Team:Freiburg/Safety/safety forms

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<p id="h1">Safety forms</p>
 +
 
 +
<p id="h2">Safety forms were approved on October 2nd, 2013 by the iGEM Safety Committee.</p>
-
<p id="h1">Safety questions</p>
 
<p><font size="5">A</font>t the beginning of our research we wanted to be aware of all the probable hazards concerning our project. This included that we tried to identify safety issues. Therefore we concentrated on pathogenicity of the microorganisms and cell lines of interest, the datasheets of chemicals probably used during the project (e.g. DNA stains) and the engineered devices and systems. Thus, we orientated on the hints of the iGEM 2013 safety page as well as the safety constraints for genetic engineering given by the “Stabsstelle Sicherheit” of the University of Freiburg. At this point we are obliged to Dr. M. Zurbriggen, who gave us safety instructions before starting our investigations.
<p><font size="5">A</font>t the beginning of our research we wanted to be aware of all the probable hazards concerning our project. This included that we tried to identify safety issues. Therefore we concentrated on pathogenicity of the microorganisms and cell lines of interest, the datasheets of chemicals probably used during the project (e.g. DNA stains) and the engineered devices and systems. Thus, we orientated on the hints of the iGEM 2013 safety page as well as the safety constraints for genetic engineering given by the “Stabsstelle Sicherheit” of the University of Freiburg. At this point we are obliged to Dr. M. Zurbriggen, who gave us safety instructions before starting our investigations.
</p>
</p>
-
<p>
 
-
In the next passages, biosafety questions concernig to our project will be answered. There are also some recommendations to other teams, what they could do to optimize biosafety in their labs.
 
-
</p>
 
-
<p id="h3">Would any of your project ideas raise safety issues in terms of: </p>
+
<p id="h3"> 1. Please describe the chassis organism(s) you will be using for this project. If you will be using more than one chassis organism, provide information on each of them: </p>
-
<ul>
 
-
<li><b>researcher safety,</b></li>
 
-
<li><b>public safety, or</b></li>
 
-
<li><b>environmental safety?</b></li>
 
-
</ul>
 
-
<p>
+
<table id="tabelle">
-
Our project deals with targeted multiple gene regulation based on the bacterial and archaeal adaptive immune system CRISPR. This tool shall facilitate the daily lab routine of molecular biologists and might enable great progress in almost all genetically characterized disciplines such as cancer research or tissue engineering. Therefore, single biobricks are transformed into E. coli or transfected into eukaryotes such as HEK 293 cells and HeLa cells under safe laboratory conditions. Here, we work on high safety standards given by the University of Freiburg. Furthermore, we try to avoid the use of any highly toxic, acidic or other unhealthy substances. Thus, safety issues do not raise in idem terms.
+
<table border="3" frame="box">
-
</p>
+
-
<p id="h3">Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes, </p>
+
<tr> <th> # </th> <th> Species </th> <th> Strain no/name </th> <th> Risk Group </th> <th> Risk group source link </th> <th> Disease risk to humans? If so, which disease? </th> </tr>
 +
<tr> <td> 1 </td> <td> <i>E.coli</i> (K12) </td> <td> TOP10 </td> <td> 1 </td> <td> <a id="link" href="http://apps2.bvl.bund.de/strainwww/protected/main/strain.do?method=detail&theId=49&d-49653-p=null"> http://apps2.bvl.bund.de/strainwww/protected/main/strain.do?method=detail&theId=49&d-49653-p=null </a> </td> <td> "Yes. May cause
 +
irritation to skin, eyes,
 +
and respiratory tract,
 +
may affect kidneys.
 +
" </td> </tr>
 +
<tr> <td> 2 </td> <td> human </td> <td> HEK 293T </td> <td> 2 (1, according to german guidelines) </td> <td> <a id="link" href="http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=73&d-49653-p=22"> http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=73&d-49653-p=22 </a> </td> <td> </td> </tr>
 +
<tr> <td> 3 </td> <td> human </td> <td> HeLa </td> <td> 2 (1, according to german guidelines) </td> <td> <a id="link" href="http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=22&d-49653-p=null"> http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=22&d-49653-p=null </a> </td> <td> </td> </tr>
 +
<tr> <td> 4 </td> <td> hamster </td> <td> CHO-K1 </td> <td> 1 </td> <td> <a id="link" href="http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=13&d-49653-p=12"> http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=13&d-49653-p=12 </a> </td> <td> </td> </tr>
 +
<tr> <td> 5 </td> <td> mouse </td> <td> NIH/3T3 </td> <td> 1 </td> <td> <a id="link" href="http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=33&d-49653-p=null"> http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=33&d-49653-p=null </a> </td> <td> </td> </tr>
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<ul>
 
-
<li><b>did you document these issues in the Registry? </b></li>
 
-
<li><b>how did you manage to handle the safety issue?</b> </li>
 
-
<li><b>how could other teams learn from your experience? </b></li>
 
-
</ul>
 
 +
</table>
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<p>
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<p></p><p></p><p></p>
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Our constructs do not raise any safety issues. They should not be able to increase or give pathogenicity to the applied microorganisms or cell lines. The single parts do not encode for any toxins. The devices as they are used in our project should also represent no hazard for public health or environmental safety. In their current state misuse according to bioterrorism should also be unlikely.
+
<p id="h3"> 2. Highest Risk Group Listed </p>
 +
 
 +
<p>[  ]1 <br>
 +
[x] Greater than 1 </p>
 +
 
 +
<p></p><p></p>
 +
<p></p>
 +
 
 +
<p id="h3"> 3. List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.) </p>
 +
 
 +
<table id="tabelle">
 +
<table border="3" frame="box">
 +
 
 +
<tr> <th> Part number </th> <th> name </th> <th> "Where did you get the
 +
physical DNA for this
 +
part (which lab,
 +
synthesis company, etc)"
 +
</th> <th> "What species does
 +
this part originally
 +
come from?"
 +
</th> <th> "What is the
 +
Risk Group of
 +
the species?"
 +
</th> <th> "What is the function of
 +
this part, in its parent species?" </th> </tr>
 +
<tr> <td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150000"> BBa_K1150000 </a> </td> <td> dcas9 </td> <td> AddGene </td> <td> <i>Streptococcus pyogenes </i></td> <td> 2 </td> <td> specific immune response </td> </tr>
 +
<tr> <td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150001"> BBa_K1150001 </a> </td> <td> vp16 </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Herpes simplex</i> virus </td> <td> 2 </td> <td> gene transcription stimulator </td> </tr>
 +
<tr> <td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150002"> BBa_K1150002 </a> </td> <td> krab </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Homo sapiens</i> </td> <td> 1 </td> <td> repressor of transcriptional activity </td> </tr>
 +
<tr> <td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150003"> BBa_K1150003 </a> </td> <td> g9a-sd </td> <td> AG Jeltsch, University of Stuttgart </td> <td> <i>Mus musculus</i> </td> <td> 1 </td> <td> histone methyl transferase </td> </tr>
 +
<tr> <td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150004"> BBa_K1150004 </a> </td> <td> phyb </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Arabidopsis thaliana</i> </td> <td> 1 </td> <td> light signaling transducer </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150005"> BBa_K1150005 </a> </td> <td> pif6 </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Arabidopsis thaliana</i> </td> <td> 1 </td> <td> interacting factor of phyB </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150006"> BBa_K1150006 </a> </td> <td> uvr8 </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Arabidopsis thaliana</i> </td> <td> 1 </td> <td> light signaling transducer </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150007"> BBa_K1150007 </a> </td> <td> cop1 </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Arabidopsis thaliana</i> </td> <td> 1 </td> <td> light signaling transducer </td> </tr>
 +
<td> BBa_K1150008 </td> <td> cip </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Arabidopsis thaliana</i> </td> <td> 1 </td> <td> light signaling transducer </td> </tr>
 +
<td> BBa_K1150009 </td> <td> cry2 </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Arabidopsis thaliana</i> </td> <td> 1 </td> <td> light signaling transducer </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150010"> BBa_K1150010 </a> </td> <td> NLS </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> AAV2 </td> <td> 2 </td> <td> nuclear localization sequence </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150011"> BBa_K1150011 </a> </td> <td> SV40 </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> Simian-Virus 40 </td> <td> 2 </td> <td> viral promoter </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150012"> BBa_K1150012 </a> </td> <td> bGH-terminator </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> <i>Bos taurus</i> </td> <td> 1 </td> <td> terminator of the bovine growth hormone gene </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150013"> BBa_K1150013 </a> </td> <td> short linker </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> synthesized by Sigma-Aldrich as Oligos </td> <td> </td> <td> short, flexible linker </td> </tr>
 +
<td> BBa_K1150015 </td> <td> CMV </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> Cytomegalievirus </td> <td> 2 </td> <td> viralpromoter </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150016"> BBa_K1150016 </a> </td> <td> HA-Tag </td> <td> AG Wilfried Weber, University of Freiburg </td> <td> synthesized by Sigma-Aldrich as Oligos </td> <td> </td> <td> Protein sequence tag </td> </tr>
 +
<td> <a id="link" href="http://parts.igem.org/Part:BBa_K1150034"> BBa_K1150034 </a> </td> <td> RNA-plasmid </td> <td> AddGene </td> <td> <i>Streptococcus pyogenes, Homo sapiens</i> </td> <td> 2 </td> <td> specific immune response </td> </tr>
 +
</table>
 +
<p></p><p></p><p></p>
 +
 
 +
<p id="h3"> 4. Do the biological materials used in your lab work pose any of the following risks? Please describe.
 +
</p><p></p><p></p><p></p>
 +
<p id="h4"> a. Risks to the safety and health of team members or others working in the lab?
</p>
</p>
-
<p id="h3">Is there a local biosafety group, committee, or review board at your institution? </p>
+
<p> The biological materials used in our project are not related to pathogenicity and/or toxicity. The <i>E. coli</i> that we use to amplify our plasmids is the safe lab strain K12 which is not pathogenic. The mammalian cell lines we use in cell culture are rated in Germany as RG1. Nevertheless we handle these cells with great caution under sterile conditions. Therefore we do not see expanded risk for our team members and other lab workers. </p>
 +
<p></p><p></p>
 +
<p id="h4"> b. Risks to the safety and health of the general public, if released by design or by accident? </p>
 +
<p> DNA constructs used in this work as well as the genetically modified cells are thought for laboratory work. They should not be able to harm of any organism. Due to this fact we see no risk to safety and health of anyone when coming in contact with our constructs. </p><p></p><p></p>
 +
<p id="h4"> c. Risks to the environment, if released by design or by accident? </p>
 +
<p> As described above there is no risk for the environment by release. Even if our DNA constructs are taken up by organisms there is no risk to the environment as the constructs do not transfer any pathogenicity factors or biological fitness advantages. </p><p></p><p></p>
 +
<p id="h4"> d. Risks to security through malicious misuse by individuals, groups, or countries? </p>
 +
<p> Our system cannot be used for malicious purposes. It is a tool for scientific research and cannot be misused. </p>
 +
<p></p><p></p><p></p>
 +
<p id="h3"> 5. If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? (Consider the different categories of risks that are listed in parts a-d of the previous question.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available? (Note: This is meant to be a somewhat open-ended discussion question.)
 +
</p>
-
<ul>
+
<p> Our main protein, Cas9 and its related RNAs, are originating from a pathogen (Streptococcus pyogenes). This bacterium is classified, according to German regulations, to be safety level 2. As we are only dealing with a protein and RNAs, which are not described as a part of any pathogenic or toxic factor of this bacterium, they can be considered as harmless.
-
<li><b>If yes, what does your local biosafety group think about your project? </b></li>
+
This is in accordance to German regulations for biotechnology and the German safety forms. <br><br>
-
</ul>
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An up-scale of this system would not raise any additional dangers and could be performed under S1 conditions as our small-scale approaches. This is also in accordance to German safety regulations. </p>
 +
<p></p><p></p><p></p>
 +
<p id="h3"> 6. Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.
 +
</p>
 +
<p> No, our project does not include any additional safety features. </p>
 +
<p></p><p></p><p></p>
 +
<p id="h3"> 7. What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.
 +
</p>
 +
<p> Every team member has received a safety introduction. The introduction was performed by the safety instructor of our facility, Dr. Matias Zurbriggen. It included the handling of hazardous materials and equipment, biological waste disposal and behavior in cases of emergency. Our project was classified as S1 security level by the safety institution of our university. The regulations of the university can be found in the attached file. </p>
 +
<p></p><p></p><p></p>
 +
<p id="h3"> 8. Under what biosafety provisions will / do you work?
 +
</p>
 +
<p></p><p></p>
 +
<p id="h4">
 +
a. Please provide a link to your institution biosafety guidelines.
 +
</p>
 +
<p>The general guidelines of the University of Freiburg:<p>
 +
  <a id="link" href="https://static.igem.org/mediawiki/2013/b/b3/Guidelines_University_Freiburg_2013.pdf"> Guidelines  </a><br>
 +
  Guidelines, University of Freiburg
 +
</p>  </p> <p></p><p></p><p></p>
 +
<p id="h4">
 +
b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review.
 +
</p>
 +
<p> We discussed our projects extensively with the “Stabsstelle Sicherheit”, the independent biosafety institution of our university. They did not raise any security concerns about our project. For further information see <br>
 +
<a id="link" href="http://www.sicherheit.uni-freiburg.de"> http://www.sicherheit.uni-freiburg.de </a>
 +
. </p>
 +
 +
<p id="h4">
 +
c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.
 +
</p>
 +
<p> In Germany all biosafety aspects are regulated by the GenTG (Gesetz zur Regulierung der Gentechnik – Gene technology regulation law.) and the GenTSV (Verordnung über die Sicherheitsstufen und Sicherheitsmaßnahmen bei gentechnischen Arbeiten in gentechnischen Anlagen – Regulations for the security levels and security measurements in biotechnological work in biotechnological facilities). <br><br>
 +
<a id="link" href="http://www.gesetze-im-internet.de/gentg/index.html "> http://www.gesetze-im-internet.de/gentg/index.html  </a> <br>
 +
<a id="link" href="http://www.gesetze-im-internet.de/gentsv/"> http://www.gesetze-im-internet.de/gentsv/ </a>  </p>
 +
<p></p><p></p>
 +
 +
<p id="h4"> d. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? (Check the summary table on page 3, and the fuller description that starts on page 9.) If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features [see <a id="link" href="https://2013.igem.org/Safety"> 2013.igem.org/Safety </a> for help].
 +
</p>
 +
 +
 +
<p> Our lab has been approved for work under safety level 2. All the work we  are doing for this project has been classified as safety level 1 (considering German regulations) experiments as we are not using any pathogen or toxicity related organism or genetic part. According to the WHO our lab can be rated as biosafety level 2.  </p>
 +
<p id="h4"> e. What is the Risk Group of your chassis organism(s), as you stated in question 1? If it does not match the BSL rating of your laboratory, please explain what additional safety measures you are taking.
 +
</p>
 +
 +
<p> We are using Top10 OneShot™ K12 <i>E. coli</i> cells as vector chassis. They are rated level 1 as they do not cause any harm to people if handled correctly.
 +
As a test chassis we were using different mammalian cell lines. We were using CHO (Chinese hamster ovary), HEK293T- (human embryonic kidney), NIH/3T3 cells and HeLa (Henrietta Lacks) cells.  In Germany these cell lines are classified as BSL 1, in contrast to the USA regulations were they are classifies as BSL 2. The lab in which the team works is accredited to BSL 2. Thus, additional safety measurments are not necessary.
 +
<p></p><p></p><p></p><p></p>
 +
<div id="h2"><p></p>
 +
Safety forms part 2<p></p> </div>
 +
<p> As we are working in mammalian cells we are dealing with a lot of parts that have their origin in mammals. Additionally our main protein, the dCas9 is a part of the adaptive immune system of <i>Streptococcus pyogenes</i>. As these organisms are more complex to handle and are partially under RG2 regulations we filled out several Safety Forms part 2, where we discuss the risks and the alternatives to the usage of parts that are not originate grom RG2 organisms. </p><p>
 +
Find the informations in the following files. </p>
<p>
<p>
-
Yes, among other things, the department “Stabsstelle Sicherheit” is responsible for questions and issues concerning biosafety at the University of Freiburg.  
+
  <a id="link" href="https://static.igem.org/mediawiki/2013/a/a3/BGH_safety_Freiburg_2013.pdf"> bGH terminator  </a>
-
</p>
+
  </p>
-
<p id="h3">
+
<p>
-
Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?
+
  <a id="link" href="https://static.igem.org/mediawiki/2013/d/d2/Cas9_safety_Freiburg_2013.pdf "> Cas9  </a>
-
</p>
+
 
 +
</p> 
 +
<p>
 +
  <a id="link" href="https://static.igem.org/mediawiki/2013/9/90/CHO_K1_safety_Freiburg_2013.pdf"> CHO-K1 </a>
 +
 
 +
</p> 
 +
<p>
 +
  <a id="link" href="https://static.igem.org/mediawiki/2013/5/5a/CMV_safety_Freiburg_2013.pdf ">CMV promoter  </a>
 +
 
 +
</p> 
 +
<p>
 +
  <a id="link" href="https://static.igem.org/mediawiki/2013/0/0b/CrRNA_safety_Freiburg_2013.pdf"> crRNA  </a>
 +
</p> 
<p>
<p>
-
In our lab, we were especially concerned with carcinogenity. To protect our team we banned ethidium bromide completely and used next generation DNA stains concerned to be less carcinogenic. Additionally, nitrile gloves were used while cutting agarose gels containing DNA intercalating substances. Another hazard is the UV light, which can lead to mutations in DNA. Eyes and skin were protected due to lab coats and UV shield helmets. Theses safety precautions proved themselves in practice and are recommended to all other iGEM-teams.  
+
  <a id="link" href="https://static.igem.org/mediawiki/2013/4/46/G9a_safety_Freiburg_2013.pdf "> G9a  </a>
-
</p>
+
 
 +
</p>
<p>
<p>
-
Systems can be made safer through genetically switches. Our constructs are induced or silenced by stimuli, e.g. light of special wavelengths or hormones. Without the right stimulus the system is not running. Other possibilities are constructs that regulate themselves or self destruction mechanisms in case of dysfunctions.  
+
  <a id="link" href="https://static.igem.org/mediawiki/2013/0/00/HEK293T_safety_Freiburg_2013.pdf"> HEK293T  </a>
-
</p>
+
 
 +
</p> 
 +
<p>
 +
  <a id="link" href="https://static.igem.org/mediawiki/2013/9/91/Hela_safety_Freiburg_2013.pdf "> HeLa  </a>
 +
 
 +
</p>
 +
<p>
 +
  <a id="link" href="https://static.igem.org/mediawiki/2013/a/a2/KRAB_safety_Freiburg_2013.pdf"> KRAB  </a>
 +
 
 +
</p> 
 +
<p>
 +
  <a id="link" href="https://static.igem.org/mediawiki/2013/1/1e/NIH_3T3_safety_Freiburg_2013.pdf "> NIH/3T3  </a>
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  <a id="link" href="https://static.igem.org/mediawiki/2013/8/82/NLS_safety_Freiburg_2013.pdf"> NLS  </a>
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  <a id="link" href="https://static.igem.org/mediawiki/2013/b/b5/PH1_safety_Freiburg_2013.pdf "> H1 promoter </a>
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  <a id="link" href="https://static.igem.org/mediawiki/2013/8/8f/PU6_safety_Freiburg_2013.pdf"> U6 promoter  </a>
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  <a id="link" href="https://static.igem.org/mediawiki/2013/5/58/TracrRNA_safety_freiburg_2013.pdf"> tracrRNA  </a>
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</p> 
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  <a id="link" href="https://static.igem.org/mediawiki/2013/5/5a/SV40_safety_Freiburg_2013.pdf "> SV40 promoter  </a>
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</p> 
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<p>
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  <a id="link" href="https://static.igem.org/mediawiki/2013/4/4c/VP16_safety_Freiburg_2013.pdf"> VP16  </a><br>
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Latest revision as of 23:19, 28 October 2013

Safety forms

Safety forms were approved on October 2nd, 2013 by the iGEM Safety Committee.

At the beginning of our research we wanted to be aware of all the probable hazards concerning our project. This included that we tried to identify safety issues. Therefore we concentrated on pathogenicity of the microorganisms and cell lines of interest, the datasheets of chemicals probably used during the project (e.g. DNA stains) and the engineered devices and systems. Thus, we orientated on the hints of the iGEM 2013 safety page as well as the safety constraints for genetic engineering given by the “Stabsstelle Sicherheit” of the University of Freiburg. At this point we are obliged to Dr. M. Zurbriggen, who gave us safety instructions before starting our investigations.

1. Please describe the chassis organism(s) you will be using for this project. If you will be using more than one chassis organism, provide information on each of them:

# Species Strain no/name Risk Group Risk group source link Disease risk to humans? If so, which disease?
1 E.coli (K12) TOP10 1 http://apps2.bvl.bund.de/strainwww/protected/main/strain.do?method=detail&theId=49&d-49653-p=null "Yes. May cause irritation to skin, eyes, and respiratory tract, may affect kidneys. "
2 human HEK 293T 2 (1, according to german guidelines) http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=73&d-49653-p=22
3 human HeLa 2 (1, according to german guidelines) http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=22&d-49653-p=null
4 hamster CHO-K1 1 http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=13&d-49653-p=12
5 mouse NIH/3T3 1 http://apps2.bvl.bund.de/cellswww/protected/main/cell.do?method=detail&theId=33&d-49653-p=null

2. Highest Risk Group Listed

[ ]1
[x] Greater than 1

3. List and describe all new or modified coding regions you will be using in your project. (If you use parts from the 2013 iGEM Distribution without modifying them, you do not need to list those parts.)

Part number name "Where did you get the physical DNA for this part (which lab, synthesis company, etc)" "What species does this part originally come from?" "What is the Risk Group of the species?" "What is the function of this part, in its parent species?"
BBa_K1150000 dcas9 AddGene Streptococcus pyogenes 2 specific immune response
BBa_K1150001 vp16 AG Wilfried Weber, University of Freiburg Herpes simplex virus 2 gene transcription stimulator
BBa_K1150002 krab AG Wilfried Weber, University of Freiburg Homo sapiens 1 repressor of transcriptional activity
BBa_K1150003 g9a-sd AG Jeltsch, University of Stuttgart Mus musculus 1 histone methyl transferase
BBa_K1150004 phyb AG Wilfried Weber, University of Freiburg Arabidopsis thaliana 1 light signaling transducer
BBa_K1150005 pif6 AG Wilfried Weber, University of Freiburg Arabidopsis thaliana 1 interacting factor of phyB
BBa_K1150006 uvr8 AG Wilfried Weber, University of Freiburg Arabidopsis thaliana 1 light signaling transducer
BBa_K1150007 cop1 AG Wilfried Weber, University of Freiburg Arabidopsis thaliana 1 light signaling transducer
BBa_K1150008 cip AG Wilfried Weber, University of Freiburg Arabidopsis thaliana 1 light signaling transducer
BBa_K1150009 cry2 AG Wilfried Weber, University of Freiburg Arabidopsis thaliana 1 light signaling transducer
BBa_K1150010 NLS AG Wilfried Weber, University of Freiburg AAV2 2 nuclear localization sequence
BBa_K1150011 SV40 AG Wilfried Weber, University of Freiburg Simian-Virus 40 2 viral promoter
BBa_K1150012 bGH-terminator AG Wilfried Weber, University of Freiburg Bos taurus 1 terminator of the bovine growth hormone gene
BBa_K1150013 short linker AG Wilfried Weber, University of Freiburg synthesized by Sigma-Aldrich as Oligos short, flexible linker
BBa_K1150015 CMV AG Wilfried Weber, University of Freiburg Cytomegalievirus 2 viralpromoter
BBa_K1150016 HA-Tag AG Wilfried Weber, University of Freiburg synthesized by Sigma-Aldrich as Oligos Protein sequence tag
BBa_K1150034 RNA-plasmid AddGene Streptococcus pyogenes, Homo sapiens 2 specific immune response

4. Do the biological materials used in your lab work pose any of the following risks? Please describe.

a. Risks to the safety and health of team members or others working in the lab?

The biological materials used in our project are not related to pathogenicity and/or toxicity. The E. coli that we use to amplify our plasmids is the safe lab strain K12 which is not pathogenic. The mammalian cell lines we use in cell culture are rated in Germany as RG1. Nevertheless we handle these cells with great caution under sterile conditions. Therefore we do not see expanded risk for our team members and other lab workers.

b. Risks to the safety and health of the general public, if released by design or by accident?

DNA constructs used in this work as well as the genetically modified cells are thought for laboratory work. They should not be able to harm of any organism. Due to this fact we see no risk to safety and health of anyone when coming in contact with our constructs.

c. Risks to the environment, if released by design or by accident?

As described above there is no risk for the environment by release. Even if our DNA constructs are taken up by organisms there is no risk to the environment as the constructs do not transfer any pathogenicity factors or biological fitness advantages.

d. Risks to security through malicious misuse by individuals, groups, or countries?

Our system cannot be used for malicious purposes. It is a tool for scientific research and cannot be misused.

5. If your project moved from a small-scale lab study to become widely used as a commercial/industrial product, what new risks might arise? (Consider the different categories of risks that are listed in parts a-d of the previous question.) Also, what risks might arise if the knowledge you generate or the methods you develop became widely available? (Note: This is meant to be a somewhat open-ended discussion question.)

Our main protein, Cas9 and its related RNAs, are originating from a pathogen (Streptococcus pyogenes). This bacterium is classified, according to German regulations, to be safety level 2. As we are only dealing with a protein and RNAs, which are not described as a part of any pathogenic or toxic factor of this bacterium, they can be considered as harmless. This is in accordance to German regulations for biotechnology and the German safety forms.

An up-scale of this system would not raise any additional dangers and could be performed under S1 conditions as our small-scale approaches. This is also in accordance to German safety regulations.

6. Does your project include any design features to address safety risks? (For example: kill switches, auxotrophic chassis, etc.) Note that including such features is not mandatory to participate in iGEM, but many groups choose to include them.

No, our project does not include any additional safety features.

7. What safety training have you received (or plan to receive in the future)? Provide a brief description, and a link to your institution’s safety training requirements, if available.

Every team member has received a safety introduction. The introduction was performed by the safety instructor of our facility, Dr. Matias Zurbriggen. It included the handling of hazardous materials and equipment, biological waste disposal and behavior in cases of emergency. Our project was classified as S1 security level by the safety institution of our university. The regulations of the university can be found in the attached file.

8. Under what biosafety provisions will / do you work?

a. Please provide a link to your institution biosafety guidelines.

The general guidelines of the University of Freiburg:

Guidelines
Guidelines, University of Freiburg

b. Does your institution have an Institutional Biosafety Committee, or an equivalent group? If yes, have you discussed your project with them? Describe any concerns they raised with your project, and any changes you made to your project plan based on their review.

We discussed our projects extensively with the “Stabsstelle Sicherheit”, the independent biosafety institution of our university. They did not raise any security concerns about our project. For further information see
http://www.sicherheit.uni-freiburg.de .

c. Does your country have national biosafety regulations or guidelines? If so, please provide a link to these regulations or guidelines if possible.

In Germany all biosafety aspects are regulated by the GenTG (Gesetz zur Regulierung der Gentechnik – Gene technology regulation law.) and the GenTSV (Verordnung über die Sicherheitsstufen und Sicherheitsmaßnahmen bei gentechnischen Arbeiten in gentechnischen Anlagen – Regulations for the security levels and security measurements in biotechnological work in biotechnological facilities).

http://www.gesetze-im-internet.de/gentg/index.html
http://www.gesetze-im-internet.de/gentsv/

d. According to the WHO Biosafety Manual, what is the BioSafety Level rating of your lab? (Check the summary table on page 3, and the fuller description that starts on page 9.) If your lab does not fit neatly into category 1, 2, 3, or 4, please describe its safety features [see 2013.igem.org/Safety for help].

Our lab has been approved for work under safety level 2. All the work we are doing for this project has been classified as safety level 1 (considering German regulations) experiments as we are not using any pathogen or toxicity related organism or genetic part. According to the WHO our lab can be rated as biosafety level 2.

e. What is the Risk Group of your chassis organism(s), as you stated in question 1? If it does not match the BSL rating of your laboratory, please explain what additional safety measures you are taking.

We are using Top10 OneShot™ K12 E. coli cells as vector chassis. They are rated level 1 as they do not cause any harm to people if handled correctly. As a test chassis we were using different mammalian cell lines. We were using CHO (Chinese hamster ovary), HEK293T- (human embryonic kidney), NIH/3T3 cells and HeLa (Henrietta Lacks) cells. In Germany these cell lines are classified as BSL 1, in contrast to the USA regulations were they are classifies as BSL 2. The lab in which the team works is accredited to BSL 2. Thus, additional safety measurments are not necessary.

Safety forms part 2

As we are working in mammalian cells we are dealing with a lot of parts that have their origin in mammals. Additionally our main protein, the dCas9 is a part of the adaptive immune system of Streptococcus pyogenes. As these organisms are more complex to handle and are partially under RG2 regulations we filled out several Safety Forms part 2, where we discuss the risks and the alternatives to the usage of parts that are not originate grom RG2 organisms.

Find the informations in the following files.

bGH terminator

Cas9

CHO-K1

CMV promoter

crRNA

G9a

HEK293T

HeLa

KRAB

NIH/3T3

NLS

H1 promoter

U6 promoter

tracrRNA

SV40 promoter

VP16

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