Difference between revisions of "Team:BostonU/Mammalian synbio/Significance"

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<h3>Significance</h3>
 
<h3>Significance</h3>
−
<h4>Safety and Concerns</h4>
+
<p>This summer, our team was particularly excited to characterize our foundational systems in HEK293T mammalian cells. We were extremely interested in understanding best practices involved in successfully executing this type of research, both in the iGEM and broader synthetic biology communities.</p>
−
<p>One obvious concern that spans both the scientific and ethical realms are the safety issues that come from working with mammalian cells. Our iGEM team has thoroughly addressed these and hope to spread awareness through protocols and tips here (link to protocols page, specifically mammalian). Every member of our team completed BSL1 and BSL2 laboratory safety training through BU. With regards to BSL1 we utilized K12 E. Coli cells, an abundantly worked with and minimally threatening chassis. WIth regards to BSL2, we worked only with HEK293T cells. These cells were contained within one specific tissue culturing room. More specifically they were stored in a CO2-regulated incubator that had no other types of cells in it or they were worked with in our sterile, O2-regulated hood equipped with UV light to sterilize after being worked in. Working with the cells it was imperative to sterilize anything that came from outside the hood, but more importantly in concerns to disposing of the cells the priority was to kill all remaining cells and contain any media or other reagents they had come in contact with inside a tightly closed flask which was all disposed of as biohazardous waste. Safety protocols for this type of cell culturing are very important not only to verify that the cells are viable but more importantly that when they are no longer useful for research that they are properly disposed of and do not escape into any outside environments.</p>
+
<p>While working with mammalian cells offers great insight into developing synthetic tools within a eukaryotic system, one growing concern that spans both the scientific and ethical realms are the safety issues that arise from working with this chassis. </p>
  
−
<p>A concern that comes up further down this process is: if this split protein methodology is feasible, is it safe for human therapeutic use? This question was especially pertinent to our team as we were already testing in mammalian cells and had seen some promising results. What we want to emphasize is that our research is fundamentally trying to make safer a process which could be life saving in the future. Genomic editing and manipulation techniques are only getting better everyday, however, our project is not specifically focusing on making them better. We are focusing on the actual implementation of them. For example, right now it is possible that SaCas9 will recognize the wrong sequence and cut out the wrong gene, which could be potentially fatal. But with our system in place, it would hopefully be less likely that this would happen. There would be less chances for SaCas9 to even bind and carry out its functionalities since most of the time in vivo it would be split apart and inert. So already the protein is inactive for most of the time, however if it was required to be active the inducible dimerization provides for a simple and specific way to bring together the protein to carry out its genomic editing properties. We want to emphasize that this research is meant to directly address the natural, random problems that arise with any protein, and attempt to make them more regular and reliable with temporal control.</p>
 
  
−
<p>While we are excited to do foundational research in mammalian synthetic biology for such downstream genome editing and manipulation, we realized that this type of research is not heavily conducted in the iGEM community. However, many teams do intensive research for potential health and therapeutic applications and we feel it is important for this to get translated into mammalian systems for more precise downstream applications. We understand that there are many limitations to utilizing mammalian cells, but believe it is important to continue and intensify the dialogue in iGEM to promote it. </p>
+
<h4>How can scientists most safely conduct research using mammalian cells?</h4>
 +
<p>Since many graduate students and postdoctoral researchers working in our shared lab facilities at BU often use mammalian cells, we followed all of the necessary safety training that they had completed. Every member of our team completed BSL1 and BSL2 laboratory safety training through BU Environmental Health & Safety. We furthermore all completed a training module in Bloodborne Pathogens. </p>
  
−
<p>To better understand the climate in iGEM, we initially analyzed how many previous iGEM teams have used mammalian cells over the years.</p>
+
<p>Our cloning was done at a BSL1 level utilizing K12 E. coli cells, which are widely-used and minimally-threatening; however, our entire lab space is actually BSL2 certified, and as such we followed all BSL2 procedures whenever in the shared lab space. We recognized from our training sessions that Personal Protective Equipment was crucial for the safety of all researchers, so all team members wore long pants, close-toed shoes, lab coats, and gloves at all times, and additionally wore safety goggles and face shields whenever necessary.</p>
  
−
<p>It is clear above that the amount of teams using mammalian chassis are fairly static over the years, while other chassis like E. Coli increase greatly. It is obvious that there are many problems and impediments in using mammalian cells, and although we do not expect teams to immediately start working with them, we find it imperative to dig deeper and not only understand what the problems are but provide feasible feedback to potentially fix it.</p>
+
<p>Additionally, to use HEK293T cells, we exclusively worked in a BSL2 tissue culture room. We stored our HEK293T cells in a CO2-regulated incubator that did not have any other cell types or virus stored in it. When actively working with these cells, we put them into our sterile, O2-regulated tissue culture hood; when we finished, using the hood, we turned on the hood’s UV light to sterilize all surfaces. In all cases, we were absolutely sure to spray down all surfaces in the hood prior to use with 70% EtOH, as well as any equipment coming into the hood (including our gloves!).</p>
  
 +
<p>It was also a priority to make sure that all cells were properly killed and disposed as biohazardous waste. We added bleach to a final concentration of 10% to all cell solutions, and waited for at least 20 minutes before rinsing with water. We disposed of all sharps in biological sharps containers, and disposed of all other hazardous waste in biohazard waste bins. These containers were properly secured before EH&S arrived to remove them, so as to not compromise safety of all personnel as well.</p>
 +
 +
<p>Not only did all of our aforementioned procedures ensure that our cells did not escape into any outside environment, but also helped us validate that our cells were viable and not contaminated for proper experimental use. Our iGEM team has thoroughly addressed proper safety protocols and furthermore best practices for executing mammalian research (particularly with HEK293T cells!) Visit our PROTOCOLS AND TIPS <a href="https://2015.igem.org/Team:BostonU/Notebook/Protocols">here!</a> </p>
 +
 +
<h4>How safe are engineered mammalian synthetic biology systems, and moreover, how safe is our summer research project?</h4>
 +
 +
<p>We recognized that not only is there a question of safety when working with mammalian cells, but furthermore there is a question of safety regarding engineered mammalian cells, especially for therapeutic use. </p>
 +
 +
<p>Our team investigated this issue in the context of our own project: if our conditionally dimerizable protein methodology was feasible, would it be safe for downstream use? Perhaps the most scary question that we faced was: what would be the implications of engineering genome editing proteins such as saCas9 in order to make them easier for use in mammalian cells?</p>
 +
 +
<p>With regards to our specific research project, we want to emphasize that our conditionally dimerization workflow is simply a foundational advance that can improve the ability of researchers to exert more reliable and temporal control of their proteins. Currently, there are many limitations with proteins like cas9 that hinder them from creating “perfect synthetic systems” including off-target effects. Our ability to temporally control proteins does not truly address this problem, and as such we would not recommend that our system be used in an actual therapeutic context. Gaining temporal control of a protein such as cas9 could perhaps help with timing of activity, as a perfect dimerizable system would render the protein entirely inactive when not dimerized. </p>
 +
 +
<p><u>We are not actively pushing for this to become a medical technology anytime soon; rather, we have identified applications for both of our systems in which these advances can complement current biological studies, including both creating sophisticated gene expression control and studying disease progression in mouse models.</u></p>
 +
 +
<p>Genetic manipulation and genome editing techniques are quickly improving in the field of synthetic biology, and while much improvement is still necessary before some of these tools are ready for therapeutic use, it is important to acknowledge that certain groups may prematurely deploy these technologies. We note that proper regulation of such translational technologies is an important issue that spans many stakeholder groups and has truly significant consequences for everyone. </p>
 +
 +
<h4>What are concerns with carrying out mammalian synthetic biology research, particularly within the iGEM community?</h4>
 +
 +
<p>Throughout our experience this summer working with mammalian cells, we came to realize that research using this chassis is not heavily conducted in the iGEM community. Many teams every year do intensive research for potential health and therapeutic applications, and we noted that some systems could be better improved by either testing in conjunction <i>with</i> mammalian cells or actually testing parts <i>in</i> mammalian cells. These experiments could lead to important downstream applications.</p>
 +
 +
<p>We first tried to better understand the climate of mammalian research in iGEM. We analyzed how many previous iGEM teams have used mammalian cells over the years.</p>
 +
 +
<h4>Mammalian Graph</h4>
 +
 +
<p>The graph showed us that the number of teams which used mammalian chassis year to year was fairly static, while other chassis, in particular E. coli, increase greatly. We recognized that E. coli is typically the easiest chassis to work with, in terms of cost and convenience. Additionally, it has been widely used by researchers since the birth of synthetic biology.</p>
 +
 +
<p>We think that the research done in iGEM every year using E. coli is absolutely fantastic - looking through the past iGEM teams helped us see so many creative and interesting iGEM projects! However, through our analysis, we also see opportunities for more creativity and other synthetic biology applications when expanding to the eukaryotic space, and we can only tap into this potential if relevant chassis are more accessible to iGEM teams. While we do not expect all iGEM teams to immediately or entirely switch over to doing research in mammalian cells, we believe it is important to continue intensifying the dialogue about using mammalian (and other non-E. coli chassis) in order to promote these areas of research.</p>
 +
 +
<p>We thought about the problem of why there isn’t much mammalian research done in iGEM, and quickly identified some key problems and impediments that that prevent accessibility. We found it imperative to dig deeper and not only understand what these problems were, but how we could identify feasible steps that could mitigate them. Read on further!</p>
  
 
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Revision as of 17:33, 16 September 2015




Significance Current Challenges Proposed Solutions

Significance

This summer, our team was particularly excited to characterize our foundational systems in HEK293T mammalian cells. We were extremely interested in understanding best practices involved in successfully executing this type of research, both in the iGEM and broader synthetic biology communities.

While working with mammalian cells offers great insight into developing synthetic tools within a eukaryotic system, one growing concern that spans both the scientific and ethical realms are the safety issues that arise from working with this chassis.

How can scientists most safely conduct research using mammalian cells?

Since many graduate students and postdoctoral researchers working in our shared lab facilities at BU often use mammalian cells, we followed all of the necessary safety training that they had completed. Every member of our team completed BSL1 and BSL2 laboratory safety training through BU Environmental Health & Safety. We furthermore all completed a training module in Bloodborne Pathogens.

Our cloning was done at a BSL1 level utilizing K12 E. coli cells, which are widely-used and minimally-threatening; however, our entire lab space is actually BSL2 certified, and as such we followed all BSL2 procedures whenever in the shared lab space. We recognized from our training sessions that Personal Protective Equipment was crucial for the safety of all researchers, so all team members wore long pants, close-toed shoes, lab coats, and gloves at all times, and additionally wore safety goggles and face shields whenever necessary.

Additionally, to use HEK293T cells, we exclusively worked in a BSL2 tissue culture room. We stored our HEK293T cells in a CO2-regulated incubator that did not have any other cell types or virus stored in it. When actively working with these cells, we put them into our sterile, O2-regulated tissue culture hood; when we finished, using the hood, we turned on the hood’s UV light to sterilize all surfaces. In all cases, we were absolutely sure to spray down all surfaces in the hood prior to use with 70% EtOH, as well as any equipment coming into the hood (including our gloves!).

It was also a priority to make sure that all cells were properly killed and disposed as biohazardous waste. We added bleach to a final concentration of 10% to all cell solutions, and waited for at least 20 minutes before rinsing with water. We disposed of all sharps in biological sharps containers, and disposed of all other hazardous waste in biohazard waste bins. These containers were properly secured before EH&S arrived to remove them, so as to not compromise safety of all personnel as well.

Not only did all of our aforementioned procedures ensure that our cells did not escape into any outside environment, but also helped us validate that our cells were viable and not contaminated for proper experimental use. Our iGEM team has thoroughly addressed proper safety protocols and furthermore best practices for executing mammalian research (particularly with HEK293T cells!) Visit our PROTOCOLS AND TIPS here!

How safe are engineered mammalian synthetic biology systems, and moreover, how safe is our summer research project?

We recognized that not only is there a question of safety when working with mammalian cells, but furthermore there is a question of safety regarding engineered mammalian cells, especially for therapeutic use.

Our team investigated this issue in the context of our own project: if our conditionally dimerizable protein methodology was feasible, would it be safe for downstream use? Perhaps the most scary question that we faced was: what would be the implications of engineering genome editing proteins such as saCas9 in order to make them easier for use in mammalian cells?

With regards to our specific research project, we want to emphasize that our conditionally dimerization workflow is simply a foundational advance that can improve the ability of researchers to exert more reliable and temporal control of their proteins. Currently, there are many limitations with proteins like cas9 that hinder them from creating “perfect synthetic systems” including off-target effects. Our ability to temporally control proteins does not truly address this problem, and as such we would not recommend that our system be used in an actual therapeutic context. Gaining temporal control of a protein such as cas9 could perhaps help with timing of activity, as a perfect dimerizable system would render the protein entirely inactive when not dimerized.

We are not actively pushing for this to become a medical technology anytime soon; rather, we have identified applications for both of our systems in which these advances can complement current biological studies, including both creating sophisticated gene expression control and studying disease progression in mouse models.

Genetic manipulation and genome editing techniques are quickly improving in the field of synthetic biology, and while much improvement is still necessary before some of these tools are ready for therapeutic use, it is important to acknowledge that certain groups may prematurely deploy these technologies. We note that proper regulation of such translational technologies is an important issue that spans many stakeholder groups and has truly significant consequences for everyone.

What are concerns with carrying out mammalian synthetic biology research, particularly within the iGEM community?

Throughout our experience this summer working with mammalian cells, we came to realize that research using this chassis is not heavily conducted in the iGEM community. Many teams every year do intensive research for potential health and therapeutic applications, and we noted that some systems could be better improved by either testing in conjunction with mammalian cells or actually testing parts in mammalian cells. These experiments could lead to important downstream applications.

We first tried to better understand the climate of mammalian research in iGEM. We analyzed how many previous iGEM teams have used mammalian cells over the years.

Mammalian Graph

The graph showed us that the number of teams which used mammalian chassis year to year was fairly static, while other chassis, in particular E. coli, increase greatly. We recognized that E. coli is typically the easiest chassis to work with, in terms of cost and convenience. Additionally, it has been widely used by researchers since the birth of synthetic biology.

We think that the research done in iGEM every year using E. coli is absolutely fantastic - looking through the past iGEM teams helped us see so many creative and interesting iGEM projects! However, through our analysis, we also see opportunities for more creativity and other synthetic biology applications when expanding to the eukaryotic space, and we can only tap into this potential if relevant chassis are more accessible to iGEM teams. While we do not expect all iGEM teams to immediately or entirely switch over to doing research in mammalian cells, we believe it is important to continue intensifying the dialogue about using mammalian (and other non-E. coli chassis) in order to promote these areas of research.

We thought about the problem of why there isn’t much mammalian research done in iGEM, and quickly identified some key problems and impediments that that prevent accessibility. We found it imperative to dig deeper and not only understand what these problems were, but how we could identify feasible steps that could mitigate them. Read on further!