Team:TecMonterrey/Security.html

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        <p>One of the most important parts of science and scientific progress is commonly forgotten by researchers. Working and communication between teams is not only beneficial for both in technical terms, but also opens a bridge that is essential for science; collaboration. </p>
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<b>Security and Safety Considerations</b>
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<p>Since the designing phase of our project, we were aware that every project may have safety implications and that it is our responsibility to address them in an appropriate manner. </p>
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        <p>This year we crossed words with a fellow Latin American team. Team: Buenos_Aires started the conversation and our ideas began to flow we talked about how we were achieving hypoxia for our hypoxic promoters characterization and how they were measuring their arsenic sensitive promoters. After some discussion we concluded that we could improve each other’s measurements and agreed to make the collaboration. </p>
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<p>From the beginning of the brainstorming, the main idea involved the expression of therapeutic proteins that would act as toxins for tumor cells. At this point, we realized that we needed more specificity if healthy tissue was to be unharmed. In the hypothetic case that our bacteria were liberated into the environment and some way it survived and maintained its plasmid without a selectivity marker, it would be a catastrophe. This imaginary scenario led us to the idea of implementing safety locks in the design of our project. </p>
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        <p>When we received the part Team: Buenos_Aires we proceeded with transforming its DNA into <em>Escherichia coli TOP10</em> chemocompetent cells. Selection of transformants was done with Tetracicline and we continued with a restriction analysis using EcoRI and PstI enzymes. The resulting agarose gel is shown by duplicate in the following pictures. </p>
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<p>Our first task to design our genes in a way their products wouldn't harm us, was to think about which therapeutic proteins we were going to produce as tumor toxins. After bibliographic research we found out that the soluble part of the protein TRAIL (BBa_K1166004) and TAT-Apoptin (BBa_K1166005) were proteins that have antitumoral activity with high specificity leaving healthy tissue in its majority unharmed. </p>
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<p>After we decided which proteins were going to be expressed, we also realized that its specificity could be enhanced if we managed to somehow produce them solely in the tumor. After weeks of bibliographic research, we learnt that in general, tumors exhibit an environment with a lower amount of oxygen concentration (hypoxia). From this research, we decided the regulation for our project to be controlled by a hypoxic induction promoter, for which we created FNR hypoxic regulation part (BBa_K1166001) and a new hypoxic promoter HIP-1 (BBa_K1166000), along with the characterization of an existing hypoxia promoter (BBa_K905000) complemented with our FNR regulation part. </p>  
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        <img src="https://static.igem.org/mediawiki/2013/3/31/BA_1.png" alt="Image BA" height="800" width="950">
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<p>We believe that these precautions along with good practices at the execution of the project will work to maintain and highlight safety in our lab and for our society. </p>
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<p>Also, during the execution phase of our project we followed the top security requirements for the materials we were using. First of all, it is important to cover a brief description of both the materials used in the project, and the organisms employed and their characteristics. It is important to understand that we are always exposed to risk and no experiment is 100% risk free. Nevertheless, our job is to minimize the probability of being exposed to any danger. </p>
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        <p>After transformants were proven to have the correct plasmid by restriction analysis, we started induction with sodium arsenite. Different concentrations ranging from 0 ppb to 1000ppb were used as induction and measurements were done at 1, 2, 4, 6, 8 hours. </p>
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<p>During the realization of the project we did the following procedures: </p> 
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        <img src="https://static.igem.org/mediawiki/2013/b/b1/BA_2.png" alt="Image BA" height="200" width="950">
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<ol><li>Miniprep (plasmidic DNA extraction) </li>
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        <p>In each column, 10mL cultures of <em>E.coli</em> transformed with the measurement device were grown until 0.5 OD600. After that, cultures were induced with different concentrations (in ppb) of sodium arsenite. Measurements of RFP fluorescence (AU) and OD600 were taken every time specified in the <b> time (h) </b> column and AU/OD600 is reported. A public water source and water from a nearby lake were also assayed. </p>
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<li>Agarose gel DNA electroforesis</li>
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<li>Restriction enzyme analysis</li>
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        <img src="https://static.igem.org/mediawiki/2013/9/95/BA_3.png" alt="Image BA" height="500" width="950">
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<li><em>Escherichia coli</em> Transformation by CaCl2</li>
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<li>Inducible promoter induction with L-Arabinose, IPTG and hypoxia</li>
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        <p>Using the last measured fluoresence/OD600 value plotted against the concentration of arsenite used as inductor, a calibration curve was obtained. </p>
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<li>SDS-PAGE</li>
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<li>Western Blot</li>
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        <p>With the use of the newly obtained calibration curve we also measured two samples that we obtained from our neighborhood. We took samples from a nearby lake and from a public water source. Following the same procedure, we grew <em> E.coli </em> cells until .5 OD600 and induced with the samples we obtained (after using a 0.0002 mm syringe microfilter) until 8 hours of induction. After measuring AU/OD600 for 8 hours we obtained the following data: </p>
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<li>Cell lysis and Protein Extraction</li>
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<li>Mammalian cells toxicity/internalization assay</li></ol>
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        <img src="https://static.igem.org/mediawiki/2013/2/29/BA_4.png" alt="Image BA" height="127" width="450">
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<p>The realization of the named protocols implies the use of some hazardous materials. These substances require special treatment and disposal. In our protocols we use the following dangerous substances: </p>
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        <p>Taking the latter into account we might be more careful from where do we have contact with water. These results encourage us to inform the responsible authorities in the future about the current status of the water and have the samples analyzed by an already established method. </p>
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<ol><li>Acrylamide</li>
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<li>Ethidium Bromide</li>
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<li>Acetic acid</li>
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<li>Chlorine</li>
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<li>HCl</li>
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<li>NaAC</li>
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<li>NaOH</li>
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<li>β-Mercaptoethanol</li>
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<li>SDS</li>
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<li>Coomassie Blue</li></ol>  
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<p>For us to use this kind of substances some security protocols are needed to be followed.  </p>
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<ol><li>Miniprep (plasmidic DNA extraction) uses solutions with EDTA, these solutions are made with nitrile gloves to prevent any possible contact with the skin, as it is known that EDTA has suspected effects on the reproductive system. Also solutions using NaOH and NaAc are made under a laminar flow hood to prevent any kind of dangerous inhalation. </li>
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<li>During the Agarose gel electrophoresis the dangerous part come when the researcher has to use Ethidium Bromide to reveal the gel. Ethidium Bromide intercalates into the DNA double strand, as a strong intercalator is mutagenic and carcinogen. To work with Ethidium Bromide, it is needed to designate a special workplace; also you need to restrict the number of people to the minimum. To keep the biosafety we followed all of the recommendations, also we used nitrile gloves that are less permeable to EtBr than latex gloves. Finally, we designated a special disposal bin for all EtBr–related residues. </li>
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<li>In the <em>E. coli</em> Transformation by CaCl2 protocol, we are exposed to the <em>E. coli</em> non-transformed strains. To prevent any escape to the environment of these strains, we perform every transformation under a biosecurity chamber. Any residues generated in this protocol that had contact with biological material are then disposed in a biohazard bag that is sealed and autoclaved in 121°C and 15psi for 15 minutes. </li>
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<li>Induction protocols consist in adding a specific substance to a cell culture for it to start the production of a specific protein. In our case, our inductors are L-Arabinose, IPTG and hypoxia. To prevent any damage by inhalation or any possible fire, the preparation of methanol is done in a gas extraction cabinet. Also, its sterilization is done by filtration to prevent any fire or explosion. </li>  
 +
<li>SDS-PAGE is very usefull tool at the time when we need to make a protein analysis by size, nevertheless it is full of hazardous materials. To begin with the SDS-PAGE, you need to prepare the polyacrylamide gel. This is done by adding TEMED and PSA to Acrylamide. TEMED and PSA are harmful for the respiratory tract, so for its use we need to have precaution. Acrylamide is known for being a strong neurotoxin, so nitrile gloves are used. It is important to assign a separate special workspace for any procedure using these substances. </li></ul>  
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<p>To prepare the protein sample, B-mercaptoethanol and SDS are needed, and both have toxic effects, especially B-mercaptoethanol. To keep our biosafety standards, we minimize the time of exposure to any of this materials, also we work using always nitrile gloves, and in our designated space. </p> 
 +
<p>After the preparation of the SDS-PAGE and the running of the gel, you need to stain with Coomassie Blue. As some of the acrylamide may have not polymerized yet, we keep our gloves on, especially because of the Coomassie staining. Coomassie Blue is a known flammable toxin that targets some of our organs. </p> 
 +
<p>For the Coomassie Blue to stain with more definition it is recommended to use a “fixating solution”, which contains methanol, acetic acid and water. For the preparation of this solution, an air-extraction cabinet is needed. Finally, we dispose of all the generated wastes in a bin we labeled “PAGE wastes”, separated from our normal wastes. </p>  
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Revision as of 02:46, 28 September 2013

<!doctype html> Security and Safety Considerations

Since the designing phase of our project, we were aware that every project may have safety implications and that it is our responsibility to address them in an appropriate manner.

From the beginning of the brainstorming, the main idea involved the expression of therapeutic proteins that would act as toxins for tumor cells. At this point, we realized that we needed more specificity if healthy tissue was to be unharmed. In the hypothetic case that our bacteria were liberated into the environment and some way it survived and maintained its plasmid without a selectivity marker, it would be a catastrophe. This imaginary scenario led us to the idea of implementing safety locks in the design of our project.

Our first task to design our genes in a way their products wouldn't harm us, was to think about which therapeutic proteins we were going to produce as tumor toxins. After bibliographic research we found out that the soluble part of the protein TRAIL (BBa_K1166004) and TAT-Apoptin (BBa_K1166005) were proteins that have antitumoral activity with high specificity leaving healthy tissue in its majority unharmed.

After we decided which proteins were going to be expressed, we also realized that its specificity could be enhanced if we managed to somehow produce them solely in the tumor. After weeks of bibliographic research, we learnt that in general, tumors exhibit an environment with a lower amount of oxygen concentration (hypoxia). From this research, we decided the regulation for our project to be controlled by a hypoxic induction promoter, for which we created FNR hypoxic regulation part (BBa_K1166001) and a new hypoxic promoter HIP-1 (BBa_K1166000), along with the characterization of an existing hypoxia promoter (BBa_K905000) complemented with our FNR regulation part.

We believe that these precautions along with good practices at the execution of the project will work to maintain and highlight safety in our lab and for our society.

Also, during the execution phase of our project we followed the top security requirements for the materials we were using. First of all, it is important to cover a brief description of both the materials used in the project, and the organisms employed and their characteristics. It is important to understand that we are always exposed to risk and no experiment is 100% risk free. Nevertheless, our job is to minimize the probability of being exposed to any danger.

During the realization of the project we did the following procedures:

  1. Miniprep (plasmidic DNA extraction)
  2. Agarose gel DNA electroforesis
  3. Restriction enzyme analysis
  4. Escherichia coli Transformation by CaCl2
  5. Inducible promoter induction with L-Arabinose, IPTG and hypoxia
  6. SDS-PAGE
  7. Western Blot
  8. Cell lysis and Protein Extraction
  9. Mammalian cells toxicity/internalization assay

The realization of the named protocols implies the use of some hazardous materials. These substances require special treatment and disposal. In our protocols we use the following dangerous substances:

  1. Acrylamide
  2. Ethidium Bromide
  3. Acetic acid
  4. Chlorine
  5. HCl
  6. NaAC
  7. NaOH
  8. β-Mercaptoethanol
  9. SDS
  10. Coomassie Blue

For us to use this kind of substances some security protocols are needed to be followed.

  1. Miniprep (plasmidic DNA extraction) uses solutions with EDTA, these solutions are made with nitrile gloves to prevent any possible contact with the skin, as it is known that EDTA has suspected effects on the reproductive system. Also solutions using NaOH and NaAc are made under a laminar flow hood to prevent any kind of dangerous inhalation.
  2. During the Agarose gel electrophoresis the dangerous part come when the researcher has to use Ethidium Bromide to reveal the gel. Ethidium Bromide intercalates into the DNA double strand, as a strong intercalator is mutagenic and carcinogen. To work with Ethidium Bromide, it is needed to designate a special workplace; also you need to restrict the number of people to the minimum. To keep the biosafety we followed all of the recommendations, also we used nitrile gloves that are less permeable to EtBr than latex gloves. Finally, we designated a special disposal bin for all EtBr–related residues.
  3. In the E. coli Transformation by CaCl2 protocol, we are exposed to the E. coli non-transformed strains. To prevent any escape to the environment of these strains, we perform every transformation under a biosecurity chamber. Any residues generated in this protocol that had contact with biological material are then disposed in a biohazard bag that is sealed and autoclaved in 121°C and 15psi for 15 minutes.
  4. Induction protocols consist in adding a specific substance to a cell culture for it to start the production of a specific protein. In our case, our inductors are L-Arabinose, IPTG and hypoxia. To prevent any damage by inhalation or any possible fire, the preparation of methanol is done in a gas extraction cabinet. Also, its sterilization is done by filtration to prevent any fire or explosion.
  5. SDS-PAGE is very usefull tool at the time when we need to make a protein analysis by size, nevertheless it is full of hazardous materials. To begin with the SDS-PAGE, you need to prepare the polyacrylamide gel. This is done by adding TEMED and PSA to Acrylamide. TEMED and PSA are harmful for the respiratory tract, so for its use we need to have precaution. Acrylamide is known for being a strong neurotoxin, so nitrile gloves are used. It is important to assign a separate special workspace for any procedure using these substances.

    6. To prepare the protein sample, B-mercaptoethanol and SDS are needed, and both have toxic effects, especially B-mercaptoethanol. To keep our biosafety standards, we minimize the time of exposure to any of this materials, also we work using always nitrile gloves, and in our designated space.

    After the preparation of the SDS-PAGE and the running of the gel, you need to stain with Coomassie Blue. As some of the acrylamide may have not polymerized yet, we keep our gloves on, especially because of the Coomassie staining. Coomassie Blue is a known flammable toxin that targets some of our organs.

    For the Coomassie Blue to stain with more definition it is recommended to use a “fixating solution”, which contains methanol, acetic acid and water. For the preparation of this solution, an air-extraction cabinet is needed. Finally, we dispose of all the generated wastes in a bin we labeled “PAGE wastes”, separated from our normal wastes.

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