The first safety concern we had was, understandably, creating a kill-switch. Kill-switches are are common practice for synthetic biology and for a good reason, and are made even more important when it comes to a project like ours, where you want your final GMO product to spread as much as possible, safely. Our entire project was designed and executed side-by-side with our Integrated Human Practices, trying to gain as much feedback as we could from specialists about the safety and biocontainment of our P. agglomerans.
We designed a pH-based kill-switch, but as we didn’t get to the testing stage of releasing it into the wild, it didn’t get to be put to work for real. Additionally, we gathered all the information we have on designing a paratransgenesis kill-switch on our Conceptual Framework.
The elimination module of a paratransgenesis should, ideally, eliminate the target. And the stronger the effect the better, right? Well, not really. As we discussed in our Elimination page, when picking paratransgenesis effectors you must keep in mind how broad-working they are. Ideally, it should harm pathogens as much as possible while harming humans (and other non-pathogenic organism) as least as possible.
Our only effector with a Risk Group classification was DN59, against the dengue virus, characterized as Risk Group 2. However, it’s classified as so because it is derived from one of the virus’ protein - but modified to block that protein, which made it completely harmless to humans and very troubling for the virus.
Another important aspect of designing a safe, implementable paratransgenesis strategy is choosing the right chassis. That are many things that factor into that, but from a safety perspective, it needs to pose as little harm to humans as possible. We go into all of that in detail in our Conceptual Framework section.
P. agglomerans, our chosen chassis, is mostly a Risk Group 1 organism. We say “mostly”, because it really depends on the strain and on the literature source you base yourself on. Some strains can cause infections in immunocompromised individuals (but pose no threat to healthy adults), while others pose no risk at all to humans. Our strain was isolated from the eucalyptus tree, and it does not cause infection to humans at all, so we considered it to be a Risk Group 1 organism. Regardless, since we had access to a Biosafety Level 2 lab, we chose to perform all our experiments with P. agglomerans there.
Tiago filling out the Safety Form. You can never be too safe!
Our proof of concept experiment of colonizing the mosquito midgut with transformed bacteria (you can read more about it here) was initially suggested by Prof. Guimarães (you can read about our interview with her here), and she oversaw the entire thing. The experiments were performed in Prof. Guimarães’ lab, which is a Biosafety Level 2 lab and has the proper infrastructure to handle the mosquitoes. In fact, since Prof. Guimarães researches genetically modified mosquitoes, they need extra safety measures. Two of her students, Helena Araújo and Rafaella Sayuri Loshino, gave us the appropriate safety training and instructed us on how to properly dissect the mosquitoes. They also accompanied us the entire time, helping us perform the experiment.
As this kind of experiment is something novel to iGEM as a whole (and even for Prof. Guimarães’ lab, too - they had never tried to colonize mosquito midguts with GMO bacteria), we made sure that her lab had the appropriate safety infrastructure, and we checked with the iGEM Safety Committee before starting.
Brazilian Regulations & Shipment
Impractical rules - “House Rule goes”
In Brazil, unfortunately, Biosafety legislation is still far from perfect. It was designed at a different era with a different scientific context in mind - and Brazilian researchers know this, and often find themselves trying to work despite these regulations rather than with them. And we understand and agree with the necessity of biosafety regulations, we are not here to advocate for completely unregulated science. But the problem that arises with regulation that gets in the way of things is pretty evident in Brazil: if it’s too unreasonable, people won’t follow it. What ends up happening, for example, is that “house rules” override these legislation - there are official regulations by Brazil’s Biosafety Technical Committee (CTNBio, we talked to some of them in our Integrated Practices), but research institutions are free create (and follow) their own biosafety norms based off of the national standard. The institutional biosafety norms, at least in our case, are very carefully crafted by a team of specialists, and these norms are often nearly identical to the national regulations, so they do a good job at guaranteeing that researches are not at risk. But that still sets a dangerous precedent - although institutionally defining and following safety norms is always a good thing, the official legislation should prevent it from depending on a person’s (or a group of people’s) morals and opinions, that’s the whole point of laws.
So, in Brazil, the scenario is basically two things: Brazilian researchers are met with piles and piles of bureaucracy that they must always be dodging to try to get work done, and Brazilian researchers are at the same time at more risk, because the regulations end up getting bent to the point of losing effectiveness.
For our project, our team adhered to the institutional safety norms for the experiments, and we also defined precautions and norms for ourselves to try to ensure we were working in safe conditions. In our Integrated Human Practices, we talked to specialists about the national regulations regarding the release of GMOs, to try to shape our project to fit these guidelines. But, unfortunately, making the national biosafety regulations better and more effective is a very difficult and long-termed task.
During the process of shipping our parts to the registry, we met with another example of outdated, research-limiting regulations. The international regulations for shipping of biological material (mainly, IATA’s Dangerous Goods Regulations) are very strict - but also very thorough. Their strictness is understandable, biosafety is a very important issue, and the shipping of biological materials should be very carefully handled. But no matter how strict, they still cover a lot within these regulations - meaning that whatever biological material you wanna send, you are able to send it, you just need to fill out the corresponding paperwork. Brazil’s shipping regulations on biological materials, however, is very lacking in comparison. For example, rather than determining norms for shipping of infectious agents (like IATA does), Brazil’s Health Regulatory Agency ANVISA just bans shipping it completely. That didn’t stop us from shipping the registry DNA samples, but it hampers Brazilian research unimaginably. Also, shipping of ALL biological material in Brazil must be done under the name of Institutions, not individuals, which also added a world of unnecessary bureaucracy for us. For international shipments of goods from Brazil, senders are required to provide an NCM (Mercosul Common Number), a number ID that identifies which kind of product is being shipped. However, since Brazil shipping regulations don’t take biosafety regulation under consideration, the closest description of our DNA samples we could find was “Miscellaneous Unspecified Goods from Animal Sources” (despite our samples being from E coli). And we weren’t the only Brazilians facing these issue, either. We were communicating extensively with teams AQA_Unesp and Amazonas_Brazil when preparing our DNA samples, to try to help each other out with the loads of pointless bureaucracies.
Shipment instructions for future Brazilian teams
First of all, the shipment must be made under the name of an Institution, so you will need to provide a CNPJ (if your PI or someone you know has a CNPJ and are willing to send it for you, that works too). You will need a signed declaration (in Portuguese and English) that states the detailed description of the product (Exempt Biological Material: DNA (non-infectious, non-hazardous, non-regulated) and its purpose (research only), and stating that you are aware that your package will be submitted, without exception, to an X-ray machine, and that the submission to the X-ray machine will not compromise your shipment’s functionality (yes, we are aware of the contradiction of that sentence; the Federal Police, however, isn’t). The declaration will need to be printed in letterhead paper (“papel timbrado”). Your package will need to be sent through EMS, and will need to be identified with NCM 05119999 (“Miscellaneous Unspecified Goods from Animal Sources”. Yes, we realize the source of your DNA is E coli, which is not an animal. No, there is no better identification). They will tell you that your package must adhere to IATA regulations, but according to section 220.127.116.11.3.1 of IATA’s Dangerous Goods Regulations (DGR) manual (and according to section 8.1 of ANVISA’s Manual for Shipment of Biological Materials), exempt biological materials do not need to adhere to any specific shipping package regulations. The package regulations they claim are necessary basically consists of: two layers of leak-proof packaging, with an outer layer of a sturdy material such as a cardboard box. Although it isn’t necessary, we recommend following these guidelines just for good measure, specially since the shipping materials iGEM provides already meet the criteria (leak-proof layer 1: the sealed plate; leak-proof layer 2: the ziplock bag; sturdy layer: the cardboard box).