Team:Arizona State/Safety

From 2011.igem.org

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==Researcher Safety==
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<p>All the organisms utilized in the Wang Lab comply with biosafety level 1 (BSL1) and do not possess the potential to cause disease in individuals. All organisms, despite having no association with disease, are treated as potential pathogens, thus personal protective equipment such as gloves, laboratory coats, and protective eyewear/goggles are used to prevent contact with bacteria and yeast samples in the lab.</p>
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<p>Standard [[Team:Arizona State/Lab/Protocols|protocols]] were followed for all genetic manipulation, including PCR, plasmid assembly (restriction, ligation, and transformation), and DNA extraction. These protocols standardize specific safety procedures encountered in day to day labwork.</p>
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The following organisms are present in the lab and directly utilized in our project:
 +
* Escherichia coli K12 MG1655<sup>[http://www.genome.wisc.edu/resources/strains.htm], [http://www.ncbi.nlm.nih.gov/bioproject/225]</sup>
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* Escherichia coli BL21(DE3)<sup>[http://www.ncbi.nlm.nih.gov/bioproject/30681]</sup>
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* Escherichia coli NEB-10 Beta<sup>[http://www.neb.com/nebecomm/products/productc3020.asp]</sup>
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* Bacillus halodurans C-125<sup>[http://www.ncbi.nlm.nih.gov/bioproject/235]</sup>
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* Listeria innocua CLIP11262<sup>[http://www.ncbi.nlm.nih.gov/bioproject/86]</sup>
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<p>Strains L. innocua CLIP11262 and B. halodurans C-125 were both received from ATCC<sup>[http://www.atcc.org/]</sup>, which classifies both organisms as BSL1. The ATCC Biosafety page<sup>[http://www.atcc.org/CulturesandProducts/TechnicalSupport/BiosafetyLevels/tabid/660/Default.aspx]</sup> states that BSL1 organisms and reagents, "are not known to cause disease in healthy adult humans".</p>
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== Researcher Safety ==
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=== Biosafety training ===
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*
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<p>All members of our team were required to attend biosafety and bloodborne pathogen training according to Arizona State policy before working in the lab. This course satisfies the OSHA Bloodborne Pathogens training requirement as well as the Biosafety requirements for working with recombinant DNA. The Laboratory-Specific biosafety training checklist was followed<sup>[http://www.asu.edu/uagc/EHS/forms/asu_lab_specific_biosafety_training.pdf]</sup> to ensure all team members were adequately trained.</p>
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General Lab safety: All members of our team were required to attend biosafety training according to Arizona State policy before working in the lab. The lab space is classified as biosafety level one. As such, rules concerning proper physical attire and use of protective barriers like gloves are combined with aseptic techniques to ensure cross-contamination between lab organisms and researchers does not occur. All biological trash is autoclaved before disposal.  
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Safety issues concerning CRISPR: Our project involves manipulating the CRISPR pathway as a gene regulation tool. The CRISPR system is naturally present in many prokaryotes and nearly all archea. Introducing a CRISPR locus into bacteria lacking CRISPR has the potential to increase resistance to phage attack and plasmid uptake. This is unlikely to have a significant effect on pathogenicity, especially when the spacers are designed to target relatively benign genes products such as GFP, as in our proof of concept. Currently our constructs omit two genes (CAS1 and CAS2) implicated in the incorporation of new spacers into CRISPR arrays such that we minimize the possibility of unwanted targeting that could potentially increase pathogenicity (although there is no evidence to suggest that this would even be a favorable event).
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=== Biological Reagents===
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<p>Reagents and equipment including solid and liquid bacterial growth media, and yeast culture media, micropipette, volumetric pipette tips and centrifuge tubes are autoclave sterilized (heated to 121 degrees celsius at 15 PSI) both preceding and following use, ensuring there is no threat of waste contamination of outside the lab. This is in accordance with Arizona State University's biological waste procedures<sup>[http://cfo.asu.edu/ehs-biowaste-compliance-guideline]</sup>.</p>
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== Public and Environmental Safety ==
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=== Flammable Reagent Safety ===
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* There is no expected public safety risk from this project. Bacteria with ASU iGEM's manipulated CRISPR-Cas system will only target genes that have been specifically engineered to be targeted, none of which will be detrimental if released.
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<p>Reagents that are potentially flammable (e.g. ethanol and isopropyl alcohol) are stored in a flame protective cabinet.</p>
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== Biobrick considerations ==
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=== Corrosive and Noxious Reagent Safety ===
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* Our submitted Biobrick parts will not pose any risk to the public or to the environment.
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<p>Reagents that have corrosive and/or noxious fumes (e.g. bleach solutions and phenol-chloroform) are kept within a chemical fume hood to prevent inhalation and physical contact.</p>
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== Safety at Arizona State University ==
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== Public Safety ==
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* Environmental Health and Safety Services at ASU is aware of our project and has not expressed safety concerns to date. Our proposal will be reviewed in detail to ensure that every aspect complies with university and state safety regulations.
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<p>There is minimal risk associated with the release of the aforementioned organisms. The recombinant DNA (rDNA) experiments conducted in the laboratory provide ampicillin, kanamycin and/or chloramphenicol resistance to E.coli K12 MG1655, E.coli BL21(DE3) and E.coli NEB-10 Beta to select for plasmids. Under the circumstances that any of these genetically altered organisms were released they would have minimal potential for pathogenesis. In accordance with Arizona State University’s Environmental Health & Safety policy, “Nothing in the trash, nothing down the drain”, we autoclave and bleach sterilize all waste from recombinant DNA experiments. This reduces the likelihood of accidental release.</p>
 +
<p>Under the circumstances of “designed” release, the organisms and all rDNA products present no possibility of harming the outside population and ecosystem. Our iGEM team, faculty mentor, and department lab safety manager, find no foreseeable public health threat associated with the organisms and recombinant DNA project utilized by our team.</p>
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<html>
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== Environmental Safety ==
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<script type="text/javascript" charset="utf-8">
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<p>Our project involves manipulating the CRISPR pathway as a gene regulation tool. The CRISPR system is naturally present in many prokaryotes and nearly all archea. Introducing a CRISPR locus into bacteria lacking CRISPR has the potential to increase phage resistance, and therefore fitness, through incorporation of new spacers. This is unlikely to have a significant effect on pathogenicity, especially when the spacers are designed to target relatively benign gene products such as GFP, as in our proof of concept. Currently our E. coli and B. halodurans constructs are engineered to omit two genes (CAS1 and CAS2) implicated in the incorporation of new spacers into CRISPR arrays. We also test these constructs in BL21, a laboratory strain of E. coli used for expression that has no native CAS genes. This minimizes the possibility of unwanted targeting that could potentially increase pathogenicity (although there is no evidence to suggest that this would be a favorable event).</p>
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$(document).ready(function(){
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<p>We envision CRISPR as a tool that is used primarily in the laboratory. CRISPR arrays and CAS genes are highly susceptible to horizontal gene transfer. New spacers are constantly incorporated in wild-type strains, and CAS genes are easily lost in the absence of selection pressure. Because of this volatility the utility of CRISPR-engineered strains of bacteria in the environment seems minimal.</p>
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$("a[rel^='prettyPhoto']").prettyPhoto();
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});
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== Biosafety Regulations and Provisions ==
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</script>
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* National Guidelines:
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<div align="center"><object width="640" height="385"><param name="movie" value="http://www.youtube.com/v/9DVCyWZSEBQ?fs=1&amp;hl=en_GB"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/9DVCyWZSEBQ?fs=1&amp;hl=en_GB" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="640" height="385"></embed></object></div>
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:* National Institutes of Health<sup>[http://www.nih.gov/]</sup>
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</html>
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::* NIH Guidelines for Research Involving Recombinant DNA Molecules<sup>[http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm]</sup>
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::* NIH Risk Group Classifications<sup>[http://rpi.edu/research/office/ibc/riskgroupclassifications.html]</sup>
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:* Center for Disease Control<sup>[http://www.cdc.gov/]</sup>
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::* CDC Biosafety<sup>[http://www.cdc.gov/biosafety/]</sup>
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:* American Biological Safety Association<sup>[http://www.absa.org/]</sup>
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:* Environmental Protection Agency<sup>[http://www.epa.gov/]</sup>
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::* Toxic Substances Control Act (TSCA) Biotechnology Program<sup>[http://www.epa.gov/opptintr/biotech/index.htm]</sup>
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:* Occupational Safety and Health Administration<sup>[http://www.osha.gov/]</sup>
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::* Toxic and Hazardous Substances: Blood borne pathogens<sup>[http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=standards&p_id=10051]</sup>
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* Arizona State Environmental Health and Safety Program<sup>[http://cfo.asu.edu/ehs-biosafety]</sup>
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:* Biosafety manual<sup>[http://www.asu.edu/uagc/EHS/documents/biosafetymanual.pdf]</sup>
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:* Biowaste compliance guidelines<sup>[http://cfo.asu.edu/ehs-biowaste-compliance-guideline]</sup>
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=== Institutional Biosafety Committee ===
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}}

Revision as of 20:28, 31 August 2011


Safety


ASU Logo.png
 

Contents

Researcher Safety

All the organisms utilized in the Wang Lab comply with biosafety level 1 (BSL1) and do not possess the potential to cause disease in individuals. All organisms, despite having no association with disease, are treated as potential pathogens, thus personal protective equipment such as gloves, laboratory coats, and protective eyewear/goggles are used to prevent contact with bacteria and yeast samples in the lab.

Standard protocols were followed for all genetic manipulation, including PCR, plasmid assembly (restriction, ligation, and transformation), and DNA extraction. These protocols standardize specific safety procedures encountered in day to day labwork.

The following organisms are present in the lab and directly utilized in our project:

  • Escherichia coli K12 MG1655[http://www.genome.wisc.edu/resources/strains.htm], [http://www.ncbi.nlm.nih.gov/bioproject/225]
  • Escherichia coli BL21(DE3)[http://www.ncbi.nlm.nih.gov/bioproject/30681]
  • Escherichia coli NEB-10 Beta[http://www.neb.com/nebecomm/products/productc3020.asp]
  • Bacillus halodurans C-125[http://www.ncbi.nlm.nih.gov/bioproject/235]
  • Listeria innocua CLIP11262[http://www.ncbi.nlm.nih.gov/bioproject/86]

Strains L. innocua CLIP11262 and B. halodurans C-125 were both received from ATCC[http://www.atcc.org/], which classifies both organisms as BSL1. The ATCC Biosafety page[http://www.atcc.org/CulturesandProducts/TechnicalSupport/BiosafetyLevels/tabid/660/Default.aspx] states that BSL1 organisms and reagents, "are not known to cause disease in healthy adult humans".

Biosafety training

All members of our team were required to attend biosafety and bloodborne pathogen training according to Arizona State policy before working in the lab. This course satisfies the OSHA Bloodborne Pathogens training requirement as well as the Biosafety requirements for working with recombinant DNA. The Laboratory-Specific biosafety training checklist was followed[http://www.asu.edu/uagc/EHS/forms/asu_lab_specific_biosafety_training.pdf] to ensure all team members were adequately trained.

Biological Reagents

Reagents and equipment including solid and liquid bacterial growth media, and yeast culture media, micropipette, volumetric pipette tips and centrifuge tubes are autoclave sterilized (heated to 121 degrees celsius at 15 PSI) both preceding and following use, ensuring there is no threat of waste contamination of outside the lab. This is in accordance with Arizona State University's biological waste procedures[http://cfo.asu.edu/ehs-biowaste-compliance-guideline].

Flammable Reagent Safety

Reagents that are potentially flammable (e.g. ethanol and isopropyl alcohol) are stored in a flame protective cabinet.

Corrosive and Noxious Reagent Safety

Reagents that have corrosive and/or noxious fumes (e.g. bleach solutions and phenol-chloroform) are kept within a chemical fume hood to prevent inhalation and physical contact.

Public Safety

There is minimal risk associated with the release of the aforementioned organisms. The recombinant DNA (rDNA) experiments conducted in the laboratory provide ampicillin, kanamycin and/or chloramphenicol resistance to E.coli K12 MG1655, E.coli BL21(DE3) and E.coli NEB-10 Beta to select for plasmids. Under the circumstances that any of these genetically altered organisms were released they would have minimal potential for pathogenesis. In accordance with Arizona State University’s Environmental Health & Safety policy, “Nothing in the trash, nothing down the drain”, we autoclave and bleach sterilize all waste from recombinant DNA experiments. This reduces the likelihood of accidental release.

Under the circumstances of “designed” release, the organisms and all rDNA products present no possibility of harming the outside population and ecosystem. Our iGEM team, faculty mentor, and department lab safety manager, find no foreseeable public health threat associated with the organisms and recombinant DNA project utilized by our team.

Environmental Safety

Our project involves manipulating the CRISPR pathway as a gene regulation tool. The CRISPR system is naturally present in many prokaryotes and nearly all archea. Introducing a CRISPR locus into bacteria lacking CRISPR has the potential to increase phage resistance, and therefore fitness, through incorporation of new spacers. This is unlikely to have a significant effect on pathogenicity, especially when the spacers are designed to target relatively benign gene products such as GFP, as in our proof of concept. Currently our E. coli and B. halodurans constructs are engineered to omit two genes (CAS1 and CAS2) implicated in the incorporation of new spacers into CRISPR arrays. We also test these constructs in BL21, a laboratory strain of E. coli used for expression that has no native CAS genes. This minimizes the possibility of unwanted targeting that could potentially increase pathogenicity (although there is no evidence to suggest that this would be a favorable event).

We envision CRISPR as a tool that is used primarily in the laboratory. CRISPR arrays and CAS genes are highly susceptible to horizontal gene transfer. New spacers are constantly incorporated in wild-type strains, and CAS genes are easily lost in the absence of selection pressure. Because of this volatility the utility of CRISPR-engineered strains of bacteria in the environment seems minimal.

Biosafety Regulations and Provisions

  • National Guidelines:
  • National Institutes of Health[http://www.nih.gov/]
  • NIH Guidelines for Research Involving Recombinant DNA Molecules[http://oba.od.nih.gov/oba/rac/Guidelines/NIH_Guidelines.htm]
  • NIH Risk Group Classifications[http://rpi.edu/research/office/ibc/riskgroupclassifications.html]
  • Center for Disease Control[http://www.cdc.gov/]
  • CDC Biosafety[http://www.cdc.gov/biosafety/]
  • American Biological Safety Association[http://www.absa.org/]
  • Environmental Protection Agency[http://www.epa.gov/]
  • Toxic Substances Control Act (TSCA) Biotechnology Program[http://www.epa.gov/opptintr/biotech/index.htm]
  • Occupational Safety and Health Administration[http://www.osha.gov/]
  • Toxic and Hazardous Substances: Blood borne pathogens[http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=standards&p_id=10051]
  • Arizona State Environmental Health and Safety Program[http://cfo.asu.edu/ehs-biosafety]
  • Biosafety manual[http://www.asu.edu/uagc/EHS/documents/biosafetymanual.pdf]
  • Biowaste compliance guidelines[http://cfo.asu.edu/ehs-biowaste-compliance-guideline]
=== Institutional Biosafety Committee ===