Team:Paris Bettencourt/Script

Risk assessment :

Review board of the iGEM project , September 16th 2015

URSZULA: Could the DNA of the dead bacteria and yeast [in idli] be ingested by other bacteria [present in the environment]?

JUANMA: the DNA would not in principle go to environmental bacteria and in any case the genes that we are introducing to the bacteria do not increase the ability of the bacteria to transfer genes [to other bacteria already present in the environment]. That would be the main argument [...]

URSZULA: Yes, but in the rice DNA is not denaturated. Bacteria can be dead but DNA is maybe not denaturated [...] it should be actually with the cooking.

JUANMA: I think it goes more than 100 C for sure and DNA denaturates around 100 [...] do you have any insight on this? [talking to ALEXEY]

ALEXEY: 100 sounds right but I think there is still a small but existing probability that some bacteria get this DNA inside of them.

JUANMA: But how? No more than a natural strain in a natural environment.

ALEXEY: Not, definitely not.

JUANMA:[...] the genes that we are introducing are vitamins and they are already present in some natural strains so the genes by themselves do not constitute any harm.

URSZULA: [...] it is not about the harm but about the modification.

JUANMA: O.K.

URSZULA: What if a natural strain ingest this new [...] and survive the cooking and can transfer this gene to a natural strain and make them producing the vitamin [...] it is just a hypothetical situation. It is not about the risk itself of this DNA of these vitamins but [...] environmental risk. That you would modify natural existing bacteria.

JUANMA: O.K. I guess if there is no synthetic promoter and you have in the final strain [exclusively] ORFs [open reading frames] and natural promoters it would not be a harm as far as genes are already presented in the environment in other natural strains. So it would not increase the probability [...] to be transferred to [this specific environment] idli [microflora]. And by itself the bacteria that grow in idli are not found anywhere else than in food.

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NIKOLA: I have a question about your technique. Are they going to prepare it with baking soda or with the [bacteria and yeast] strains?

JUANMA: It would be with the natural strains. Baking soda does not change the taste of the food, it just provides bubbles [of carbonic gas]. Baking soda is just a quick and dirty way to make idli in 10 minutes

NIKOLA: But with your technique, are you going to…

JUANMA: Is the traditional way to make idli, that is letting it ferment for 8 or 10 hours.

NIKOLA: And with all the vitamins that you provide in the food, is it possible that it creates an environment for other bacteria to develop inside of it, bacteria that you don not want?

JUANMA: So you are asking if the modifications that we are introducing to these strains make it easier for other strains to grow on idli?

NIKOLA: Yes.

ALEXEY: I think no.

JUANMA: I think no.

URSZULA: And then you would cook it.

JUANMA: I think no but it is a question to take into account. I am not answering to you, we are just taking these questions to write another [more complete] report.

NIKOLA: O.K.

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SIMON: How would you bring to them the modified strains? I do not understand how you put it in the food. [Do] you give them a tube with modified bacteria?

JUANMA: It would be nice to make the risk assessment for two cases. [...] We are having two scenarios: one, where you give the farmers little bags of dried yeast and bacteria and they put it in their rice…

SIMON: Would it be like a powder?

JUANMA: Yes. And the scenario number 2 is if this is not possible. You would prepare the idli in confined space, and then this kind of regulation [EC 1829/2003] does not apply, and then you would serve the idli with dead bacteria to the people.

SIMON: Would it cost them more?

JUANMA: I am not sure about the cost but it is for sure harder to get. Because you cannot provide this kind of facilities everywhere.

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LARA: Actually the problem with Indian people is that they cannot buy anything but rice. So if you put put bacteria into rice, don’t you think this rice would augment its cost? [...] They would not be able to buy it.

JUANMA: No because they buy rice from the market, it is the exact same rice.

LARA: [...] then they would put the bacteria.

JUANMA: Yes, and we would not sell rice.

SIMON: Yes, but they would have to buy the bacteria.

JUANMA: No, [...] the powder would be given for free, in principle. as [...] golden rice was given for free to farmers under certain income in certain countries.

SIMON: Do you think that the idea of putting bacteria in your food will be received [positively] by the public opinion?

JUANMA: [...] The public perception about the food? This does not count on the risk assessment, that is the second part. [...] That is a concern that the perception by the public opinion might be bad. While “bacteria” are generally perceived as bad, “fermentation”, “idli”, “traditional food” [are positive]. Idli is perceived as a healthy food in India.

URSZULA: “Probiotics”.

JUANMA: “Probiotics” is a good term and they define the same concept.

LARA: You can [...] not lie to them but not telling all the truth.

[...]

URSZULA: This is what is done with the products that contain bacteria in it. They are probiotics, or bioactive products, or things like this, and they sell them. People do not care as long as you don not show the bad.

JUANMA: Yes.

URSZULA: I do not know in India how is the public awareness about [...] microbiology is. [...] I think they would follow the main force [...] if there is enough people convinced this is a good idea [...].

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JUANMA: I will go through the [EFSA] report and either you give your opinion about the risk or request further information. Normally this is just like the review of a paper: you send the paper and then they would request additional information [...]. We can do the first round [...] here. Let’s start by the comparative approach. To which kind of microorganisms do we compare them with?

URSZULA: With the yeast [Saccharomyces cerevisiae] and Lactobacillus plantarum, the bacteria that are not modified.

JUANMA: So we will take these two. Can you check in which hazard group are they included?

JUANMA: Yeast are actually quite impossible to infect humans.

ALEXEY: Only one case have been described [to the best of my knowledge] because this person was taking many antibiotics.

JUANMA: This only would happen with a compromised immune system.

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JUANMA: Just to remind the body of laws [that regulate this topic in the European Union]. There is a General food law (Regulation (EC) No 178/2002), GM food and feed regulation (Regulation (EC) 1829/2003), Deliberate release of GMO (Directive 2001/18/EC), Contained use of GMO (Directive 98/81/EC) and there is also a law for additives for animal nutrition (Regulation (EC) No 1831/2003). So actually it is been more than 10 years.

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JUANMA: I would like to show you an interesting experiment from the regulation point of view related to the CRISPR/Cas9 system. [...] the most interesting characteristic about this system is that it is a nuclease that can cut [custom] genomic DNA [with a very high specificity]. There are natural CRISPR/Cas9 systems in almost most organisms. Many people are using these characteristics to do the cloning inside the cell. This, as seen by the EU regulation, is called “self-cloning”. This happen when you do not introduce foreign DNA [sequences]. Even if the DNA is synthetic, even if the sequences [or functional elements] are in a different order [...] if it is not a pathogenic strain, the pre-market regulations are very limited in the E.U. and only post-market follow-up in the U.S.

SIMON: So it is all already there?

JUANMA: Imagine that lactobacillus produces naturally vitamin B2 [...]. It produces B2 under a low promoter, that is downregulated naturally. Then you would introduce a linear DNA sequences that have the guide RNA sequences to cut the flanking regions to this promoter and another DNA that has homologous regions to those. When the cell tries to repair the cut by recombination, it will use the DNA that you have put in place. If this DNA has a different sequence inside you would exchange the promoter sequence by you custom one. If this promoter was not there but was already present somewhere else in the genome [...] it is like you were shuffling the DNA of this bacteria. But you will increase the production of B2. This is a way to bypass the regulation, because by the time it was made it was not possible to do this kind of stuff.

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ALEXEY: Where can you get the information about safety? Google just gives scientific articles that say that it is safe. [...The source of this information is the directive 2000/54/EC on the protection of workers from risks related to exposure to biological agents at work. This E.U. directive made by the European Commission divides the biological agents in 4 different biosafety groups. This directives specifies:

Group 1: Unlikely to cause human disease.

Group 2: Can cause human disease and may be a hazard to employees; it is unlikely to spread to the community and there is usually effective prophylaxis or treatment available.

Group 3: Can cause severe human disease and may be a serious hazard to employees; it may spread to the community, but there is usually effective prophylaxis or treatment available.

Group 4: Causes severe human disease and is a serious hazard to employees; it is likely to spread to the community and there is usually no effective prophylaxis or treatment available.

The different E.U. state members have implemented this directive in their national laws. We took the case of the U.K. which is described in the website of the Health and Safety Executive (http://www.hse.gov.uk/pubns/misc208.pdf). Both our S. cerevisiae and L. plantarum strains are used in food products are are included in the lowest biosafety level.]

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NIKOLA: Are our immune defenses reacting against these bacteria or yeast?

JUANMA: Our immune defenses are not reacting again those microorganisms because they are not pathogens. They cannot produce an infection [...] but you can think about the interaction between these bacteria and the intestinal flora. Lactobacillus can live with us and if you introduce a GM strain [in the intestinal flora] it can create problems. In this case, the bacteria would be consumed dead.

SIMON: [...] you have to be sure that the farmers cook the food. How can you ensure that there are some [...] that do not cook the food.

NIKOLA: Some of them may not have access to a cook system.

JUANMA: I think they all have access to fire [...]. I think it might be important to assess the risk of these strains living in nature. How easy is for S. cerevisiae and L. plantarum to live in the environment and for how long?

SIMON: Maybe [...] the introduced vitamins than make them more adapted to some medium? Maybe it can change the [natural] equilibrium.

LARA: For example, in the [micro]flora [...].

JUANMA: Normally you give rodents your product during 90 days, and then you assess this [...].

JUANMA: [...] We can conclude about [...] the strain. First its pathogenicity: do you think there is enough proof in the scientific body of knowledge or you would request extra experiments?

URSZULA: I would assume the organisms to be biosafety level 1. We can assume that they are safe as they are, with the possibility that they can mutate or that someone could get an infection if their immune system is compromised [...]. This is a minimal risk [...] negligible for these strains.

JUANMA: What do you think about the survival in the environment of the modified strains? Could it make a risk to the environment? [...] does it change the strain so that their ability to survive is higher and therefore constitutes a risk for the rest of microorganisms?

SIMON: Do you mean that the overproduction of vitamins can become a risk to the environment?

JUANMA: No, but the fact that they overproduce could make them survive better in the environment?

ALEXEY: No, because they have evolved to express the optimal level of this vitamin and if you change this level they are going to be less fit for survival.

LARA: I did not understand it.

ALEXEY: They have evolved to express just the right amount of this vitamin that they use for themselves and if you use this level, it is going to decrease their fitness.

SIMON: But they do not produce at all this kind of vitamins.

JUANMA: Yes, for some of the bacteria, they don’t naturally express the vitamin. Lactobacillus does not express all vitamins.

SIMON: Maybe their optimal level is 0. [...] but it is also possible that they never got the DNA before.

ALEXEY: [...] bacteria exchange DNA quite often so there is always a probability that some would get this DNA and if it is needed at some point [...] it will become dominant.

SIMON: In this case, how would you ensure that they keep the gene is it is completely useless to them?

JUANMA: [...] they were thinking of restarting the culture every n times from bacteria that are produced in the lab.

LARA: I want to ask a question to ALEXEY. Do you assume that all [natural] bacteria have full fitness in their environment?

ALEXEY: For this particular environment, I would say yes [...] maybe it is a local maximum, but if it would be just a matter of exchanging the expression of a few genes they would have done it, because it is just a matter of a few mutations.

URSZULA: I assume that there is not a big risk of survival in the environment [...] I think additional experiments that show in co-cultures with bacteria that present in the environment that their fitness is not increased would be a good point.

JUANMA: [...] the information in indigenous on genetic mobile elements is also important. This includes sex factors, plasmids or other mechanisms of DNA transfer. Do you know if S. cerevisiae and L. plantarum have those elements?

ALEXEY: Yes, for yeast definitely yes because it can reproduce sexually. For bacterial there certainly could have bacteriophages or other mobile elements that would carry DNA.

JUANMA: What strategy or suggestion would you make to the scientific project in order to reduce the risk of this?

ALEXEY: No suggestion, I think it is fine as it is [...] because those genes would not increase the fitness of other bacteria as well.

JUANMA: Another important point is about the history of genetic modifications.

URSZULA: Do you mean in the natural strain? [...] Bacillus subtilis has been used a lot in synthetic biology, you would probably have to write a note on how this has been used in the past [...] What were the advantages in industry for the production of vitamins or other compounds and why they were accepted. It would play on your side that there is already a B. subtilis strain that was used and there were no problems.

JUANMA: I think this is a more about how you know your chassis or genetic background, [...] there is a level of uncertainty for genetic modifications.

URSZULA: More the bacteria is used, more studies are and more we know it is safe. B. subtilis is a good example.

JUANMA: [...] other considerations include toxicology [...] if the GMM produce toxins that may arise unexpectedly as a consequence of the genetic modification.

NIKOLA: Are the levels [...] of vitamins controlled in the rice? [...] You could die of hypervitaminosis.

JUANMA: [...] It is a reasonable concern. We are assuming that they will produce the vitamins at a reproducible and stable level [...].

URSZULA: If you succeed in producing vitamins and you sell the cooked product idli [...] you should absolutely control the level of vitamins. If you sell it dehydrated in a form of a power you should [...] provide instructions of use. How are you going to show that the level of vitamins that you have is this final product are actually beneficial? [...] if it is not beneficial, even a little risk is higher than the benefits.

JUANMA: That is another point: [...] the nutritional assessment of GM food. GM food can introduce nutritional imbalances as a result of both expected and unexpected results of the genetic modification. The genetic modification of the GMM may alter the general profile of the product [...] in this case is important to assess the safety for the whole product as well. Hopefully there are many studies about vitamins, both hyper or hypo vitaminosis. An expectation of the correct intake should be taking into account. If we need to consume 400 idlis per day it would not make sense.

URSZULA: Are there studies that show that these people have problems with vitamins?

JUANMA: Yes.

URSZULA: Then you should [...] define the level of vitamins you give them do that it does not make a shock for the organism. [...] a normal person could not have an effect [...] but in general with substances drastic changes can create problems. All these issues should be considered in the final report.

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NIKOLA: Can your product be considered as a medication?

JUANMA: [...] it would be a nutritional supplement.

ALEXEY: [...] vitamins are not considered medicines but supplements [...].

SIMON: I have never seen in a pharmacy medicines only constituted by vitamins.

URSZULA: You would not want to make your product a medical component because you need to agree with additional regulations.

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JUANMA: Other points that report include are [...] transfer of recombinant DNA, general ability of the GMM to survive, the effect on humans, animals and plants, the effect on biogeochemical processes [...].

URSZULA: If you have your powder, it activates with water. What if there is an accidental spill? I imagine if should be safely disposed if it is not used in idli and boiled [...]. How can you guarantee that people treat it carefully?

JUANMA: One of the way to prevent the microorganisms to live in the environment is to make an auxotrophic or xenobiotic strain that only survives when you supplement the medium [...]. Xenobiology works with chemical compounds that are two steps away from [...] compounds found in nature. This ensures that a molecule required for growth cannot be produced by a mutation of the genetic system. [...] you make this GM microorganism dependent on a synthetic compound.

URSZULA: It would increase the cost [...].

ALEXEY: [...] you would need to introduce some chemical compounds into your rice. Something that is not found naturally and cannot be degraded.

JUANMA: [...] this is normally used for processes with a downstream genetic purification.

ALEXEY: How can you make a microorganisms dependent on a certain compound?

JUANMA: [...] for example with XNAs, that are synthetic molecules of DNA that have additional synthetic bases.

JUANMA: But this would not be the case, [...] how would you prevent the synthetic idli microflora to be released to the environment? If this constitutes a risk, then you need to make the idli and cook it in a confined space.

NIKOLA: Maybe just confer to the bacteria the property of only grow in rice.

URSZULA: That would decrease the risk.

JUANMA: O.K., I think we are done with the science part. Now let’s talk about the implementation and the socio-economical issues.

Implementation and bioethics :

LARA: I specially like the fact that people can have this powdered yeast and bacteria in their home and have the independence [...] to get the product.

JUANMA: But then, as we discussed before, now you have a problem of release [...] how would prevent people from throwing the bacteria to the sink [...]. We have two cases [...].

NIKOLA: Maybe you can add some color to make the rice coloured and see that there is something in it apart from rice [...].

ALEXEY: [...] I think the project is not very feasible in the implementation [...]. These people are isolated in the village and probable the word “bacteria” sounds magic to them. It would be very hard to educate them about what are bacteria starting from 0 [...].

JUANMA: How would you best frame the project?

ALEXEY: I think it could be an interesting project for a more developed world, [...] as a novelty food [...] but the regulations are hard.