). For this endeavor, both regular LB medium and v2 salts were inapplicable since they all rely on chloride salts as the source for the cations necessary for growth. Therefore, we investigated the possibilities for a novel fermentation medium to aid the accessibility of V. natriegens for industrial application.

Experiment design

The goal of the experiment is to identify how well the wild type of V. natriegens can grow without sodium chloride if we provide consistently high sodium levels with an alternative sodium source. Therefore, the essence of the method is a dilution series in small incremental steps.

Figure 2:general layout for sodium chloride replacement
To screen a lot of possibilities in a short time we utilised platereader experiments for high throughput with stepwise replacement of NaCl
Inspired by the successes achieved with our measurement workflow for our Marburg collection we decided on plate reader experiments to achieve the highest possible throughput of combinations.

Medium composition and carbon source

We adapted the composition of common M63 low osmolarity minimal media and made adjustments to our purposes. Due to the slightly higher uptake rate and the general cost-efficiency we elected sucrose as the carbon source for our screening experiments. To simplify the minimal medium to be tested in other laboratories we refrained from adding trace elements.
We encountered troubles when first working with M63 and witnessed a reoccuring percipitate. Chemical investigation revealed that the high magnesia content leads to a formation of an insoluble magnesia phosphate salt. This needs to be taken into consideration when preparing the medium.

Alternative sodium sources

Figure 3: Dilution and alternative sodium sources
The needed medium composition was adjusted a head of time to faster distribute medium into the platereader.
Sodium citrate is an ideal candidate to replace NaCl in industrial media. A study from 1978 showed that V. natriegens could grow with sodium citrate as the sole carbon source (Austin et al., 1978). Therefore we hoped that it would help to maintain the growth rate of V. natriegens. Furthermore, we hoped that it would lead to favored uptake of sodium in correlation with the higher bioavailability of the citrate salt overall.
Sodium sulfate is another alternative sodium source to NaCl. It too is non-toxic in high concentrations, has already been tested for applications in bioreactors and it is readily available.

The sodium content of the media was derived from the available sodium in v2 salts. Due to the polyvalence of the citrate and the sulfate, the overall molarity had to be adjusted. The lower solubility of sodium sulfate was taken into account and resulted in a lower stock solution (10x NaCl = 2.4 M; 10x Na3Citrate = 0.8 M; 5x Na2SO4 = 1.2 M).

Contents of the medium:

5x M63 (1.25x the standard concentration)
12.5 g (NH4)2SO4
85 g KH2PO4
3.125 mg FeSO4 (7H2O)
→ Adjust to pH 7 with KOH
add 34.2g MgSO4 (7H2O) afterwards
→ Adjust to a final concentration of 240 mM sodium ions in the ready-to-use medium.
→ Readjust pH as needed.


NaCl was replaced in 2.5% incremental steps (2.5% → 100%) with the respective sodium alternative, sodium citrate or sodium sulfate. The scheme of the workflow is depicted in Figure XX.

The culture inside the wells was inoculated with an overnight culture (1µL) with 100% NaCl content without alternative sodium source. The preliminary result of the dilution experiments is depicted below. So far sodium citrate seems to be an outstanding choice in taking up the role of NaCl as sodium source. The growth continuously stays on par with the pure NaCl culture while the growth significantly declines with increasing sodium sulfate replacement. Those results show that the NaCl content we took as none-problematic can be addressed by easily available means such as citrate salts.

Figure 4: growth in sodium citrate as alternative Na-source:Observed changes in M63 growth medium with various concentration of sodium citrate instead of NaCl.
Figure 5: growth in sodium sulfate as alternative Na-source:Observed changes in M63 growth medium with various concentration of sodium sulfate instead of NaCl.
Further investigations have to be made in order to conclude the impact the minimal medium has on the doubling rate of V. natriegens.

For the future experiments, adaptation of V. natriegens strains to these new sodium sources could be achieved by iterative exposure to increasing concentrations. If coupled with selection for a high growth rate, it is conceivable to reach similar doubling times to NaCl media and thereby a suitable and easy fermentation medium for the industry.

B. Marchal