Difference between revisions of "Team:UCopenhagen/Safety"

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<div class="column full_size">
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<h1>Safety</h1>
  
−
<h1> Safety </h1>
 
−
<p>Please visit the <a href="https://2018.igem.org/Safety">Safety Hub</a> to find this year's safety requirements & deadlines, and to learn about safe & responsible research in iGEM.</p>
 
  
−
<p>On this page of your wiki, you should write about how you are addressing any safety issues in your project. The wiki is a place where you can <strong>go beyond the questions on the safety forms</strong>, and write about whatever safety topics are most interesting in your project. (You do not need to copy your safety forms onto this wiki page.)</p>
+
<h2>Safety Tour First</h2>
  
−
</div>
 
  
 +
<p>
 +
Before entering the laboratories, it was mandatory for us to take the Safety Tour organized by the Laboratory Coordinator at our Host Institution; Center for Synthetic Biology at University of Copenhagen. All team members, incl the ones that might not be working in the lab later on, took this safety tour to ensure safety and education for the team as a whole. Especially proper waste disposal of GMO material and personal lab safety was underlined.
 +
</p>
 +
<p>
 +
Due to our choice of bacterial strain (see more below), we were given instructions as to how we handle our strain with extra precautions it in the laboratory, including:
 +
</p><ol>
  
−
<div class="column two_thirds_size">
+
<li>Creating a buffer zone in the lab, that only members with the right protection may enter while handling the bacteria
−
<h3>Safe Project Design</h3>
+
<li>Wearing mouth, nose and eye protection
 +
<li>Wearing lab coats at all times </li></ol>
  
−
<p>Does your project include any safety features? Have you made certain decisions about the design to reduce risks? Write about them here! For example:</p>
+
<p>
 +
These precautions were decided upon in collaboration with our internal Safety Coordinator and Head of Studies Kirsten Jørgensen from the University of Copenhagen.
 +
</p>
 +
<h2>Bacteria: Evaluating the safety risks </h2>
  
−
<ul>
 
−
<li>Choosing a non-pathogenic chassis</li>
 
−
<li>Choosing parts that will not harm humans / animals / plants</li>
 
−
<li>Substituting safer materials for dangerous materials in a proof-of-concept experiment</li>
 
−
<li>Including an "induced lethality" or "kill-switch" device</li>
 
−
</ul>
 
  
−
</div>
+
<p>
 +
Our bacteria proved to be a challenge when we tried to ensure we followed proper safety guidelines.
 +
</p>
 +
<h3>Bacteria</h3>
  
−
<div class="column third_size">
 
−
<h3>Safe Lab Work</h3>
 
  
−
<p>What safety procedures do you use every day in the lab? Did you perform any unusual experiments, or face any unusual safety issues? Write about them here!</p>
+
<p>
 +
The injectisome system needed for our project could be obtained from several different bacteria (<a href="https://www.nature.com/articles/nrmicro1526">https://www.nature.com/articles/nrmicro1526</a>).
 +
</p>
 +
<p>
 +
We chose to work with  ended up working with a chassis encoding the type three secretion system<strong> (</strong>T3SS) from Enteropathogenic <em>Escherichia coli</em> (EPEC). EPEC can cause diarrhoea in humans when it adheres to the intestinal tissue and successfully injects effector proteins through its 'needle' - the T3SS.
 +
</p>
 +
<p>
 +
We chose this particular strain since it fulfilled several of our safety requirements:
 +
</p><ol>
  
 +
<li>Non-pathogenic chassis E. coli K-12
 +
<li>The strain had effector protein, promoters and transcriptional regulators removed. There was therefore no risk that the bacteria we would be working with would be able to inject its normal disease causing proteins through the T3SS.
 +
<li>The Assembly of the T3SS was inducible</li></ol>
  
−
<h3>Safe Shipment</h3>
+
<p>
 +
By choosing this particular non-pathogenic bacterial strain we tried to minimize the risk that working with it would bring.
 +
</p>
 +
<p>
 +
The natural attachment site is intestinal cells, which is why mucus-lining protection has been implemented in our safety precautions.
 +
</p>
 +
<h3>Transport restrictions </h3>
  
−
<p>Did you face any safety problems in sending your DNA parts to the Registry? How did you solve those problems?</p>
 
−
</div>
 
  
 +
<p>
 +
Our first choice was from the Salmonella enterica subspecies enterica serovar Typhi, that turned out to be on the Australia Groups list and therefore regulated. 
 +
</p>
 +
<p>
 +
The Australia Group rules apply not only to whole organisms but also, as stated on their website, to "Genetic Elements and Genetically-modified Organisms" - that includes "any gene or genes specific to any listed bacterium... [which] could endow or enhance pathogenicity."(<a href="https://australiagroup.net/en/human_animal_pathogens.html">https://australiagroup.net/en/human_animal_pathogens.html</a>)
 +
</p>
 +
<p>
 +
A chassis containing the Salmonella T3SS would therefore qualify for regulation.
 +
</p>
 +
<p>
 +
After discussion with Piers Millett from the iGEM Safety Counsel and some research on the Danish safety regulations, we were informed that acquiring and transporting the bacteria would require us to obtain extra permissions.
 +
</p>
 +
<p>
 +
We chose to substitute the Salmonella T3SS part with the EPEC version, to side step problems about permissions, since this part isn't on the Australia Group's List of human and animal pathogens and toxins for export control and is therefore not regulated in the same way. This meant that we were able to obtain a bacterial strain with a working injectisome (T3SS) for our experiments.
 +
</p>
 +
<p>
 +
Since the two injectisome systems could be considered functionally - if not genetically - equivalent, this made us question why only the Salmonella T3SS is regulated. This question prompted Piers Millett to provide the Australia Group with an overview of how our project connects to the challenge of functional equivalence in genetic elements between listed and unlisted bacterial pathogens. We have been informed that this topic may be discussed at the next formal implementation meeting of the group.
 +
</p>
 +
<h3>Working in a level 1 or 2 lab</h3>
 +
 +
 +
<p>
 +
To ensure we worked in a laboratory that followed the proper safety regulations, we needed to determine whether a level 1 or level 2 lab was necessary
 +
</p>
 +
<p>
 +
As stated on the iGEM Safety Hub:
 +
</p>
 +
<p>
 +
"<strong>Virulence factors</strong> refer to the properties (i.e., gene products) that enable a microorganism to establish itself on or within a host of a particular species and enhance its potential to cause disease. Virulence factors include bacterial toxins, <strong>cell surface proteins that mediate bacterial attachment</strong>, cell surface carbohydrates and proteins that protect a bacterium, and hydrolytic enzymes that may contribute to the pathogenicity of the bacterium." (<a href="https://2018.igem.org/Safety/White_List#FAQ">https://2018.igem.org/Safety/White_List#FAQ</a>)
 +
</p>
 +
<p>
 +
This means that the Injectisome and the connected effector proteins can be considered virulence factors and can cause disease in humans or mediate bacterial attachment to mammalian cells.
 +
</p>
 +
<p>
 +
We chose to work with a bacteria with the natural disease-causing effector proteins removed. Therefore we were allowed to work in a level 1 classified laboratory. Still, this  required us to apply for permission from Danish Working Environment Authority (Arbejdstilsynet).
 +
</p>
 +
<p>
 +
This decision was taken after a discussion between ourselves, our supervisors and our internal Safety Coordinator at University of Copenhagen, Dept. of Plant and Environmental Sciences, Kirsten Jørgensen. The decision was later confirmed by another Safety Coordinator connected to the University of Copenhagen and sent to the iGEM Safety Council to confirm that we were working in the appropriate laboratory both by direct contact with iGEM representative Piers Millett and by submitting a safety check-in form. 
 +
</p>
 +
<h3>Mammalian vs plant cells in experiments</h3>
 +
 +
 +
<p>
 +
To test our concept we wished to show secretion of protein by the injectisome and through a membrane. To avoid working with mammalian cells, we decided to only use artificially constructed lipid liposomes and onion cells to prove our concept.
 +
</p>
 +
<h2>Integrating Safety Considerations in our Design</h2>
 +
 +
 +
<p>
 +
We took several safety considerations into account when we were deciding on how our system should look and who should use it.
 +
</p>
 +
<h3>1: Safety in our system</h3>
 +
 +
 +
<p>
 +
Early in our project design process, we chose to avoid using the injectisome as a direct drug-delivery system, since this method has several integrated risks: Controlling the amount and purity of the protein as well as  ensuring a correct folded and fully functioning  protein.
 +
</p>
 +
<p>
 +
To avoid this we decided to make an optimized protein production and purification system that naturally has a step between collection by an expert and injection into a patient. The protein product should in theory be pure and have all the advantages that using the injectisome brings, but the product is still testable by experts use by being secreted directly into the collection chamber - instead of the patient's body.
 +
</p>
 +
<p>
 +
This choice has some drawbacks: The equipment and time needed to transfer the protein from collection-chamber to the patient would be increased?? However,  we decided that this method would be superior as it would remove the risk of patients being injected with non-functional protein that is in the worst case scenario toxic.
 +
</p>
 +
<p>
 +
To avoid GMO contamination of the environment`, we designed the bacteria, media etc. to be contained in closed chambers.
 +
</p>
 +
<p>
 +
No kill-switch was included in our bacteria, since the environment on Mars can act as a natural kill-switch. No E. coli bacteria would be able to survive those conditions.
 +
</p>
 +
<h3>2: Safety in our choice of user</h3>
 +
 +
 +
<p>
 +
We considered making our system accessible to the general public. The ability to e.g.  produce insulin and other protein drugs  at home seemed like a the good goal.
 +
</p>
 +
<p>
 +
We decided against this approach when we realized the risk of mishandling the system, the bacteria and environmental contamination. Since we designed our system to naturally implement a step meant to be used for testing the protein product and ensuring it is both effective and safe, making the system usable by the entire population seemed problematic. 
 +
</p>
 +
<p>
 +
To ensure the safety of the patient using the protein product we changed our target users  to  include educated personnel only.
 +
</p>
 +
<p>
 +
Any astronauts using our system on Mars would  therefore be required to undergo  training before use.
 +
</p>
 +
<h3>3: Dual-use concerns </h3>
 +
 +
 +
<p>
 +
The natural function of the injectisome in bacteria is to produce and inject disease-causing proteins into mammalian cells. Therefore our system harbors the inherent risk of being misused to produce toxic proteins.
 +
</p>
 +
<p>
 +
To evaluate this risk of dual-use, we reached out to our national institution The Centre for Biosecurity and Biopreparedness (CBB). As stated on their website, The centre "is the national authority that issues licences to research institutions, pharmaceutical companies, hospital laboratories etc. to allow them to work with biological dual use components" and was therefore the perfect candidate to evaluate our project. (<a href="https://www.biosecurity.dk/">https://www.biosecurity.dk/</a> Viewed 18:00 11/09/2018)
 +
</p>
 +
<p>
 +
Analyst at CBB Jacob Hofman-Bang, PhD (Molecular Biology) was very helpful in  answering questions and he conducted a dual-use assessment for our project.
 +
</p>
 +
<p>
 +
The conclusion of the assessment was that our Protein Printer system does not, in its current form, possess dual-use potential. Though it is stated that at a later stage: Were it is possible to produce large amounts of proteins or to produce currently impossible proteins, we should contact CBB again, as such a system would need to be reevaluated.
 +
</p>
  
 
</html>
 
</html>
 +
{{UCopenhagen/Footer}}

Revision as of 20:02, 16 September 2018

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Safety

Safety Tour First

Before entering the laboratories, it was mandatory for us to take the Safety Tour organized by the Laboratory Coordinator at our Host Institution; Center for Synthetic Biology at University of Copenhagen. All team members, incl the ones that might not be working in the lab later on, took this safety tour to ensure safety and education for the team as a whole. Especially proper waste disposal of GMO material and personal lab safety was underlined.

Due to our choice of bacterial strain (see more below), we were given instructions as to how we handle our strain with extra precautions it in the laboratory, including:

  1. Creating a buffer zone in the lab, that only members with the right protection may enter while handling the bacteria
  2. Wearing mouth, nose and eye protection
  3. Wearing lab coats at all times

These precautions were decided upon in collaboration with our internal Safety Coordinator and Head of Studies Kirsten Jørgensen from the University of Copenhagen.

Bacteria: Evaluating the safety risks

Our bacteria proved to be a challenge when we tried to ensure we followed proper safety guidelines.

Bacteria

The injectisome system needed for our project could be obtained from several different bacteria (https://www.nature.com/articles/nrmicro1526).

We chose to work with ended up working with a chassis encoding the type three secretion system (T3SS) from Enteropathogenic Escherichia coli (EPEC). EPEC can cause diarrhoea in humans when it adheres to the intestinal tissue and successfully injects effector proteins through its 'needle' - the T3SS.

We chose this particular strain since it fulfilled several of our safety requirements:

  1. Non-pathogenic chassis E. coli K-12
  2. The strain had effector protein, promoters and transcriptional regulators removed. There was therefore no risk that the bacteria we would be working with would be able to inject its normal disease causing proteins through the T3SS.
  3. The Assembly of the T3SS was inducible

By choosing this particular non-pathogenic bacterial strain we tried to minimize the risk that working with it would bring.

The natural attachment site is intestinal cells, which is why mucus-lining protection has been implemented in our safety precautions.

Transport restrictions

Our first choice was from the Salmonella enterica subspecies enterica serovar Typhi, that turned out to be on the Australia Groups list and therefore regulated.

The Australia Group rules apply not only to whole organisms but also, as stated on their website, to "Genetic Elements and Genetically-modified Organisms" - that includes "any gene or genes specific to any listed bacterium... [which] could endow or enhance pathogenicity."(https://australiagroup.net/en/human_animal_pathogens.html)

A chassis containing the Salmonella T3SS would therefore qualify for regulation.

After discussion with Piers Millett from the iGEM Safety Counsel and some research on the Danish safety regulations, we were informed that acquiring and transporting the bacteria would require us to obtain extra permissions.

We chose to substitute the Salmonella T3SS part with the EPEC version, to side step problems about permissions, since this part isn't on the Australia Group's List of human and animal pathogens and toxins for export control and is therefore not regulated in the same way. This meant that we were able to obtain a bacterial strain with a working injectisome (T3SS) for our experiments.

Since the two injectisome systems could be considered functionally - if not genetically - equivalent, this made us question why only the Salmonella T3SS is regulated. This question prompted Piers Millett to provide the Australia Group with an overview of how our project connects to the challenge of functional equivalence in genetic elements between listed and unlisted bacterial pathogens. We have been informed that this topic may be discussed at the next formal implementation meeting of the group.

Working in a level 1 or 2 lab

To ensure we worked in a laboratory that followed the proper safety regulations, we needed to determine whether a level 1 or level 2 lab was necessary

As stated on the iGEM Safety Hub:

"Virulence factors refer to the properties (i.e., gene products) that enable a microorganism to establish itself on or within a host of a particular species and enhance its potential to cause disease. Virulence factors include bacterial toxins, cell surface proteins that mediate bacterial attachment, cell surface carbohydrates and proteins that protect a bacterium, and hydrolytic enzymes that may contribute to the pathogenicity of the bacterium." (https://2018.igem.org/Safety/White_List#FAQ)

This means that the Injectisome and the connected effector proteins can be considered virulence factors and can cause disease in humans or mediate bacterial attachment to mammalian cells.

We chose to work with a bacteria with the natural disease-causing effector proteins removed. Therefore we were allowed to work in a level 1 classified laboratory. Still, this required us to apply for permission from Danish Working Environment Authority (Arbejdstilsynet).

This decision was taken after a discussion between ourselves, our supervisors and our internal Safety Coordinator at University of Copenhagen, Dept. of Plant and Environmental Sciences, Kirsten Jørgensen. The decision was later confirmed by another Safety Coordinator connected to the University of Copenhagen and sent to the iGEM Safety Council to confirm that we were working in the appropriate laboratory both by direct contact with iGEM representative Piers Millett and by submitting a safety check-in form.

Mammalian vs plant cells in experiments

To test our concept we wished to show secretion of protein by the injectisome and through a membrane. To avoid working with mammalian cells, we decided to only use artificially constructed lipid liposomes and onion cells to prove our concept.

Integrating Safety Considerations in our Design

We took several safety considerations into account when we were deciding on how our system should look and who should use it.

1: Safety in our system

Early in our project design process, we chose to avoid using the injectisome as a direct drug-delivery system, since this method has several integrated risks: Controlling the amount and purity of the protein as well as ensuring a correct folded and fully functioning protein.

To avoid this we decided to make an optimized protein production and purification system that naturally has a step between collection by an expert and injection into a patient. The protein product should in theory be pure and have all the advantages that using the injectisome brings, but the product is still testable by experts use by being secreted directly into the collection chamber - instead of the patient's body.

This choice has some drawbacks: The equipment and time needed to transfer the protein from collection-chamber to the patient would be increased?? However, we decided that this method would be superior as it would remove the risk of patients being injected with non-functional protein that is in the worst case scenario toxic.

To avoid GMO contamination of the environment`, we designed the bacteria, media etc. to be contained in closed chambers.

No kill-switch was included in our bacteria, since the environment on Mars can act as a natural kill-switch. No E. coli bacteria would be able to survive those conditions.

2: Safety in our choice of user

We considered making our system accessible to the general public. The ability to e.g. produce insulin and other protein drugs at home seemed like a the good goal.

We decided against this approach when we realized the risk of mishandling the system, the bacteria and environmental contamination. Since we designed our system to naturally implement a step meant to be used for testing the protein product and ensuring it is both effective and safe, making the system usable by the entire population seemed problematic.

To ensure the safety of the patient using the protein product we changed our target users to include educated personnel only.

Any astronauts using our system on Mars would therefore be required to undergo training before use.

3: Dual-use concerns

The natural function of the injectisome in bacteria is to produce and inject disease-causing proteins into mammalian cells. Therefore our system harbors the inherent risk of being misused to produce toxic proteins.

To evaluate this risk of dual-use, we reached out to our national institution The Centre for Biosecurity and Biopreparedness (CBB). As stated on their website, The centre "is the national authority that issues licences to research institutions, pharmaceutical companies, hospital laboratories etc. to allow them to work with biological dual use components" and was therefore the perfect candidate to evaluate our project. (https://www.biosecurity.dk/ Viewed 18:00 11/09/2018)

Analyst at CBB Jacob Hofman-Bang, PhD (Molecular Biology) was very helpful in answering questions and he conducted a dual-use assessment for our project.

The conclusion of the assessment was that our Protein Printer system does not, in its current form, possess dual-use potential. Though it is stated that at a later stage: Were it is possible to produce large amounts of proteins or to produce currently impossible proteins, we should contact CBB again, as such a system would need to be reevaluated.