Subject

Vitamin B12 is one of many vitamins essential for the human body. It plays a role in many key pathways such as DNA metabolism, fatty acid metabolism or neurotransmitter synthesis. A deficiency in Vitamin B12 may thus seriously impact health [1]. Vitamin B12 deficiency can be considered a public health problem potentially affecting thousands of individuals [2,3].

Several categories of people are susceptible to B12 deficiency. The prevalence is estimated at 20% in the general population of industrialized countries; it is estimated at 30 to 40% in elderly subjects [4].

Vitamin B12 is the only vitamin not found in plant products. As it comes essentially from animal food, Vitamin B12 deficiency directly impacts people with plant-based diets. However, there is a rising trend in diets with lower intakes of animal products such as the flexitarian diet, therefore many people are concerned. Those people must add vitamin B12 to their diet in the form of food supplements or fortified food.
However, consumers of animal food can also be affected by a vitamin B12 deficiency. It is therefore a global problem.

For all those who are concerned by a B12 deficiency, it is crucial to measure the level on B12 in the blood, to correct it appropriately.

Currently, one of the main ways of measuring one’s B12 level is through a clinical blood test. The patient must come to a laboratory or a hospital to get blood drawn and analyze it. This method is not always convenient and affordable. Other commercially available at home test kits on the market still require one to send back the test samples to a laboratory in order to analyze the sample which takes about 48-72 hrs for the results to be accessible which is not that convenient (see Entrepreunership)

After conducting an in depth research on this subject, we came to the conclusion that an at home test kit to detect the level of B12 is needed and that there’s a market for it. With the recent pandemic where laboratories were not accessible for many people, it became more necessary than ever to have an at home B12 test kit that doesn't require a professional analysis.

It is in this context that Cobatect was created. Cobatect aims to offer an easy solution to detect Vitamin B12 levels at home.

Cobatect is a synthetic biology based approach diagnostic device in which one can detect the level of vitamin B12 at the convenience of their home without the need of a professional analysis with results being accessible in about 3-4 hours of testing.

Human practices

To fully understand the relevance and impact of our project, we wanted to speak with key stakeholders from different horizons. We thought about all the people that could be impacted by our project (positively and negatively), and all the actors in the environment of this project.

Direct consumers: vegetarians

As they do not consume animal food, vegan and vegetarian people are almost certain to suffer from a B12 deficiency. We approached national vegan and vegetarian associations to gather their feedback on their needs for a B12 detection test.

We met with representatives from the french vegetarianism association (AVF) and the national observatory on plant-based diet (ONAV). During this meeting we spoke with :
Loïc Blanchet-Mazuel : physician + volunteer coordinator for the AVF
Fabien Badariotti : biologist
Paco Maginot : physician

The first thing we talked about this exchange was the relevance of our project on the market. The representatives assured us that this is an important topic, because the current clinical blood test is not reimbursed by the social security system, is not 100% reliable and not performed by all laboratories. Therefore vegans are looking for new alternatives and there is an actual need for a B12 detection kit.

According to them, if our test is financially attractive and reliable, it could have a considerable potential compared to the demand of the vegans.

After reassuring us on the relevance of our project, Fabien Badariotti, the biologist of the group gave us ideas to test the reliability of the detection component. He told us to try our system with existing analogs of B12 such as those present in spirulina. He also told us to be careful about the direct effect of vitamin B12 in the bacterium metabolism and to make sure it wouldn’t impact our measurements.

After this discussion, we decided to speak with biologists specialized in B12 testing to understand the challenges we would have to face in our system.

Specialist in B12 blood testing

We read a paper written about B12 deficiency : how to diagnose and take care of it. We wrote to the corresponding author, Dr Hernan Valdes-Socin from the university hospital of Liège in Belgium to speak with him more in depth about current B12 detection methods and their drawbacks. He is a specialist in B12 dosage in his unit.

We first talked with Dr Valdes-Socin about the number of people affected. He told us that this condition is more common than we would think, that he sees a lot of people every day for B12 blood testing. This again confirmed us that there is a need for our detection kit.

However, Dr Valdes-Socin was honest with us and told us from the start that we would face many challenges.

Firstly, we would detect total free B12 in the blood, not the one that would be directly metabolized by the cells, because not all free B12 is transported and eventually used. This could induce bias in the measurements and give less accurate information to the patient. Moreover, blood is a particular matrix, and dosing components in the blood is not the same as dosing.

Moreover, he was sceptical on whether we would have a sufficient quantity of B12 to be detected by the bacteria, since vitamins are present in very small quantities in the organism. The system might not be very precise on the quantitative aspect, and we should be careful about interferences and false positives/false negative.

Dr Valdes-Socin gave us the same warning as Fabien Badariotti, that B12 could have an impact on the growth rate of the bacteria (especially because B12 acts on DNA synthesis), or that it could be produced by the bacteria. All of this would impact our measurements.

In the end, Dr Valdes-Socin was enthusiastic about our project, and despite the challenges he was optimistic about the perspectives. Current methods of dosage have drawbacks because they come from a chemical point of view rather than a biological one. Dr Valdes-Socin thus thinks that biosensors have a great potential to detect biologically active components, and that they could fill the gap currently left by chemical testing.

Regulator

In order to be able to launch our device in the future we need to be aware about the current regulations on the market, essential requirements to consider and guidelines to follow.

This is why we have contacted Adrien Rocha, Quality insurance and Regulatory affairs specialist in medical devices at Stilla technologies, Paris.

During our interview we figured out that the main official document to rely on while working with GMO’s is Regulation 2018/848 - it is the regulation relative to the organic production and labelling of organic products. Speaking precisely about the disposal of biological waste, which is key part of user experience with our device, we need to rely on paragraph in the Regulation 2017/746, in the Annex I General Requirement 13.6 :
Devices shall be designed and manufactured in such a way as to facilitate their safe disposal and the safe disposal of related waste substances by users, or other person. To that end, manufacturers shall identify and test procedures and measures because of which their devices can be safely disposed after use. Such procedures shall be described in the instructions for use.

Another question from our part was about collecting patient’s data safely and legally if we want to connect our device with a mobile application.

Adrien Rocha: That is a matter of data protection, which you can consult Regulation (UE) 2016/679, on the protection of natural persons about the processing of personal data and on the free movement of such data

The last question was about feasibility of obtaining market authorisation of our device in European market.

Adrien Rocha: You have multiple challenges, as being CE conform to 3, if not more, European Regulation. Each of these regulations decrease your market introduction feasibility, be aware of it.

The last advice from our expert was the following: "I encourage you to define your In Vitro diagnostic medical device as such, before talking about auto-tests (the right term is self-testing, according to the Regulation2017/746), because IVD Medical Device is the legal term to refer to your product".

Public opinion

As our project is destined for the general public, we wanted to gather opinions on our project. To broadcast this survey, we were helped by one of our collaborators, team ITU. We prepared a survey to check if people would be inclined to use our system. We were able to reach 164 people.

Population sample

From the survey results we received, the majority of participants (67.9%) were of the age group 19 - 25 from many different fields of work as shown on Figure 2. The majority of these participants were female with 74.5% and 23% of male.

Knowledge on Synthetic Biology

Among the different fields of work, many belonged to scientific related fields. But it was quite surprising to see that the majority of the participants (34.7%) knew very little about synthetic biology. It was only a percentage of 14.3 that had an overall idea about synthetic biology. As a result, we chose to keep engaging the public in order to generate more awareness of synthetic biology.

Preferred Diets

To get a better overview of the sample population and their diets we questioned about the type of diet they preferred and the reasoning behind it.

Out of the 198 participants, the majority (63.3%) were found to be omnivorous with only 1.5% being vegetarians. For most participants, the reasoning behind their diets was the likeability (51.5%) followed by healthy-eating (29.6%). 9.2% mentioned their reasoning to be eco - responsibility while 5.6% reasons with affordability.

Statistics on deficiency

In order to get a better idea on the topic of deficiencies, we wanted to check how many people were affected by a deficiency in the past or have been tested for one. According to the respondents, the majority (43.9%) had not suffered from any form of deficiency whereas 40.8% had suffered from one. However, 53.6% of these participants had been tested for deficiencies in the past.

Use of autotests

At last, we wanted to get feedback on how individuals felt about the use of autotests and whether they were comfortable with it. It was rather shocking to realize that almost 90% of the respondents were not aware of the existing autotests; this may be because of the lack of marketing strategies or due to the fact that they didn't have a deficiency that needed to be checked on regularly.

On the brighter side, in terms of the conformability of participants with pricking their fingers for the autotest, almost 80% were confident and okay to do so with less than 10% that were not okay with it. So, it is safe to assume that utilizing cobatect would not be a problem.

Integrated Human Practices :

Adapting our design to other applications.

Our bio-electronic sensor works by associating the production of a metabolite triggered by Vitamin B12 (cobalamin), to an electric signal, obtained thanks to the metabolite.

As mentioned before, we had the chance to speak to different professionals who assured us of the interest of biosensors.

We spoke with Frank Yates, research director at Sup’Biotech. He confirmed that biosensors present key advantages to detect biological components. Moreover, he was very interested in our reporting system, as current reporter systems can have drawbacks. Indeed, the use of RFP is sometimes too slow as it takes time to appear and be quantifiable. Our electronic system would have the advantage of being faster. (Zeng, 2019)

Moreover, we would be able to directly obtain measurements, without having to go through further analysis with a plate reader, as is needed to quantify fluorescence.

Drawing on these exchanges, we thought about how this system could be adapted to other projects using biosensors, by switching our cobalamin riboswitch with another riboswitch or another riboregulator structure.

The first example we thought of is the adaptation of the project from team U Paris BME to our system. This example was inspired by our collaboration with them, and you can learn more about their project and our collaboration on the dedicated page. Their aim is to detect cancer at an early stage using a liquid biopsy, thanks to a toe miRNA.

In addition to our collaboration, we designed a final construction to adapt their riboswitch to our system. Contrary to our own sensing part, their riboswitch triggers the translation of the coding sequence placed after it instead of inhibiting it. Therefore, we modified our sensing design to adapt it to their project :

Structure of the construction of a Lactate dehydrogenase regulated by a toe miRNA

To go further, we decided to take a realistic exemple to show how our design can be adapted to another field than health diagnosis. To do so, we imagined a system based on a relevant environmental issue.

We thought about water quality and treatment. We found riboswitches capable of detecting fluoride, magnesium and manganese. These components are often the target of routine screening in water treatment plants. Fluoride is often added in public water to fight teeth enamel demineralization; but should not exceed certains levels. Magnesium levels in tap water also must be kept in a precise range. Manganese is an industrial mineral that can be toxic at high concentrations. These components need to be detected in precise quantities to assess the safety of the water. By modifying our sensing part, we would be able to detect them and trigger a response by Shewanella.

We chose 3 parts from previous projects and designed constructions to adapt them to our system :

BBa_K902074 : this part is a manganese riboswitch, from team Calgary in 2012. The activation of the riboswitch triggers the translation of the sequence placed just after, so we used the same sensing structure as for the U Paris BME design (but with our constitutive promoter instead of the T7 used by U Paris)

Structure of the construction of a Lactate dehydrogenase regulated by a Manganese Riboswitch
BBa_K911001 : this part is a magnesium riboswitch, from team Cambridge in 2012. The activation of the riboswitch inhibits the translation of the sequence after it, as for our sensing system. Therefore, we used the same structure :

Structure of the construction of a Lactate dehydrogenase regulated by a Magnesium Riboswitch
BBa_K911003 : this part is a fluoride riboswitch, also from team Cambridge in 2012. This riboswitch works in the same way as the manganese one, so we used the same sensing structure :

Structure of the construction of a Lactate dehydrogenase regulated by a Fluoride Riboswitch

Furthermore, we decided to make it easier for other teams to adapt our system to their own riboswitches. To do so, we created two constructions, one for riboswitches triggering translation and one for riboswitches inhibiting translation. In place of the riboswitch, these constructions contain a space with restriction enzyme sites both for RE and Golden Gate Assembly, for an easier assembly. This space is noted as "RIZ" for Riboswitch Insertion Zone.

Construction to use for riboswitches inhibiting the translation of the coding sequence juste after it . (Details on this mechanism can be found in our project page) This construction can be found in the register : BBa_K3933018

Structure of the general construction of a Lactate dehydrogenase regulated by a sequestering Riboswitch with an insertion zone for the riboswitch
Construction to use for riboswitches triggering the translation. This construction can be found in the register : BBa_K3933019

Structure of the general construction of a Lactate dehydrogenase regulated by a Riboswitch inducing translation with an insertion zone for the riboswitch

All of these constructions show how our design can be adapted to fit other purposes than those of our project. Thanks to the interesting discussions we had with professionals, we realized how our design could go beyond the scope of the Cobactect project, and broaden its purpose.

All the constructions schemas have been created with Biorender

Sources

Pictures credit: https://www.iconfinder.com/icons/636914/multiple_avatar_group_human_peoples_team_users_icon; https://www.iconfinder.com/icons/646735/avatar_doctor_man_person_icon; https://www.iconfinder.com/icons/3203227/female_avatar_professional_female_successful_business_woman_woman_entrepreneur_women_empowerment_icon

[1] Vitamine B12-Health Professional Fact Sheet : https://ods.od.nih.gov/factsheets/VitaminB12-HealthProfessional/.

[2] Hunt, A., Harrington, D., & Robinson, S. (2014). Vitamin B12 deficiency. BMJ (Clinical research ed.), 349, g5226. https://doi.org/10.1136/bmj.g5226

[3]Allen L. H. (2009). How common is vitamin B-12 deficiency?. The American journal of clinical nutrition, 89(2), 693S–6S. https://doi.org/10.3945/ajcn.2008.26947A

[4] Carence en vitamine B12 : diagnostic et prise en charge. Dr Pauline Delannoy, Dr Laura Vranken, Dr Etienne Cavalier, Dr Hernan Valdes-Socin. https://orbi.uliege.be/bitstream/2268/252550/2/Vitamine%20B12%20TMB_425_FR_p_24-28_200915.pdf

Team:Ionis Paris/Human Practices