Team:Dundee/Safety

From 2013.igem.org

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           <h2><b>Insight to the Mop-topus and Toxi-Tweet</b> </h2>
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           <h2><b>Safety & Security</b> </h2>
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<ul style="padding-left:25px;">
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<i>Safety forms were approved on September 29, 2013 by the iGEM Safety Committee.</i><br><br>
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<li><a href ="#Q1">Question 1: Safety issues:</a></li>
 +
<li><a href ="#Q2">Question 2: Parts & devices </a></li>
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<li><a href ="#Q3">Question 3: BioBricks Parts</a></li>
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<li><a href ="#Q4">Question 4: Biosafety groups, Committee, Reviews </a></li>
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<li><a href ="#Q5">Question 5: Future iGEM Safety</a></li>
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           <h2>Aims:</h2>
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           <h2><a id="Q1">Question 1:</a> Would any of your project idea raise safety issues in terms of:</h2>
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          <p>Using mathematical tools to allow us to predict the limiting factors in the production of PP1 and its mopping applications. Working alongside the biologists to produce models which are relevant and can predict what is expected to happen during the synthetic engineering of the mop and detection bacteria.</p>
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<ul style="padding-left:25px;">
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<li><a href ="#ResearchSafety">i. Research Safety</a></li>
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<li><a href ="#PublicSafety">ii. Public Safety</a></li>
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<li><a href ="#EnvironmentalSafety">iii. Environmental Safety</a></li>
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          <h2><a id="ResearchSafety">i. Research Safety</a></h2>
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          <h2>General</h2>
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          <p> We all attended a general health and safety induction at the beginning of our iGEM project and were given a safety tour of our lab. The tour included guidance with regards to disposal of sharps, biohazardous material and trace chemicals. The team were also trained in the relevant fire safety procedures; the locations of fire blankets, fire exits, etc. The team members wore disposable gloves and lab coats at all times when working in the wet lab and eliminated risk of contamination spreading outside the laboratory environment by ensuring they removed these items upon exit. Good laboratory practice, such as regular hand-washing and frequent cleaning of workbenches was enforced. <br><br>
 +
 +
At all times, Standard Operating Procedures (SOPs) for both equipment used in our project and general safety were closely followed. Protective goggles, masks and ear defenders were used when needed (e.g. for SDS-PAGE and exposure to UV light source, while sonicating cells etc.). While working in the lab, we were supervised by our instructors, advisors or lab technicians from Dundee University’s College of Life Sciences Learning & Teaching staff to ensure that we were safely carrying out procedures.</p>
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           <h2>Development of Moptopus:</h2>
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           <p> The current method for detecting toxic levels of microcystin is to take a sample of water from different regions of the site being investigated and then to carry out high performance liquid chromatography (HPLC). This process currently takes approximately 24 hours, we hope to reduce this to a more suitable 1 hour.</p><br>
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           <h2>Chemical</h2>
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          <p>Assuming the cyanobacteria undergo binary fission and grow unbounded we were able to determine how the problem increases over 24 hours in comparison to 1 hour detection.
+
           <p> To reduce risk to our health, we decided to use Qiagen kits (mini-prepping, gel extraction, PCR purification, etc.) rather than phenol based protocols. Ethidium bromide (EtBr) is an intercalating agent (it inserts itself into the DNA helix, unravelling the structure) commonly used as a fluorescent tag in molecular biology labs for agarose gel electrophoresis. By distorting the helical structure of DNA, Ethidium bromide is considered to be a mutagen. In order to avoid risk of exposure to ethidium bromide, we took it upon ourselves to use the GelRed staining for agarose gel electrophoresis instead.<br><br>
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          where MC(t) is the number of microcystin at time t b0 is the initial number of algae</p><br>
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          <p>The ratio for time t=24:1 is 8.4million:1. To put this into perspective this is the same as the height of the empire state building compared with the length of 7 E.coli bacterium. This model therefore emphasises that the 1 hour detection period is much more efficient and worth pursuing.</p>
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As a safety precaution, all harmful chemicals were used within the sterile environment of a fume cupboard and upon contamination were disposed of promptly and correctly according to the relevant Material Safety Data Sheet (MSDS).
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            <img id="image-6" src="http://placehold.it/600x300/8066DB/000000&text=Mop-topus">
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          <h2>Biosafety</h2>
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          <p> We used safe bacteria types in the lab: <i>E. coli</i> and <i>Bacillus subtilis</i>. <i>E. coli</i> is a Gram-negative bacterium which is naturally found in the colon of warm-blooded organisms such as humans. Some strains of <i>E. coli</i> are the cause of serious food poisoning in humans, but the majority are harmless. On the other hand, <i>B. subtilis</i> is a Gram-positive bacterium and has the ability to form a tough, protective endospore. This allows the <i>B. subtilis</i> to tolerate extreme environmental conditions. <i>B. subtilis</i> inhabits the human gut, and is thought to be a natural gut commensal.<br><br>
 +
We used a few different bacterial strains throughout our project: <i>E. coli</i> MG1655, MC1061, JM110 and DH5α. These are all disabled, non-pathogenic, non-toxicogenic, non-colonising, laboratory-adapted K12 strains, which are widely used for research purposes and present little hazard to human health. The <i>B. subtilis</i> strains which we used were NRS3086 strain (derived from the 1086 strain) and 3610.<br><br>
 +
 +
Although the bacterial strains we used are non-pathogenic, it is still important that we took measures to prevent any contamination. Any protocols which involved the transfer of bacterial cells or bacterial colonies between plates or tubes were carried out in sterile conditions. Otherwise, bacterial cells were stored in lidded containers/universal tubes with the caps sealed tightly. All of the biological waste was disposed of in accordance to lab waste disposal protocols, which involves autoclaving biological waste prior to discarding it. Reusable containers that had been in contact with live cells were soaked in Virkon solution to be disinfected before disposal and to ensure that no live cultures were poured into the sinks.<br><br>
 +
Given that our project’s primary concern is the toxin microcystin, we made sure to be very safe regarding its use. The volumes at which we used microcystin for experiments were at levels so low they could present no hazard to human health.
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          <h2><a id="PublicSafety">ii. Public Safety</a></h2>
 +
          <p> While carrying out our project, we took utmost care to ensure that neither biological nor chemical materials were released from our lab accidentally. However, if unintentional release were to occur, the bacterial strains that we used would pose little to no danger to human health. With regards to the <i>E.coli</i> K12 strain derivatives which we used, the lack of danger to people is due to its poor ability to colonize the gut and establish infections. <i>E. coli</i> K12 also lacks the ability to produce large quantities of toxins that affect humans. The <i>B. subtilis</i> strains used were of  minimal danger to the public as well - this organism has never been associated with human infection and is in fact used as a food source in Japan (Netto). <br><br>
-
        <h2>The Toxi-Tweet System:</h2>
+
We used ampicillin resistant genes within our plasmids as a selectable marker for bacterial transformations. As we are fully aware of the issues surrounding horizontal gene transfer and multi-drug resistant bacteria, we followed university protocols regarding GMO waste disposal. The ultimate goal of our project, as with any other iGEM project, is for our modified bacteria to be used practically in the environment to treat algal blooms by removing microcystin. Our final step would be to remove antibiotic resistance from our plasmid prior to release of our bacteria into the environment.
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        <p>We considered different limiting factors of our mop bacteria.  The factor discussed in this section is the maximum number of PP1 which can fit either on the surface of B.subtilis, or in the periplasm of E.coli.  We considered the volumes of the bacteria and PP1 and used a cube approximation that took into account volume which was wasted, in packing, by the spherical shape of the protein. For this model we assumed there were no other surface proteins and protein production was not limited by any factors.</p><br>
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        <p>Calculations show the maximum number of PP1 which can fit on the surface of B.subtilis is between 60 000 -70 000. From the average we can calculate that the number of bacterial mops required to clean a toxic level of microcystin in a litre of water is 1.40x1010.</p><br>
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        <p>In E.coli, PP1 which would bind microcystin is free-flowing in the periplasm. The volume of the periplasm is much greater than the surface of B.subtilis. Therefore E.coli has the capacitive potential to be a more efficient mop. The maximum number of PP1 which can be packed into the periplasm is between 150 000 -200 000. Consequently, less bacterial mops are required to clean the same level of microcystin: 0.52x1010.</p><br>
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        <p>When we have accurate numbers from the biology team on how many PP1 are attached to the surface or in the periplasm for B.subtilis and E.coli respectively, we can compare these numbers and compute the efficiency of our PP1 expressing bacteria.</p><br>
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        <h2>Progress and Future Plans </h2><br>
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</p>
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         <p>An Ordinary Differential Equation (ODE) uses a function f(t) to describe how the output changes as a result of changing the input dx(t)/dt. For example how PP1 concentration changes with time in a single cell. In order to model transcription and translation of PP1 we used a system of ODEs , which is more than one ODE where the outputs are coupled.</p><br>
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        <p>We used law of mass action to obtain a system of ODEs to describe the production of mRNA to PP1. mRNA and PP1 are coupled in the sense we need mRNA before we can produce any PP1. Also, the mRNA is not used up. We also took into consideration the degradation rates of mRNA and PP1 which are denoted as .</p><br>
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        <li>k1 – rate mRNA production - 4.98x10-9</li>
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          <h2><a id="EnvironmentalSafety">iii. Environmental Safety</a></h2>
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        <li>kd1 – rate mRNA degradation – 1x10-2</li>
+
          <p>The use of genetically modified organisms in the environment is a contentious issue, however regardless of your opinion it is obvious that they must be kept isolated from the wild in case they harm the environment they have been engineered to protect, improve or enhance.<br><br>
-
        <li>k2 – rate PP1 production – 4x10-2</li>
+
 
-
        <li>kd2 rate PP1 degradation – 4x10-4</li>
+
Since ToxiMop is an environmental clean-up tool, it was obvious to us that we could not just blindly inoculate water bodies with it, so we came up with a clever way to mop and avoid releasing our bacteria in to the environment. Where in the past, mechanical filter systems have been proposed to contain synthetic bacteria, we created the ToxiTeabag a vessel for our bacteria to interact with water while still remaining isolated from the rest of the environment. The ToxiTeabag does exactly what it says on the tin, it is a dialysis bag filled with ToxiMop bacteria. Bacteria are too large to escape the through the holes in the bag but water, but more importantly microcystin is permitted to flow freely allowing it to be mopped up by PP1 expressed by the bacteria.
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          <h2><a id="Q2">Question 2:</a> Are any parts or devices in our project associated with (or known to cause):</h2>
 +
<ul>
 +
<li><a href ="#Pathnogenicity">i. Pathogenicity, infectivity, or toxicity?</a></li>
 +
<li><a href ="#Threats">ii. Threats to environmental quality?</a></li>
 +
<li><a href ="#SecurityConcerns">iii. Security Concerns?</a></li>
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</ul>
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        <br> <p><i><b>Figure 1.</b> How mRNA and PP1 are produced over 20 minute cell division time. Note scaling on PP1 compared to mRNA.</i></p><br>
 
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          <h2><a id="Pathogenicity">i. Pathogenicity, infectivity, or toxicity</a></h2>
 +
          <p> The strains we used, which were derived from <i>E. coli</i> K12 lack many of the virulence factors required for infection. <i>B. subtilis</i> has never been associated with human infection. It has GRAS status which means it is “generally recognized as safe”.</p>
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           <h2><a id="Threats ">ii. Threats to environmental quality</a></h2>
 +
          <p> Since ToxiMop is an environmental clean-up tool, it was obvious to us that we could not just blindly inoculate water bodies with it, so we came up with a clever way to mop and avoid releasing our bacteria in to the environment.<br><br>
-
          <div class="span6">
+
Where in the past, mechanical filter systems have been proposed to contain synthetic bacteria, we created the ToxiTeabag – a vessel for our bacteria to interact with water while still remaining isolated from the rest of the environment. The ToxiTeabag does exactly what it says on the tin, it is a dialysis bag filled with ToxiMop bacteria. Bacteria are too large to escape the through the holes in the bag but water, but more importantly microcystin is permitted to flow freely allowing it to be <a href="2013.igem.org/Team:Dundee/Project">mopped up by PP1 expressed by the bacteria</a>.
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          <p><br><i>Figure 2. A steady state is when the quantities describing a system are independent of time – they reach an equilibrium i.e dx/dt = 0. The steady state for (mRNA, PP1) is (0.04, 0.04) corresponding to a non-dimensionalised system. This plot demonstrates that during a 20 minute cell division period mRNA reaches the steady state but PP1 does not.</i></p><br>
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          <h2><a id="Threats ">iii. Security concerns</a></h2>
 +
           <p>Even with these measures in place, <i>E. coli</i> and <i>B. subtilis</i> are not environmentally dangerous bacteria, with each being found as natural inhabitants of soil. Their presence would not obviously pose any threat to the environment. Nonetheless, the effects of a genetically modified organism in the environment may not be predicted accurately, thus their unprotected release in to the wild would be irresponsible.<br><br>
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        <div class="span6">
+
Due to the non-pathogenic, non-toxicogenic, and non-colonising nature of <i>E. coli</i> K12 and <i>B. subtilis</i> strains which we utilised and the harmless nature of our parts, we do not foresee any security concerns with our project. Our laboratory has secure entry to prevent unauthorised access. In the wrong hands, nothing harmful could be done with our parts or bacterial strains.</p>
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        <br><p><i>Figure 3. This plot shows that given a time longer than cell division time both the mRNA and PP1 eventually reach their steady states.</i></p><br>
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          <h2><a id="Q3">Question 3:</a> Do any of the new BioBricks parts (or devices) that you made this year raise any safety issues?</h2>
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          <p> Our biobricks do not raise any direct safety concerns.</p>
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          <h2><a id="Q4">Question 4:</a>  Is there a local biosafety group, committee, or review board at your institution?</h2>
 +
          <p> Yes there is. Comprehensive risk assessments must be carried out prior to the start of any laboratory project and any accidents or spillages of micro-organisms must be reported right away.
 +
We were given a general lab safety induction by the Health and Safety board at the University of Dundee’s College of Life Sciences, which included guidance in waste disposal of biohazardous material. Documents describing Standard Operating Procedures and risk assessments were made available to us online. We also received informal training in the form of various protocols including miniprep, gel extraction, PCR and cell transformation.
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          <h2><a id="Q5">Question 5:</a> Do we have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?</h2>
 +
          <p> Perhaps future iGEM teams could be sent an information pack on lab safety and security issues so that every team member all around the world has had the same training before beginning their iGEM project. <br><br>
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Latest revision as of 18:07, 3 October 2013

iGEM Dundee 2013 · ToxiMop

Question 1: Would any of your project idea raise safety issues in terms of:

General

We all attended a general health and safety induction at the beginning of our iGEM project and were given a safety tour of our lab. The tour included guidance with regards to disposal of sharps, biohazardous material and trace chemicals. The team were also trained in the relevant fire safety procedures; the locations of fire blankets, fire exits, etc. The team members wore disposable gloves and lab coats at all times when working in the wet lab and eliminated risk of contamination spreading outside the laboratory environment by ensuring they removed these items upon exit. Good laboratory practice, such as regular hand-washing and frequent cleaning of workbenches was enforced.

At all times, Standard Operating Procedures (SOPs) for both equipment used in our project and general safety were closely followed. Protective goggles, masks and ear defenders were used when needed (e.g. for SDS-PAGE and exposure to UV light source, while sonicating cells etc.). While working in the lab, we were supervised by our instructors, advisors or lab technicians from Dundee University’s College of Life Sciences Learning & Teaching staff to ensure that we were safely carrying out procedures.

Chemical

To reduce risk to our health, we decided to use Qiagen kits (mini-prepping, gel extraction, PCR purification, etc.) rather than phenol based protocols. Ethidium bromide (EtBr) is an intercalating agent (it inserts itself into the DNA helix, unravelling the structure) commonly used as a fluorescent tag in molecular biology labs for agarose gel electrophoresis. By distorting the helical structure of DNA, Ethidium bromide is considered to be a mutagen. In order to avoid risk of exposure to ethidium bromide, we took it upon ourselves to use the GelRed staining for agarose gel electrophoresis instead.

As a safety precaution, all harmful chemicals were used within the sterile environment of a fume cupboard and upon contamination were disposed of promptly and correctly according to the relevant Material Safety Data Sheet (MSDS).

Biosafety

We used safe bacteria types in the lab: E. coli and Bacillus subtilis. E. coli is a Gram-negative bacterium which is naturally found in the colon of warm-blooded organisms such as humans. Some strains of E. coli are the cause of serious food poisoning in humans, but the majority are harmless. On the other hand, B. subtilis is a Gram-positive bacterium and has the ability to form a tough, protective endospore. This allows the B. subtilis to tolerate extreme environmental conditions. B. subtilis inhabits the human gut, and is thought to be a natural gut commensal.

We used a few different bacterial strains throughout our project: E. coli MG1655, MC1061, JM110 and DH5α. These are all disabled, non-pathogenic, non-toxicogenic, non-colonising, laboratory-adapted K12 strains, which are widely used for research purposes and present little hazard to human health. The B. subtilis strains which we used were NRS3086 strain (derived from the 1086 strain) and 3610.

Although the bacterial strains we used are non-pathogenic, it is still important that we took measures to prevent any contamination. Any protocols which involved the transfer of bacterial cells or bacterial colonies between plates or tubes were carried out in sterile conditions. Otherwise, bacterial cells were stored in lidded containers/universal tubes with the caps sealed tightly. All of the biological waste was disposed of in accordance to lab waste disposal protocols, which involves autoclaving biological waste prior to discarding it. Reusable containers that had been in contact with live cells were soaked in Virkon solution to be disinfected before disposal and to ensure that no live cultures were poured into the sinks.

Given that our project’s primary concern is the toxin microcystin, we made sure to be very safe regarding its use. The volumes at which we used microcystin for experiments were at levels so low they could present no hazard to human health.

ii. Public Safety

While carrying out our project, we took utmost care to ensure that neither biological nor chemical materials were released from our lab accidentally. However, if unintentional release were to occur, the bacterial strains that we used would pose little to no danger to human health. With regards to the E.coli K12 strain derivatives which we used, the lack of danger to people is due to its poor ability to colonize the gut and establish infections. E. coli K12 also lacks the ability to produce large quantities of toxins that affect humans. The B. subtilis strains used were of minimal danger to the public as well - this organism has never been associated with human infection and is in fact used as a food source in Japan (Netto).

We used ampicillin resistant genes within our plasmids as a selectable marker for bacterial transformations. As we are fully aware of the issues surrounding horizontal gene transfer and multi-drug resistant bacteria, we followed university protocols regarding GMO waste disposal. The ultimate goal of our project, as with any other iGEM project, is for our modified bacteria to be used practically in the environment to treat algal blooms by removing microcystin. Our final step would be to remove antibiotic resistance from our plasmid prior to release of our bacteria into the environment.

iii. Environmental Safety

The use of genetically modified organisms in the environment is a contentious issue, however regardless of your opinion it is obvious that they must be kept isolated from the wild in case they harm the environment they have been engineered to protect, improve or enhance.

Since ToxiMop is an environmental clean-up tool, it was obvious to us that we could not just blindly inoculate water bodies with it, so we came up with a clever way to mop and avoid releasing our bacteria in to the environment. Where in the past, mechanical filter systems have been proposed to contain synthetic bacteria, we created the ToxiTeabag – a vessel for our bacteria to interact with water while still remaining isolated from the rest of the environment. The ToxiTeabag does exactly what it says on the tin, it is a dialysis bag filled with ToxiMop bacteria. Bacteria are too large to escape the through the holes in the bag but water, but more importantly microcystin is permitted to flow freely allowing it to be mopped up by PP1 expressed by the bacteria.

Question 2: Are any parts or devices in our project associated with (or known to cause):

i. Pathogenicity, infectivity, or toxicity

The strains we used, which were derived from E. coli K12 lack many of the virulence factors required for infection. B. subtilis has never been associated with human infection. It has GRAS status which means it is “generally recognized as safe”.

ii. Threats to environmental quality

Since ToxiMop is an environmental clean-up tool, it was obvious to us that we could not just blindly inoculate water bodies with it, so we came up with a clever way to mop and avoid releasing our bacteria in to the environment.

Where in the past, mechanical filter systems have been proposed to contain synthetic bacteria, we created the ToxiTeabag – a vessel for our bacteria to interact with water while still remaining isolated from the rest of the environment. The ToxiTeabag does exactly what it says on the tin, it is a dialysis bag filled with ToxiMop bacteria. Bacteria are too large to escape the through the holes in the bag but water, but more importantly microcystin is permitted to flow freely allowing it to be mopped up by PP1 expressed by the bacteria.

iii. Security concerns

Even with these measures in place, E. coli and B. subtilis are not environmentally dangerous bacteria, with each being found as natural inhabitants of soil. Their presence would not obviously pose any threat to the environment. Nonetheless, the effects of a genetically modified organism in the environment may not be predicted accurately, thus their unprotected release in to the wild would be irresponsible.

Due to the non-pathogenic, non-toxicogenic, and non-colonising nature of E. coli K12 and B. subtilis strains which we utilised and the harmless nature of our parts, we do not foresee any security concerns with our project. Our laboratory has secure entry to prevent unauthorised access. In the wrong hands, nothing harmful could be done with our parts or bacterial strains.

Question 3: Do any of the new BioBricks parts (or devices) that you made this year raise any safety issues?

Our biobricks do not raise any direct safety concerns.

Question 4: Is there a local biosafety group, committee, or review board at your institution?

Yes there is. Comprehensive risk assessments must be carried out prior to the start of any laboratory project and any accidents or spillages of micro-organisms must be reported right away. We were given a general lab safety induction by the Health and Safety board at the University of Dundee’s College of Life Sciences, which included guidance in waste disposal of biohazardous material. Documents describing Standard Operating Procedures and risk assessments were made available to us online. We also received informal training in the form of various protocols including miniprep, gel extraction, PCR and cell transformation.

Question 5: Do we have other ideas on how to deal with safety or security issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?

Perhaps future iGEM teams could be sent an information pack on lab safety and security issues so that every team member all around the world has had the same training before beginning their iGEM project.