Team:Dundee/Safety

From 2013.igem.org

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At all times, Safety Operating Procedures (SOPs) for both equipment used in our project and general safety were closely followed. Protective goggles and masks were used when needed (e.g. for SDS-PAGE and exposure to UV light source). While working in the lab, we were supervised by our instructors, advisors or lab technicians from the Dundee University’s School of Life Sciences Learning & Teaching staff to ensure that we were safely carrying out procedures.</p>
At all times, Safety Operating Procedures (SOPs) for both equipment used in our project and general safety were closely followed. Protective goggles and masks were used when needed (e.g. for SDS-PAGE and exposure to UV light source). While working in the lab, we were supervised by our instructors, advisors or lab technicians from the Dundee University’s School of Life Sciences Learning & Teaching staff to ensure that we were safely carrying out procedures.</p>
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          <h2>Chemical</h2>
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          <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 (nucleic acid staining) in molecular biology labs for agarose gel electrophoresis. By distorting the helical structure of DNA, ethidium bromide is considered to be a mutagen and carcinogen. In order to avoid risk of exposure to ethidium bromide, we took it upon ourselves to use the GelRed staining method for agarose gel electrophoresis instead.</p>
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        <h2>The Toxi-Tweet System:</h2>
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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>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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        <li>kd1 – rate mRNA degradation – 1x10-2</li>
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        <li>k2 – rate PP1 production – 4x10-2</li>
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        <li>kd2 – rate PP1 degradation – 4x10-4</li>
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          <h2>Biosafety</h2>
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          <p> We used safe bacteria types in the lab: E. coli and and B. subtilis. Escherichia 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, Bacillus 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. Bacillus subtilis inhabits the human gut, and is thought to be a natural gut commensal.<br><br>
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We used a few different bacterial strains throughout our project: E. coli MG1655, JM110, 1061 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 absolutely no hazard to human health. <br><br>
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Although the bacterial strains we used are non-pathogenic (and therefore not labelled as a biohazard as such), 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; close to a Bunsen flame. 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. Re-usable containers that had been in contact cells were soaked in Virkon solution to be disinfected.
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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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          <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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        <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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Revision as of 15:32, 15 August 2013

iGEM Dundee 2013 · ToxiMop

Dundee 2013

Safety & Security

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

  • i. Research Safety
  • ii. Public Safety
  • iii. Environmental Safety

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 waste disposal of sharps, biohazardous material and trace chemicals. 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, Safety Operating Procedures (SOPs) for both equipment used in our project and general safety were closely followed. Protective goggles and masks were used when needed (e.g. for SDS-PAGE and exposure to UV light source). While working in the lab, we were supervised by our instructors, advisors or lab technicians from the Dundee University’s School 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 (nucleic acid staining) in molecular biology labs for agarose gel electrophoresis. By distorting the helical structure of DNA, ethidium bromide is considered to be a mutagen and carcinogen. In order to avoid risk of exposure to ethidium bromide, we took it upon ourselves to use the GelRed staining method for agarose gel electrophoresis instead.

Biosafety

We used safe bacteria types in the lab: E. coli and and B. subtilis. Escherichia 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, Bacillus 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. Bacillus 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, JM110, 1061 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 absolutely no hazard to human health.

Although the bacterial strains we used are non-pathogenic (and therefore not labelled as a biohazard as such), 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; close to a Bunsen flame. 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. Re-usable containers that had been in contact cells were soaked in Virkon solution to be disinfected.

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