Difference between revisions of "Team:Arizona State/Human Practices"

 
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<h2>Description</h2>
 
<h2>Description</h2>
 
<p>The lack of characterization of N-acyl homoserine lactones (AHLs) requires a comprehensive review of the safety of these molecules. The need for increased understanding extends to both the designed "Sender" parts and the AHL molecules themselves. Because quorum sensing is used by a myriad of bacterial species to induce virulence or biofilm formation, among other things, it has many implications towards activating a wide-range of bacteria. Our project aimed to investigate this broad issue by: </p>
 
<p>The lack of characterization of N-acyl homoserine lactones (AHLs) requires a comprehensive review of the safety of these molecules. The need for increased understanding extends to both the designed "Sender" parts and the AHL molecules themselves. Because quorum sensing is used by a myriad of bacterial species to induce virulence or biofilm formation, among other things, it has many implications towards activating a wide-range of bacteria. Our project aimed to investigate this broad issue by: </p>
<p>1. Consulting with experts in the field</p>
+
<p> 1. Consulting with experts in the field</p>
<p>2. Compiling a list of bacteria (pathogens, soil, water) that may possess orthogonality with AHLs produced by our Senders</p>
+
<p> 2. Compiling a list of bacteria (pathogens, soil, water) that may crosstalk with AHLs produced by our Senders</p>
 
<p> 3. Designing a AHL safe disposal plan</p>
 
<p> 3. Designing a AHL safe disposal plan</p>
<p> 4. Writing a report that provides suggestions for future research/use of AHLs. </p>
+
<p> 4. Writing a white paper that provides suggestions for future research/use of AHLs </p>
 +
<p> 5. Adding safety information to the Parts Pages of the Senders we constructed
 
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<div class="container">
 
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<ul>
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<li> Could it be harmful to our lab personnel? </li>
 
<li> Could it be harmful to our lab personnel? </li>
 
<li> Would inserting these genes into a different species lead to a new highly pathogenic strain? </li>
 
<li> Would inserting these genes into a different species lead to a new highly pathogenic strain? </li>
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<img src="https://static.igem.org/mediawiki/2016/0/09/T--Arizona_State--hpGeneWiz.png">
 
<img src="https://static.igem.org/mediawiki/2016/0/09/T--Arizona_State--hpGeneWiz.png">
 
<p>From the Skype call, our team aimed to clarify information about the dangers of AHLs. The following points were the biggest takeaways:</p>
 
<p>From the Skype call, our team aimed to clarify information about the dangers of AHLs. The following points were the biggest takeaways:</p>
<ul>
+
 
 +
<ul style="font-size:15px; margin-left:12px">
 
<li>GeneWiz checks for protein sequences, but not the products that the proteins create</li>
 
<li>GeneWiz checks for protein sequences, but not the products that the proteins create</li>
 
<li>The possibility of AHLs activating pathogens has not been brought up before, but it’s the customer’s responsibility if they are dealing with potentially harmful chemicals/toxins.</li>  
 
<li>The possibility of AHLs activating pathogens has not been brought up before, but it’s the customer’s responsibility if they are dealing with potentially harmful chemicals/toxins.</li>  
<li>They check for toxins and strains that are on a list provided by the FBI (the two documents are attached) </li>  
+
<li>They check for toxins and strains that are on a list provided by the FBI (the two documents are attached) </li>
</div>
+
<br>
<div class="column full_size">
+
<h3>Twist Bioscience</h3>
 +
<p>We consulted with Twist Bioscience to ask about their screening process when synthesizing genes. This was done through an email chain, shown below:</p>
 +
<img src="https://static.igem.org/mediawiki/2016/c/c8/T--Arizona_State--twistemail.png">
 +
<p>The following takeaways were gathered from this email response:</p>
 +
<ul>
 +
<li> Twist follows 2010 US HHS/ASPR Screening Framework Guidance for Providers of Double Stranded DNA and the follow-on IGSC Harmonized Screening Guidance, which can be found <a href="https://www.dropbox.com/sh/smq1shyxw3vt93j/AAAy3Bs4BuKscNHAadySp05ia?dl=0">here</a>.</li>
 +
<li>Twist focuses its screening on organisms specially regulated by CDC Select Agent and Commerce Control List</li>
 +
<li>Twist "does not focus on screening for sequences that, only in complex combination or in a unique context (e.g. exposed to AHL signaling as you suggest), would be capable of causing harm."</li>
 +
<li>"The Intelligence Advanced Research Projects Agency (IARPA) recently funded a three year project <a href="https://www.iarpa.gov/index.php/research-programs/fun-gcat/fun-gcat-baa">FunGCAT</a> focused intently on producing tools capable of estimating risk from primary DNA sequence." </li>
 +
<li>AHLs do not trigger the bioscreening protocol, as they are "second-order in that predicting the effect requires knowing the mix of organisms, conditions and metabolites present at the time."</li>
 +
</ul>
 +
<h3>iGEM Safety Committee</h3>
 +
<p>We contacted Dr. Megan Palmer, who " leads programs in safety and responsible innovation for the international Genetically Engineered Machine (iGEM) competition." After briefly discussing our project, she forwarded our questions to the iGEM Safety Committee, which sent the following response:</p>
 +
<img src="https://static.igem.org/mediawiki/2016/9/92/T--Arizona_State--palmeremail1.png">
 +
<img src="https://static.igem.org/mediawiki/2016/7/78/T--Arizona_State--palmeremail2.png">
 +
<p>We learned the following from this email chain:</p>
 +
<ul>
 +
<li>Some current researchers assume that AHLs degrade in water.</li>
 +
<li>Virulence factors are often only relevant after onset of infection, so the effect of AHLs prior to infection may be very weak.</li>
 +
<li> The international IAP:Global Network of Science Academies review of trends in science and technology relevant to the BWC was written in 2015, which discussed the importance of reviewing biofilm formation in potential pathogens as well as potentially inhibiting Receivers or Senders using small molecules.</li>
 +
<li>AHL quorum sensing should not be classified as dual-use research of concern(weaponized biology), and the benefits should outweigh potential dangers. </li>
 +
<li>Abide by the  ‘golden rule’, keeping bacterial pathogens sensible to AHLs away when working with it.</li>
 +
<li>Overall, do these parts come from a dangerous organism? No. Do these parts do something risky? Yes - when used in a certain way, with certain other parts. </li>
 +
</ul>
 +
<h2>List of Pathogens that Crosstalk</h2>
 +
<p>According to Davis, Muller, and Haynes, there are over 100 species of bacteria that are known to produce unique AHL inducers. This includes over 68 proteobacteria that are LuxI/R homologs, which was taken from a sample size of 512 bacteria with completed genomes (Case 2008). Our concern is that some of these bacteria are pathogenic, and that AHL exposure could cause activation of virulence or biofilm formation, among other consequences. We assembled a list of 14 pathogenic bacteria that may potentially activate due to AHL molecules generated by our Senders.  </p>
 +
<center><img src="https://static.igem.org/mediawiki/2016/f/f4/T--Arizona_State--crosstalklist.png"></center>
 +
<p>Some organisms of note include Burkholderia cepacia, which works together with other bacteria to activate Cystic Fibrosis, Nautella sp. R11, which causes bleaching in coral, and Yersinia pestis, which notably caused the Bubonic Plague.  </p>
  
<p>iGEM teams are leading in the area of Human Practices because they conduct their projects within a social/environmental context, to better understand issues that might influence the design and use of their technologies.</p>
+
<h2>AHL Safe Disposal Plan</h2>
<p>Teams work with students and advisors from the humanities and social sciences to explore topics concerning ethical, legal, social, economic, safety or security issues related to their work. Consideration of these Human Practices is crucial for building safe and sustainable projects that serve the public interest. </p>
+
<p>After investigating the risks offered by AHLs, we developed a plan to safely dispose of AHLs. This topic has been investigated before by S.A. Borchardt, who found that "the results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity". In other words, bleach exposure was capable of destroying 3-oxo AHLS, but all other AHLs were unable to fully deteriorate. </p>
<p>For more information, please see the <a href="https://2016.igem.org/Human_Practices">Human Practices Hub</a>.</p>
+
<p>Our disposal plan focused on standard EH&S sanitation methods, which included a 10% bleach treatment and autoclaving. We tested four AHL systems with bleach and autoclaving: Cer, Lux, Las, and Rpa. The samples were bleached for 10, 20 and 30 minutes, and then added to a 96-well plate with F2620. This protocol is located under the protocol header. For the autoclaving experiments, a standard liquid cycle was run (120°C for 15 minutes) on a liquid extraction of AHLs, which were then added to F2620 and measured in the plate reader. The figures below are the result of an 8-hour read measuring GFP levels. The Cer, Las, Lux, Rpa systems as well as Esa AHL deactivation was also tested through autoclaving. Esa was added to this test because there was room on the 96-well plate and it had shown good induction results on the previous F2620 tests.</p>
</div>
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<div class="row">
  
 
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<div class="column half_size">
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<center><img height="250px" src="https://static.igem.org/mediawiki/2016/a/a4/T--Arizona_State--lasbleach.png"></center>  
<div class="highlight">
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<h5>Note</h5>
+
<p>You must fill out this page in order to be considered for all <a href="https://2016.igem.org/Judging/Awards">awards</a> for Human Practices:</p>
+
<ul>
+
<li>Human Practices silver medal criterion</li>
+
<li>Human Practices gold medal criterion</li>
+
<li>Best Integrated Human Practices award</li>
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<li>Best Education and Public Engagement award</li>
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</ul>
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</div>
 
</div>
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            <div class="col-sm-6 center">
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<center><img height="250px" src="https://static.igem.org/mediawiki/2016/e/e3/T--Arizona_State--luxbleach.png"></center><br>
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</div></div>
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<div class="row">
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            <div class="col-sm-6 center">
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<center><img height="250px" src="https://static.igem.org/mediawiki/2016/4/49/T--Arizona_State--cerbleachdata.png"></center>
 
</div>
 
</div>
 
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            <div class="col-sm-6 center">
<div class="column half_size">
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<center><img height="250px" src="https://static.igem.org/mediawiki/2016/e/e1/T--Arizona_State--rpableachdata.png"></center><br>  
<h5>Some Human Practices topic areas </h5>
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</div></div>
<ul>
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<div class="row">
<li>Philosophy</li>
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            <div class="col-sm-6 center">
<li>Public Engagement / Dialogue</li>
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<center><img height="250px" src="https://static.igem.org/mediawiki/2016/0/07/T--Arizona_State--lasautoclave.png"></center>  
<li>Education</li>
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<li>Product Design</li>
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<li>Scale-Up and Deployment Issues</li>
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<li>Environmental Impact</li>
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<li>Ethics</li>
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<li>Safety</li>
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<li>Security</li>
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<li>Public Policy</li>
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<li>Law and Regulation</li>
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<li>Risk Assessment</li>
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</ul>
+
 
</div>
 
</div>
 +
            <div class="col-sm-6 center">
 +
<center><img height="250px" src="https://static.igem.org/mediawiki/2016/3/34/T--Arizona_State--luxautoclave.png"></center><br>
 +
</div></div>
 +
<div class="row">
 +
            <div class="col-sm-6 center">
 +
<center><img height="250px" src="https://static.igem.org/mediawiki/2016/4/41/T--Arizona_State--cerautoclave.png"></center>
 +
</div>
 +
            <div class="col-sm-6 center">
 +
<center><img height="250px" src="https://static.igem.org/mediawiki/2016/e/e2/T--Arizona_State--rpaautoclave.png"></center><br>
 +
</div></div>
  
 +
</br>
  
<div class="column half_size">
+
<p>The results demonstrate that all systems, Lux, Las, Rpa and Cer were able to effectively induce F2620 after being extracted. However, only the Las system was effectively neutralized by a 10% bleach solution. All 3 treatments of bleach showed negligible induction through the GFP/OD measurements. This is consistent with the results found by the Borchardt paper, which showed that a minimal concentration of bleach (0.14mM) was capable of breaking down 3-oxo AHLs. However, the Lux results were not nearly as consistent, which showed decreased induction when treated with bleach, but no evidence of complete AHL inactivation. What is also interesting is the decreased induction by Cer after being in bleach. The Cer AHL is not a 3-oxo, but rather has an unsaturated acyl tail. However, all other QS systems retained activity after bleach.</p>
<h5>What should we write about on this page?</h5>
+
<p>On this page, you should write about the Human Practices topics you considered in your project, and document any special activities you did (such as visiting experts, talking to lawmakers, or doing public engagement).</p>
+
</div>
+
  
 +
<div class="row">
  
<div class="column half_size">
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</div>
<h5>Inspiration</h5>
+
<p>According to the autoclave results, a standard 15 min liquid procedure is able to degrade nearly all AHLs including Rpa, Car, Las, and Lux AHL molecules. All 4 systems produced nearly no F2620 induction after autoclave treatment. The extreme pressure and temperature generated by the autoclave was more than enough to remove any threat posed by these AHL samples. However, Esa was still able to induce after being autoclaved.  This means that further research may be required to determine an exact method to inactivate all AHLs.</p>
<p>Read what other teams have done:</p>
+
<p>In summary, our data suggests that autoclaving will ensure at least some inactivation of AHLs, while bleach may have no effect at all on the degradation of AHLs. The data we have portrayed here is not conclusive, and further testing of disposal methods and AHL activity will be required before determining an all-inclusive disposal method. However in general, the human practices project does conclude that AHL degradation is an issue that needs to be considered. That gene synthesis companies and biosafety committees have not properly considered AHLs as a potentially harmful agent, while many studies show AHLs activate virulence factors in pathogens. Also that the widespread use and release of AHLs into the environment without proper disposal may cause significant activation and issues with pathogens. </p>
<ul>
+
<h2>White Paper</h2>
<li><a href="https://2014.igem.org/Team:Dundee/policypractice/experts">2014 Dundee </a></li>
+
<p>After gathering information and conducting the safe disposal experiment, we summarized our findings and provided detailed suggestions in a collective white paper. This can be found <a href="https://2016.igem.org/Team:Arizona_State/WhitePaper">here</a>.</p>
<li><a href="https://2014.igem.org/Team:UC_Davis/Policy_Practices_Overview">2014 UC Davis </a></li>
+
<h2>Parts Pages Safety Information</h2>
<li><a href="https://2013.igem.org/Team:Manchester/HumanPractices">2013 Manchester </a></li>
+
<p>At the bottom of each parts page that we submitted to the registry, we included a safety section, which lists potential crosstalk partners, proper disposal of the AHL, and other considerations. </p>
<li><a href="https://2013.igem.org/Team:Cornell/outreach">2013 Cornell </a></li>
+
</ul>
+
 
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</html>
 
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{{Arizona_State_Footer}}
 
{{Arizona_State_Footer}}

Latest revision as of 07:10, 12 November 2016

Human Practices

Description

The lack of characterization of N-acyl homoserine lactones (AHLs) requires a comprehensive review of the safety of these molecules. The need for increased understanding extends to both the designed "Sender" parts and the AHL molecules themselves. Because quorum sensing is used by a myriad of bacterial species to induce virulence or biofilm formation, among other things, it has many implications towards activating a wide-range of bacteria. Our project aimed to investigate this broad issue by:

1. Consulting with experts in the field

2. Compiling a list of bacteria (pathogens, soil, water) that may crosstalk with AHLs produced by our Senders

3. Designing a AHL safe disposal plan

4. Writing a white paper that provides suggestions for future research/use of AHLs

5. Adding safety information to the Parts Pages of the Senders we constructed

Consulting with Experts

Integrated Device Technologies

We contacted Integrated Device Technologies (IDT) to gather information on the possible threats associated with the Sender sequences that our team designed. This response was gathered over email, as shown below:

They asked the following questions in regards to how safe a gene might be:

  • Could it be harmful to our lab personnel?
  • Would inserting these genes into a different species lead to a new highly pathogenic strain?
  • Could an accidental transfer to a different species lead to a highly dangerous pathogen?

GeneWiz

We contacted GeneWiz about the possible impact of AHL molecules on inducing quorum sensing in nature. This was done through an email response and a Skype call. The initial email response is shown below:

From the Skype call, our team aimed to clarify information about the dangers of AHLs. The following points were the biggest takeaways:

  • GeneWiz checks for protein sequences, but not the products that the proteins create
  • The possibility of AHLs activating pathogens has not been brought up before, but it’s the customer’s responsibility if they are dealing with potentially harmful chemicals/toxins.
  • They check for toxins and strains that are on a list provided by the FBI (the two documents are attached)

  • Twist Bioscience

    We consulted with Twist Bioscience to ask about their screening process when synthesizing genes. This was done through an email chain, shown below:

    The following takeaways were gathered from this email response:

    • Twist follows 2010 US HHS/ASPR Screening Framework Guidance for Providers of Double Stranded DNA and the follow-on IGSC Harmonized Screening Guidance, which can be found here.
    • Twist focuses its screening on organisms specially regulated by CDC Select Agent and Commerce Control List
    • Twist "does not focus on screening for sequences that, only in complex combination or in a unique context (e.g. exposed to AHL signaling as you suggest), would be capable of causing harm."
    • "The Intelligence Advanced Research Projects Agency (IARPA) recently funded a three year project FunGCAT focused intently on producing tools capable of estimating risk from primary DNA sequence."
    • AHLs do not trigger the bioscreening protocol, as they are "second-order in that predicting the effect requires knowing the mix of organisms, conditions and metabolites present at the time."

    iGEM Safety Committee

    We contacted Dr. Megan Palmer, who " leads programs in safety and responsible innovation for the international Genetically Engineered Machine (iGEM) competition." After briefly discussing our project, she forwarded our questions to the iGEM Safety Committee, which sent the following response:

    We learned the following from this email chain:

    • Some current researchers assume that AHLs degrade in water.
    • Virulence factors are often only relevant after onset of infection, so the effect of AHLs prior to infection may be very weak.
    • The international IAP:Global Network of Science Academies review of trends in science and technology relevant to the BWC was written in 2015, which discussed the importance of reviewing biofilm formation in potential pathogens as well as potentially inhibiting Receivers or Senders using small molecules.
    • AHL quorum sensing should not be classified as dual-use research of concern(weaponized biology), and the benefits should outweigh potential dangers.
    • Abide by the ‘golden rule’, keeping bacterial pathogens sensible to AHLs away when working with it.
    • Overall, do these parts come from a dangerous organism? No. Do these parts do something risky? Yes - when used in a certain way, with certain other parts.

    List of Pathogens that Crosstalk

    According to Davis, Muller, and Haynes, there are over 100 species of bacteria that are known to produce unique AHL inducers. This includes over 68 proteobacteria that are LuxI/R homologs, which was taken from a sample size of 512 bacteria with completed genomes (Case 2008). Our concern is that some of these bacteria are pathogenic, and that AHL exposure could cause activation of virulence or biofilm formation, among other consequences. We assembled a list of 14 pathogenic bacteria that may potentially activate due to AHL molecules generated by our Senders.

    Some organisms of note include Burkholderia cepacia, which works together with other bacteria to activate Cystic Fibrosis, Nautella sp. R11, which causes bleaching in coral, and Yersinia pestis, which notably caused the Bubonic Plague.

    AHL Safe Disposal Plan

    After investigating the risks offered by AHLs, we developed a plan to safely dispose of AHLs. This topic has been investigated before by S.A. Borchardt, who found that "the results demonstrated that 3-oxo acyl homoserine lactone activity was rapidly lost upon exposure to oxidized halogens; however, acylated homoserine lactones lacking the 3-oxo group retained activity". In other words, bleach exposure was capable of destroying 3-oxo AHLS, but all other AHLs were unable to fully deteriorate.

    Our disposal plan focused on standard EH&S sanitation methods, which included a 10% bleach treatment and autoclaving. We tested four AHL systems with bleach and autoclaving: Cer, Lux, Las, and Rpa. The samples were bleached for 10, 20 and 30 minutes, and then added to a 96-well plate with F2620. This protocol is located under the protocol header. For the autoclaving experiments, a standard liquid cycle was run (120°C for 15 minutes) on a liquid extraction of AHLs, which were then added to F2620 and measured in the plate reader. The figures below are the result of an 8-hour read measuring GFP levels. The Cer, Las, Lux, Rpa systems as well as Esa AHL deactivation was also tested through autoclaving. Esa was added to this test because there was room on the 96-well plate and it had shown good induction results on the previous F2620 tests.






    The results demonstrate that all systems, Lux, Las, Rpa and Cer were able to effectively induce F2620 after being extracted. However, only the Las system was effectively neutralized by a 10% bleach solution. All 3 treatments of bleach showed negligible induction through the GFP/OD measurements. This is consistent with the results found by the Borchardt paper, which showed that a minimal concentration of bleach (0.14mM) was capable of breaking down 3-oxo AHLs. However, the Lux results were not nearly as consistent, which showed decreased induction when treated with bleach, but no evidence of complete AHL inactivation. What is also interesting is the decreased induction by Cer after being in bleach. The Cer AHL is not a 3-oxo, but rather has an unsaturated acyl tail. However, all other QS systems retained activity after bleach.

    According to the autoclave results, a standard 15 min liquid procedure is able to degrade nearly all AHLs including Rpa, Car, Las, and Lux AHL molecules. All 4 systems produced nearly no F2620 induction after autoclave treatment. The extreme pressure and temperature generated by the autoclave was more than enough to remove any threat posed by these AHL samples. However, Esa was still able to induce after being autoclaved. This means that further research may be required to determine an exact method to inactivate all AHLs.

    In summary, our data suggests that autoclaving will ensure at least some inactivation of AHLs, while bleach may have no effect at all on the degradation of AHLs. The data we have portrayed here is not conclusive, and further testing of disposal methods and AHL activity will be required before determining an all-inclusive disposal method. However in general, the human practices project does conclude that AHL degradation is an issue that needs to be considered. That gene synthesis companies and biosafety committees have not properly considered AHLs as a potentially harmful agent, while many studies show AHLs activate virulence factors in pathogens. Also that the widespread use and release of AHLs into the environment without proper disposal may cause significant activation and issues with pathogens.

    White Paper

    After gathering information and conducting the safe disposal experiment, we summarized our findings and provided detailed suggestions in a collective white paper. This can be found here.

    Parts Pages Safety Information

    At the bottom of each parts page that we submitted to the registry, we included a safety section, which lists potential crosstalk partners, proper disposal of the AHL, and other considerations.