Team:SDU-Denmark/safety-b

Biosafety

Would any of our project ideas raise safety issues

Do any of the new BioBrick parts (or devices) that was made this year raise any safety issues?

analysis of parts and devices

SopII – No homogenity when blasted. The gene is mentioned in Halobacterium salinarum R1 [1] and in Natronomonas pharaonis DSM 2160[2] as being protein coding. The protein is a light dependant iontransporter, and is often seen with the halophilic bacteria (halophilic: organisms that thrive in high concentrations of salt). So it might make non extremophiles more competitive in environments with high salt concentrations. The word: “might“ should be underlined, as it of course takes several genes / proteins to make it more durable in high salt concentrated areas . This clearly is a risk. If a bacterium were to gain the advantage, to survive in high salt concentrations, it would mean we could create diverse bacteria which more easily can survive, and proliferate in completely different environment, than we usually meet them. The outcome of “old” bacteria proliferating in another environment than usual is not easy to foresee.

HtrII – (sequence?). It contains three domains, which gives a chemotaxis-like property towards Methyl, Aspartate and related amino acids. One of the domains (cl01054, which is the one out of three) is commonly observed in bacteria. [3] The fact that it is often represented in bacteria creates a lower safety risk, as it will less likely transfer its genes to bacteria which don’t have this specific chemotaxis already.

Tsr - The protein is often present in various cell-types. It serves as a methyl-accepting chemotaxis protein. In some certain bacteria it is required for morphogenesis of rhabdomere [4]. As in the case with htrII, the same lack of issues arises; many different bacteria already have this property and/or specific gene.

CheW – No homogeneity when blasted. The CheW protein is yet another chemotaxis protein. In halobacterium it is only to link with CheA (which can enable the flagella-motor), but, at the moment, we were not able to figure out whether or not this specific protein is able to interfere with other pathways. By the looks of it, it will not interfere with other pathways [5]. This uncertainty creates a few problems. The fact that not every pathway is known, could lead to unknowingly give ‘wild’ bacteria an advantage. If this were to happen, people could criticize us, for not doing science – as we are not aware of what we are doing, and should not toy with something which we cannot foresee the consequences of.

CheA – is another chemotaxis protein, and is known to appear in many different bacteria. The same safety issue, or lack of such, which is described for the CheW-gene is also present here [6].

CheY – contains signal receiving domains. Present in bunch of bacteria with flagella. The same safety issue, or lack of such, which is described for the CheW-gene is also present here [7].

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Is there a local biosafety group, committee, or review board at our institution?

Which laws and guidelines we have to consider in Denmark

Which laws and guidelines we have to consider in Denmark

The scope of this part of the paper is to draw attention to some of the laws and guidelines, which we have to consider in Denmark, when we are dealing with genetically modified microorganisms (GMM's). Our project is defined as an 'contained use' project, which means that the organisms we are handling are contained from the environment at large. The following laws are based on the ”Bekendtgørelsen om Genteknologi og Arbejdsmiljø” (litt. The order on gene-technology and working environment) of 2008, which follows the rules laid down by the European Union in 1990 in the ”directive on the contained use of genetically modified micro-organisms”.

Risk-assessment One of the first, and indeed one of the weightiest points in the directive on GMM safety, is to ensure the public health and the preservation of the environment. And...

To that end [to avoid adverse effects on human health and the environment which might arise from the contained use of GMM’s], the user shall carry out an assessment of the contained uses as regards the risks to human health and the environment that those contained uses may pose, using as a minimum the elements of assessment and the procedure set out in Annex III, Sections A and B.

Article 4.2

It is required of us to make an throughout risk-assessment, so that we may judge if our use of GMM's poses a threat towards the well being or safety of human beings, animals, plants, or the environment. To help perform this assessment, the UN has laid down a minimum standard of elements required to make an adequate assessment of the potential harm of an accident resulting in the release of the GMM's into the environment. The following is an list of the minimum required elements:

   -Assessment of potential harmful effects, defined as:
       -disease in human beings animals or plants
       -harmful effects resulting from inability to cure disease
       - harmful effects resulting from organisms establishing itself in nature
       -harmful effects resulting from the organism, through natural processes confers part of it's genome, such as heightened resistance, to other organisms in nature

   -resulting from:
       -the host-organism to be modified
       -the parts inserted into or otherwise used to alter the organism
       -the vector
       -the donor-organism
       -the resulting modified organism

   -characteristics for the organism's activity
   -How potent the potential harmful effects are
   -The likelihood of harmful effects being realised

Based on this risk-assessment it is possible to rank the project according to the risk, ranking from level 1 to 4, in accordance to the procedure giving by the UN.

Personal safety To be allowed to work in a level 1 laboratory, it is required that there at all times is a suitable instructed person present. At level 2, all personnel in the laboratory is required to have been suitable instructed in lab safety and procedure. Al access to the lab by non-members of this group or the lab-staff is to be restricted. All members of our team has in the time prior to the work in the laboratory received a lab-safety-course, thus fulfilling the requirement. See appendix x for the actual safety guidelines laid down by our local work-safety group.

Substitution Further, it is not allowed to work with any host, donor or vector-system, should another, safer, system, containing the same basic features, be available. If it is possible to find a suitable system, compatible with the intended work, that is safer for humans, animals and plants, or the environment at large, it must always substitute the other, more dangerous system. It is in other words prohibited to take unnecessary risks, or use unnecessarily risky setups. Should a possible substitute system be unreasonably difficult or expensive to acquire, then the risks and benefits must be weighted out against each other, favouring safety above economical issues. As we're working with an relatively harmless strains of E.coli (mg16 and TOP10), it has not been necessary to locate a safer, compatible host, donor or system, but we have nonetheless attempted to locate such systems for wholesomeness, although without luck.

Assessment by local bio-safety group

Assessment by local bio-safety group Arbejdsmiljøgruppen is the local bio-safety group associated with the University of Southern Denmark. During an interview with a representative from this group we explained the project, it's scope, parts and procedure. The following is a number of questions concerning the safety and security issues relating to our project, and the essence of their replies

If they perceived an increased risk due to work being performed by relatively inexperienced students The project is not considered any more dangerous due to the fact that most of the work in the lab is performed by relative inexperienced students. As long as the lab's safety protocol is followed, and the fact that the risk-assessment of the work safety group put our project firmly on level 1, they believe that there should be little to no risk to lab personnel or the outside environment. As all students participating in the lab has successfully completed the lab safety course provided by the arbejdsmiljøgruppe, they perceived no increased risk.

If they perceived any danger should the bacteria get out of the lab They perceived no danger to the environment or the well being of animals, plants or human being should the bacteria be released into the environment. This is due to the extremely fragile nature of the E. coli strain that we are using in our project. Should it somehow find its way outside of the lab, it would die within a very short time.

If there exists an emergency safety protocol in case of accident (i.e. unintentional release of GMM's into environment) The emergency protocol is still a work in progress, but although it is unfinished it should not pose a breach in safety, as we're only working with an level 1 GMM, which due to it's extremely fragile nature cannot survive outside of laboratory environment. This coupled with adherence to the standard laboratory safety protocol, should at all times ensure the safety of the environment.

Overall assessment The work safety group [ufærdig] They see no apparent way of weaponizing or in any other way using our project for malign purposes. None of the modifications made to the bacteria has in any way made it more pathogenic. Further the bacteria we work with are unable to survive and reproduce outside of laboratory conditions. Thus they perceived no security issues with our project.

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Our ideas on how to deal with safety issues that could be useful for future iGEM competitions! How could parts, devices and systems be made even safer through biosafety engineering!

watermarking standard

To increase public safety we propose to introduce a water-marking standard.

Following the example of Craig Venter, who, in may 2010, created the first watermark in a bacteria, containing several readable messages, we propose to create a watermarking standard to help protect the intellectual rights of the developing team, as well as the safety of the community at large.

We believe that a watermark should contain the following:

   -the name of the organism
   -the name of the developing team
   -a link to the parts-registry containing:
       -a full description of the organism
       -the risk-assessment performed by the creating team, if available
       -the intended use of the organism
       -copyright information
       -information on how to neutralise organism, and, if available, kill-code

We have set the following criteria for a good watermark:

   -It should contain all the above information
   -It should not interfere with the other functions of the bacteria
   -It should be persistent in the bacteria, i.e. not be removed from the genome due to natural evolution
   -It should be easy to find, easy to read and easy to insert by the developing team

notes The watermark must be inserted into the genome, and not in a plasmid, as it would in theory be able to pass it's watermark on unto other naturally occurring bacteria.

The watermark should be relatively small, as it should not interfere with the functionality of the bacteria. >< Should be connected to a vital function as not to be removed due to natural evolution, perhaps linked to the bacterias metabolism.

Standarlized location or function in the bacteria targeted for insertion of watermark, to make it as easy as possible to locate watermark in rogue bacteria. Should be possible to develop specialized enzyme to cut genome at the desired location, making the creation of a standardized watermarking kit possible.

A full description of the modified organism

A full description of the modified organism should ideally contain the following information

A. characteristics of the host and donor organisms 1. Name(s) of the organism(s) in question

2. Origin of organism(s) in question

3. Information on the reproductive cyclus of the parental organisms as well as the host

4. Description of any previous genetic modification

5. Stability

6. Details concerning pathogenesis, virulence, infectivity or toxicity

7. characteristics of endogene vectors:

-sequence

-mobilisation

-specificity

-the presence of resistance-genes

8. host spectrum,

9. potentially significant physiological traits and the stability of these traits

10. natural habitat

11. significant role in environmental processes

12. Competition or symbiosis with other naturally occurring organisms

13. Ability to create survival structures (i.e. the ability to create spores)

B. characteristics of the genetically modified organism

1. origin of the genetic material used to modify the organism, as well as the intended functions of this material

2. Description of the modification, including the method of vector insertion in the host organism, as well as the method used to create the genetically modified production-organism

3. the function of the genetic modification

4. origin and characteristics of the vector

5. structure and size of vector in the genetically modified production-organism

6. stability of the organism with respect to genetic traits

7. mobiliseringshyppigheden of the inserted vector and/or the organism’s ability to transfer genetic material

8. activity of the expressed protein

C. Health concerns

1. Toxic or allergenic properties

2. Product risks

3. The genetic modified organism’s pathogenic properties compared with the donor – or the host organisms or possibly the donor organism

4. Colonization ability

5. If the organism is pathogenic to humans, who are immune competent:

a) Cause illness and the pathogenic mechanism, including invasiveness and virulence

b) infectivity

c) infective dose

d) host range, possibility of change

e) possibility for survival outside the human host

f) The presence of vectors or other distribution areas

g) Biological stability

h) resistance patterns against antibiotics

i) allergencity

j) chance for suitable disease treatment

D. Environmental concerns

1. factors that might affect the organism’s ability for survival, reproduction and it’s ability to spread in the environment.

2. techniques for detection, identification and surveillance of the modified organism

3. techniques for detection of transfer of genetic material to other organisms

4. known and expected habitats of the modified organism

5. description of ecosystems into which the organism could spread in the event of an accident

6. expected result of interaction between the modified organism and naturally occurring bacteria that would be affected in the event of an accident

7. known and expected effects on animals and plants, with regards to pathogenesis, virulence, infectivity, toxicity, allergenicity, colonisation

8.known or expected contribution to bio-geo-chemic processes

9. methods for decontamination of the area in the event of an accident

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Copyright information

It should contain all relevant copyright information, as to protect the intellectual property of the creating team. But what exactly is to be considered the subject of copyright? Is it the individual parts? Their functions? Or can one only claim copyright for the entire system?

Information on how to neutralize bacteria

This clause is intended as security measure. Should the bacteria be released into the environment, the parts-registry site should contain information on how to neutralize the bacteria.

If the bacteria has an kill-code inserted, the site should describe how to enact the self-destruct mechanism.

kill-code

-Why should we consider inserting kill-codes in genetically modified organisms -What should an efficient kill-code contain -Which bacteria should have a kill-code inserted -When should one enact a kill -Should it be mandatory or optional -Availability of the kill-code

Why should we consider kill-codes

Why should we consider inserting an self-destruct device into modified bacteria? No system is completely safe. Accidents, no matter how statistically unlikely, will occur. This is especially true when human beings are involved.

An efficient kill-code should

   -be activated by an efficient signal
   -be persistent
   -terminate the bacteria within a very short time span
   -not interfere with other functions in the bacteria

Efficient signal

What is to be considered an efficient signal? It should be a signal that

-We can control

-We can induce at will

-That is unlikely to affect other organisms in any harmful way

Would it be sensible to use a naturally occurring signal? Would be beneficial should the bacteria be released into the environment, where the naturally occurring signal would help destroy the rogue bacteria within the shortest possible time span. Of course, it would only be usable if the laboratory does not itself emit, or at least is able to shield the organism, from the activating signal. An example could be that the kill-code is activated by light of a certain wave-length. If the sun emits this wave-length of light, it should destroy any bacteria that might have been released into nature. It is very easy to shield the organism from the light of the sun in the laboratory, and thus we will not accidentally destroy controlled organisms. Thus we could satisfy the three criteria I have listed above: we can control light of a certain wave-length, at least in a laboratory environment. We can induce this light at will and, thirdly, this light will not harm any other organisms. One of the major cons of using a naturally occurring signal is that it would be almost impossible to use the organism, in case it would serve any environmental purposes.

Persistence

The kill-code would be left useless should the bacteria dispose of the code through natural evolution within a very short time. Should the bacteria accidentally or, being subject to malign use, intentionally be released into the environment, we would be unable to enact the built-in kill-code, if the code is not linked in some manner to a vital part of the bacterium's genome. If the code is linked to an essential part of the bacterium's genome, it should be unable to dispose of the code without self-termination, thus ensuring persistence. Without the requirement for persistence, the kill-code would give a false sense of security, not knowing if the code is still present in the organism in question.

Termination within a short time span

The shorter the amount of time before the signal is enacted, 'till the rogue bacteria is destroyed, the less harm it is likely to cause. Non-interference with other functions

Which type of bacteria should contain kill-codes

We suggest level 3 and 4 bacteria would be edible for insertion of a kill-code. Level 1 bacteria pose little to no threat to human beings or the environment, and insertion of a kill-code would not be relevant. Level 2 organisms too would not pose any notable threat, and insertion of a kill-code would be overkill. Level 3 and 4 organisms however pose moderate to serious threat to human beings, animals, plants and the environment at large. Should any of these organisms escape into the outside world, they would cause considerable harm to the milieu.

Watermark generator

Hej

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