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

Laws and guidelines to be considered 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” (eng. 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 rise 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 a 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 a list of the minimum elements required:

1. Assessment of potential harmful effects, defined as
a) Disease in human beings animals or plants
b) Harmful effects resulting from inability to cure disease
c) Harmful effects resulting from organisms establishing itself in nature
d) Harmful effects resulting from the organism, through natural processes confers part of its genome, such as heightened resistance, to other organisms in nature
2. Resulting from
a) The host-organism to be modified
b) The parts inserted into or otherwise used to alter the organism
c) The vector
d) The donor-organism
e) The resulting modified organism
3. Characteristics for the organism's activity
4. How potent the potential harmful effects are
5. The likelihood of harmful effects being realized
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. See appendix I for the risk-assessment we made for our project.

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. All access to the lab by non-members of this group or the lab-staff is to be restricted.

All members of our team have in the time prior to the work in the laboratory received a lab-safety-course, thus fulfilling the requirement. See appendix II 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, favoring safety above economical issues.

As we're working with relatively harmless strains of E. coli (mg1655 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

The Group

“Arbejdsmiljøgruppen” (eng. The Working Environment Group) 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, its 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 Working Environment Group, 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 a level 1 GMM, which due to its 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

We have at all times upheld the laws and regulations imposed upon us by UN and by the Working Environment Group. We have performed a risk-assessment of our project as required by the UN, as well as following the laws regarding to personal safety and to substitution of potentially harmful host and donor organisms. The work safety group has assessed our project to be a class 1 project, as they have perceived no risk associated with our work.

They see no apparent way of weaponizing or in any other way using our project for malign purposes. Thus our project should not pose any threat to the security of the world at large. Although most of the genes inserted into our bacteria are harmless, hyper-flagellation is in fact something that increased pathogenicity, due to heightened mobility. Further the bacteria we work with are unable to survive and reproduce outside of laboratory conditions. Should it accidentally be released into the wild it would lose its plasmids within a very short time span and thus return to a non-GMO state. And as the bacteria we have been working with, namely E. coli mg.1065, is a naturally occurring bacteria, it should not pose any threat to the environment at all.

As long as the normative work safety protocols were followed they could not perceive any danger due to the work being performed by relatively inexperienced students.

Thus they perceived no security nor safety 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

The ideas behind the watermark

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

Following the example of J. 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 increase the safety of the environment, as well as the safety of the community at large.

Why we should consider watermarking

But why should we consider creating and using a watermarking standard for work in synthetic biology? Let us consider the following example.

A company has created a synthetic organism capable of absorbing harmful substances from the ground. The government, eager to help clean polluted ground, releases the bacteria into the wild, confident that the bacteria will not pose any threat to the environment. These bacteria do not, however, react as planned. Instead of absorbing only the harmful substances from the ground, they transfer the substances to other organisms, or simply run amok spreading themselves in an uncontrolled manner, causing harm instead of good. Let us imagine that this happens close to the border. The government in the neighboring state finds itself with a unexplained biological phenomenon, possibly causing great harm. Should the rogue bacteria contain engineered, watermarked parts, it would be a small matter to have the parts sequenced, thus the government would be able to easily access all the relevant information on the rogue bacteria, contact the manufacturers and would know how to stop it.

The watermark

We believe that a watermark should contain the following:

I. A 12 nucleotide ‘license’

We have set the following criteria for a good watermark:

I. It should contain the creating team’s ‘license’

II. It should not interfere with the other functions of the part

III. It should be persistent in the plasmid, i.e. not be removed from the plasmid due to natural evolution

IV. It should be easy to find, easy to read and easy to insert by the developing team

We have set the following criteria to the team page in the parts-registry:

I. information on the creating team

II. the name of the part

III. a description of the part

IV. the risk-assessment performed by the creating team

V. information on how to neutralize organism, and, if available, kill-code

Placing the watermark

´ The initial thought was to insert the watermark into the genome of the bacteria, as to increase the stability of the watermark in the bacteria. However, as all parts inserted into the bacteria are placed in plasmids, it would make no sense to insert the watermark into the genome. The bacteria could transfer their plasmids to other bacteria, and retain their watermark, and the watermark would have become useless, persisting in bacteria that have no modified material at all.

Also the thought was to mark the bacteria, and not the parts. Thus we could have a single watermark to cover the entire modified organism. However, this presents us with many of the above problems. Should anyone encounter a rogue bacteria which has lost some of its plasmids, the watermark would be useless. It would only cover the entire modified system, and should some, or all, the plasmids have been discarded by the bacteria, the watermark would be useless. We therefore propose to mark every plasmid, before and after the coding sequence. The watermark will therefore be split into two separate components. The team creates a watermark for every part, and inserts it into the plasmid, before and after the coding sequence.

We chose to divide the code into two separate parts for two reasons. Firstly, being split into two equally long parts at each end of the coding sequence ensures symmetry, which again should help make insertion of a watermark easy, as this will reduce the complexity of the primers we need to design to insert them. Secondly this ensures that identification is done easily, since the combination of nucleotides of the sequence from E to X and from S to P is known. Thus we effectively increase the sequence we search for from 6 to 26 nucleotides. Also this means that the risk of a naturally occurring combination of nucleotides, identical to the synthetically created one decreases.

E X Start End S P |------|----------|-------------------------------------------|---------------|---------| watermark 1 coding sequence watermark 2 license part 1 license part 2

Sequencing the part should therefore yield the watermark, which could then be accessed, read and understood.

Alternative placement

We also thought about the possibility of placing the watermark between the cutting sites of E and X, and S and P respectively. Since this area is a spacing area, it might be a good spot to insert the nucleotide combination we want. Difficulties this might cause include disturbing ... (hvad præcist? – LC snakkede om at kombinationen blev brugt til at forstærke et eller andet..). Another idea was to expand the restriction sites E-X and S-P, but we are uncertain whether this might disturb the functionality of the part.

Size and design

Placing the watermark after the restriction sites, it should be relatively small, as to not interfere with the functionality of the part into which it is inserted. We propose that the watermark should be 12 nucleotides, divided in two groups of six which will allow for 4096 combinations each.

The watermark must not contain any restriction-enzymes or stop codons. If the watermark accidentally contained a restriction-enzyme or a stop codon, the watermark would interfere with the function of the part into which it is inserted, and would likely render the part useless. This severely restricts the number of combinations we can use.

Ideally we would have liked to use more nucleotides, as we would have been able to generate more combinations. But the watermark should be as small as possible as not to interfere with the functions (i.e. cause a frame shift) of the part and not make the design of primers unduly complicated. We could make relatively long watermarks to satisfy our need for a very large number of possible combinations, but it would make the design of the necessary primers extremely complicated, and would go against our goal of making the insertion of watermarks as small and easy a procedure as possible. We believe that the best compromise between the amount of combinations and the ease of insertion would be at around 12 nucleotides.

License

Every team, and lab / company which works with synthetic biology will be assigned a specific “license plate”, unique to them and being their ‘ID’ on parts-registry. An extension to parts registry’s search function should then be added. If its database contains information on each “license”, one could easily find information on a foreign organism, in case they found it in the wild (that is, anywhere else than the lab), and had it sequenced afterwards. Upon entering the parts-registry, one should then be able to enter the license and gain access to information on the parts that the team / lab / company has created. Anyone should be able to enter the license code into the parts-registry and gain access to all information on the creating team, the parts created by the team, and of course some contact information, in order to seek advice.

Procedure

Retrieval of the watermark should be easy, in the event of a rogue bacteria spreading havoc in the environment. Standardized placement of the watermark within the part should make it possible to easily retrieving the watermark through sequencing. Thus, if a rogue bacteria is discovered, it should be a simple matter to sequence the part, and obtain the license. Then it is just a matter of consulting the parts-registry to access all available information on the part and on how to contact the creating team. Getting a bacteria sequenced is a technology which is easy to access, and a lot of companies worldwide offers sequencing of bacteria, in addition it is a relative speedy process, which further makes the identification of a malign bacteria, and its properties relatively easy.

===== 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

[https://www.retsinformation.dk/Forms/R0710.aspx?id=12325 [3]]

Once again please note that this is only intended as a guideline on how to characterize the part in the most wholesome manner.

Risk-assessment in conjunction with the use of this part in a particular organism.

Should the part, or a number of parts, be inserted into an organism the team should perform a risk-assessment and make it available on the parts-registry. In some countries, it is mandatory to submit a risk-assessment prior to engaging in a project involving synthetic biology, so we believe that any risk-assessments should be made public through parts-registry.


Inclusion of copyright information?

We do not believe in any form of copyright prohibition. We believe in an open-source approach to the field of synthetic biology, as in iGEM. Any copy-right prohibitions would only stall the progress in this most vital field of science. We believe that any and all information on created parts, and experience with these parts in particular organisms, should be shared freely.


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.

===== Anticipated problems =====
Code deterioration

The code will deteriorate over time due to mutation. This could prove to be a serious problem should watermarking become an integrated part of synthetic biology. Should the genetic watermark deteriorate to the point where one is no longer able to read it, it would not constitute any kind of safety measure, being able to tell that the part was likely to have been made artificially being the only thing we would be able to tell.

Knowing that data will deteriorate, it may be impossible to determine whether the watermark found in a rogue bacteria is authentic or a degenerate. The deterioration is however slow and arbitrary. Our code is so small, that the change that any nucleotide associated with the watermark is going to mutate is very limited. The chances of finding the authentic code intact should be very good.

The open source approach

Sharing all this information on creating new synthetic parts that can be inserted into living organisms, also means that people with harmful agendas have access to this knowledge. This naturally means that wrongful actions are possible by use of synthetic biology, but we do not think that this should stand in the way of all the possibilities synthetic biology holds. Also, the more we know about how to create synthetic DNA strands, the better we are equipped if any harmful incident should occur. As of now, it might also just be easier to drop a bomb or send out letters of anthrax.

Should a bacteria be used for a malign purpose it would be quite easy to insert a false watermark to blame others. So we need to keep in mind that a plot could be made against someone. However, if anyone was interested in harming as many as possible, this person probably wouldn’t care about watermarking at all. This also means that we cannot expect watermarking to play a part in any legal case.


==== 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 environment.

Appendix I: risk assessment of the project

Risk-assessment

Title:

Synthesis of hyper-flagellated phototaxic E. coli

Purpose:

To create a bacteria that can induce a micro-flow regulated by light

Host:

Bacteria:

E.coli.

Strains:

Mg1655

Donor:

Coding regions amplified from naturally occurent organism.

Photosensor: N. Pharaonis, S. Enterica serovar typhimurium. These create a fusion protein. Sr2 + Htr2 fra N. P. tar CheW from S. E.

Retinal: D. Melanogaster fra cDNA gen ninaB?

Flagella: I. Coli: gen flhDC

Vektors:

pSB3T5

pSB3C5

pSB3K3

pSB1A2

Insert:

Risk-assessment

Host:

Bacteria:

E. coli is naturally occurring and the strains used for amplifying vector-DNA/proteins is not reported pathogenic Al material used in bacterial work is autoclaved and/or Inactivated with Iodofor

Stains:

A cell-culture from a higher eucaryot which does not contain any endogene vectors that would be able to mobilize parts of the transferred genetic material The strains used have not been reported pathogenic The cellular strain is very fragile and is unable to procreate or survive outside of laboratory conditions, as they need the correct temperature, humidity, pH, CO2, O2 and nourishment Al material used in bacterial work is autoclaved and/or inactivated with iodofor

Donor:

Naturally occurring healthy genes of insect origin and it is not believed to be able to transform/infect human cells in vitro/vivo. The risk is therefore considered to be minimal. The S.E. gene has homology in E. coli and is therefore not considered to pose any threat. Vector: Vectors are of pUC or pOt2 origin and nothing from the vector has human recombinations/infection potential and the risk of working with these strains are therefore believed to be minimal. Our vector is in addition equipped with resistance to antibiotics and cannot exist without it. Should discard the resistance if not within a antibiotic environment.

Insert:

Is naturally occurring genes with well-defined tags and it is believed that they cannot transform/infect human cells in vitro/vivo. The fusion-protein has had limited testing, but is also considered safe. The risk is therefore believed to be minimal.

Health-aspects of the final GMO:

Bacteria not exposed to antibiotics will discard the plasmids within a very short timespan. The bacteria are modified with plasmids, and will return to a non-GMO state within a short time-span. The modification is not infective/self-reproductive in humans. It is not believed to pose any threat towards human health. We have at no point worked with any self-reproductive or pathogenic material

Assessment=Class 1

Watermark generator

Hej

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