http://2010.igem.org/wiki/index.php?title=Special:Contributions/LisaS&feed=atom&limit=50&target=LisaS&year=&month=2010.igem.org - User contributions [en]2024-03-28T23:52:53ZFrom 2010.igem.orgMediaWiki 1.16.5http://2010.igem.org/Team:Baltimore_US/SafetyTeam:Baltimore US/Safety2010-10-27T22:25:42Z<p>LisaS: </p>
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<div>[[Image:TitleBarBalti US.png | center]]<br />
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{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
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{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
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<br />
__TOC__<br />
== Overview ==<br />
"Not only would I like to have the participation of the Baltimore DIYbio group, but I think that the two of you have possibly set a standard on how the community can participate in an established event such as iGEM. You're contribution would be invaluable. " Agent You, FBI - WMD Directorate. July 19, 2010<br><br />
<br><br />
==== Bio-Safety, Bio-Security, Bio-Ethics ====<br />
At Baltimore-US, we strongly endorse the democratization of knowledge. However, we recognize that areas of research such as synthetic biology can have far-reaching consequences and the dissemination of knowledge and materials therefore must be balanced with self-responsibility. Because many members of our group were introduced to this topic through the DIY movement, including Jason Bobe and Mackenzie Cowell's DIY-Bio community ( http://diybio.org/ ), we have discussed the issues of safety, security, and ethics since our initial gathering. While our own group has not adopted a consensus position, we continue to discuss issued that surface when promoting increased access to recombinant DNA technologies. <br />
<br />
<br />
While much of the scientific community has held the idea of non-institutional involvement in research at arms length, we recognize that a responsible and informed citizenry has the potential to work harmoniously with the scientific, security and healthcare establishment. Indeed, citizen biotechnology could revolutionize the contributions made by individuals to the advancement of biology through both direct participation in the scientific enterprise and through more robust advocacy. We can envision benefits from these collaborative endeavors, for example the creation of pathogen and bioweapon detection grids or the home manufacture of pharmaceuticals and vaccines. An interesting paper from self-proclaimed bio-punk Meredith Patterson which advocates for the increased participation of citizens in science can be found at: http://maradydd.livejournal.com/496085.html <br><br />
<br />
<br />
The Baltimore-US team seeks to join the dialogue regarding the involvement of citizen-scientists in synthetic biology research. As part of this effort, we have taken several steps to become aware of and discuss issues in biosafety:<br />
<br />
1. We contacted the iGEM security director, Piers Millet, directly following our organizational meeting in February to address issues of biosafety as soon as we established our team.<br />
<br />
2. We hosted a discussion with Michelle Williams of ''National Journal'' which can be viewed here: http://burnafterreading.nationaljournal.com/2010/05/citizen-scientists-attract-fbi.php. The attempt by journalists and editors to frame issues of biosecurity in such as way as to create a sense of drama has reinforced our commitment to promoting the responsible incorporation of citizens into the scientific enterprise.<br />
<br />
3. We invited William You, WMD Director of the FBI, to speak to our group only days after he attended the Woodrow Wilson Center's Synthetic Biology forum. http://wilsoncenter.org/index.cfm?topic_id=1414&fuseaction=topics.event_summary&event_id=601732 <br><br />
<br />
4. We've worked with various individuals within the federal infrastructure to delineate a clearer set of standards and have proposed a Bio-C.O.R.E. (Citizen Outreach Response and Education) volunteer organization to help decentralize bio-safety/security response. <br />
<br />
5.We've recommended to all our community members to familiarize themselves with the training tools from the American Bio Safety Association located at http://www.absa.org/trainingtools.html as well.<br><br />
<br />
6. Several members of our group attended the Public Meeting for Presidential Summit on Bioethics. http://www.tvworldwide.com/events/bioethics/100708/default.cfm?id=12536&type=flv&test=0&live=0<br />
<br />
<br />
<br />
==== DIY-GEM Baltimore, USA Lab BioSafety Summary ====<br />
The DIY Bio iGEM team is a diverse group of students with a wide range of ages and varying backgrounds – all with a common interest of genetic engineering. The main Baltimore iGEM team lab is located at the Community College of Baltimore County(CCBC) under the direction of Dr. Tom Burkett. The responsibility of Bio-Safety and Bio-Security extends from the institution to the lab and to each individual team member. <br><br />
<br />
===== Institutional Responsibility =====<br />
The college is accountable for: hazard identification, a written Hazard Communication Plan (HCP), management of Material Safety Data Safety Sheets (MSDS) and safety training. <br><br />
<br />
CCBC abides by federal regulations and guidelines developed and enforced by: Center for Disease Control (CDC), National Institute for Health (NIH), Occupational Safety and Health Administration (OSHA), EnvironmentalProtection Services (EPA), Department of Transportation (DOT) and the Nuclear Regulatory Commission (NRC).<br><br />
<br />
A Hazard Communication Program (HAZCOM) has been established to insure compliance with all directives according to the Code of Federal Regulations (29 CFR 1910.1200). The purpose is to provide all those utilizing college lab facilities with a reference guide to working with hazardous chemicals. This program includes a chemical hygiene plan (CHP) detailing chemical safety information and procedures. <br><br />
<br />
Items included in the CHP are: <br><br />
* General chemical safety rules and procedures<br />
* Purchases, distribution and storage of chemicals<br />
* Environmental monitoring<br />
* Availability of medical programs<br />
* Maintenance, housekeeping and inspection procedures<br />
* Availability of protective devices and clothing<br />
* Record keeping policies<br />
* Training and employee/student information programs<br />
* Chemical labeling requirement<br />
* Accident and spill policies<br />
* Waste disposal programs<br />
* Emergency response plans<br />
* Designation of a safety officer<br />
A critical piece to the hazard communication program includes employee/student training. Science Safety Procedures apply to all campus labs. Each iGEM team member is required to complete lab safety training and signs a safety procedure agreement or risks team eviction. Included in the training are Standard or Good Microbiological Practices (GMP) which are basic practices for working with any microorganism. Also some universal lab safety rules are required practice. <br><br />
<br />
===== Laboratory Responsibility =====<br />
The lab plays a large role in the Biosafety and and BioSecurity of all campus labs including the iGem team lab. A key role is implementing the institutional and Federal and local applicable regulations and designating a Lab Safety Officer. In addition, lab accountability covers: <br><br />
* Labeling and documentation - on lab doors, cabinets/storage, waste containers and materials <br />
* Material Data Safety Sheets on any incoming or outgoing lab material. <br />
* Project Safety Analysis - where hazards are identified in every lab process and risk reduction strategy is implemented. <br />
* Housekeeping of the facilities and equipment<br />
* Emergency response also falls under the responsibility of the lab from First Aid, Protective devices, training and evacuation routes<br />
* Also very important is the lab risk analysis - evaluating the biosafety level (BSL)designation. <br><br />
The iGem lab is equipped for a BSL 2 designation, but our team lab facilities are designated as basic - BioSafety Level 1 - based on the lab design/construction, equipment, practices and operational procedures working with various agents. The recombinant DNA technology used is our labs is safe. Plasmid cloning vectors in combination with Escherichia coli K12 strain have been entirely sequenced. E.coli K12 is a non-pathogenic strain that can’t permanently colonize in healthy humans so routine genetic engineering experiments can safely be performed at BSL 1. <br>.<br />
<br />
==== Individual Team Member Responsibility ==== <br />
Each team member is responsible for their own safety while working in the lab as well as for those around them. It is required that all team members abide by government, institutional and lab regulations and policy including personnel safety practices and lab safety practices as emphasized in training. Baltimore iGem team members are committed to staying up to date on current regulation and reducing risk for Physical, Chemical and Biological hazards <br />
<br />
[[image:BioSafety_cert.JPG|400px]]<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
==Safety==<br />
<br />
==== BALTIMORE/USA iGEM TEAM SAFETY CODE ====<br />
We recognize the importance of each member's personal responsibility to the safety and security of our labs and our work. This includes the duty of not ignoring another's unsafe or possible harmful actions. We are committed to acting in a responsible manner and taking a pro-active approach to staying current with international and national laws, regulations and guidelines. We are dedicated to being informed about principles and practices designed to prevent hostile use of our labs, equipment, materials and products. It is our duty to contemplate the safety and security issues that may arise as an outcome of our projects. <br><br />
<br />
# 'Would any of your project ideas raise safety issues in terms of: researcher safety, public safety or environmental safety?'<br />
#* No, our project does not raise any safety issues in regards to researcher, public or environmental safety. We operate under the BSL-1 category and adhere to Standard Microbiological Practices as recommended by the US Dept. of Health & Human Services, CDC & NIH. Lab Safety Training has been provided to each team member and are properly supervised. <br />
# 'Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'<br />
#* No, our new parts do not raise any safety issues.<br />
# 'Is there a local biosafety group, committee, or review board at your institution?'<br />
#* Yes, a BioSafety team Committee has been assembled for our iGEM project. <br />
# 'Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions?'<br />
#* CDC Lab BioSafety certification would be useful training to iGEM team members.<br />
<br />
<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_US/SafetyTeam:Baltimore US/Safety2010-10-27T20:00:17Z<p>LisaS: </p>
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<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<br />
__TOC__<br />
== Overview ==<br />
"Not only would I like to have the participation of the Baltimore DIYbio group, but I think that the two of you have possibly set a standard on how the community can participate in an established event such as iGEM. You're contribution would be invaluable. " Agent You, FBI - WMD Directorate. July 19, 2010<br><br />
<br><br />
==== Bio-Safety, Bio-Security, Bio-Ethics ====<br />
At Baltimore-US, we strongly endorse the democratization of knowledge. However, we recognize that areas of research such as synthetic biology can have far-reaching consequences and the dissemination of knowledge and materials therefore must be balanced with self-responsibility. Because many members of our group were introduced to this topic through the DIY movement, including Jason Bobe and Mackenzie Cowell's DIY-Bio community ( http://diybio.org/ ), we have discussed the issues of safety, security, and ethics since our initial gathering. While our own group has not adopted a consensus position, we continue to discuss issued that surface when promoting increased access to recombinant DNA technologies. <br />
<br />
<br />
While much of the scientific community has held the idea of non-institutional involvement in research at arms length, we recognize that a responsible and informed citizenry has the potential to work harmoniously with the scientific, security and healthcare establishment. Indeed, citizen biotechnology could revolutionize the contributions made by individuals to the advancement of biology through both direct participation in the scientific enterprise and through more robust advocacy. We can envision benefits from these collaborative endeavors, for example the creation of pathogen and bioweapon detection grids or the home manufacture of pharmaceuticals and vaccines. An interesting paper from self-proclaimed bio-punk Meredith Patterson which advocates for the increased participation of citizens in science can be found at: http://maradydd.livejournal.com/496085.html <br><br />
<br />
<br />
The Baltimore-US team seeks to join the dialogue regarding the involvement of citizen-scientists in synthetic biology research. As part of this effort, we have taken several steps to become aware of and discuss issues in biosafety:<br />
<br />
1. We contacted the iGEM security director, Piers Millet, directly following our organizational meeting in February to address issues of biosafety as soon as we established our team.<br />
<br />
2. We hosted a discussion with Michelle Williams of ''National Journal'' which can be viewed here: http://burnafterreading.nationaljournal.com/2010/05/citizen-scientists-attract-fbi.php. The attempt by journalists and editors to frame issues of biosecurity in such as way as to create a sense of drama has reinforced our commitment to promoting the responsible incorporation of citizens into the scientific enterprise.<br />
<br />
3. We invited William You, WMD Director of the FBI, to speak to our group only days after he attended the Woodrow Wilson Center's Synthetic Biology forum. http://wilsoncenter.org/index.cfm?topic_id=1414&fuseaction=topics.event_summary&event_id=601732 <br><br />
<br />
4. We've worked with various individuals within the federal infrastructure to delineate a clearer set of standards and have proposed a Bio-C.O.R.E. (Citizen Outreach Response and Education) volunteer organization to help decentralize bio-safety/security response. <br />
<br />
5.We've recommended to all our community members to familiarize themselves with the training tools from the American Bio Safety Association located at http://www.absa.org/trainingtools.html as well.<br><br />
<br />
6. Several members of our group attended the recent Presidential Summit on BioEthics in Relation to Synthetic Biology.http://www.tvworldwide.com/events/bioethics/100708/default.cfm?id=12536&type=flv&test=0&live=0<br />
<br />
<br />
<br />
==== DIY-GEM Baltimore, USA Lab BioSafety Summary ====<br />
The DIY Bio iGEM team is a diverse group of students with a wide range of ages and varying backgrounds – all with a common interest of genetic engineering. The main Baltimore iGEM team lab is located at the Community College of Baltimore County(CCBC) under the direction of Dr. Tom Burkett. The responsibility of Bio-Safety and Bio-Security extends from the institution to the lab and to each individual team member. <br><br />
<br />
===== Institutional Responsibility =====<br />
The college is accountable for: hazard identification, a written Hazard Communication Plan (HCP), management of Material Safety Data Safety Sheets (MSDS) and safety training. <br><br />
<br />
CCBC abides by federal regulations and guidelines developed and enforced by: Center for Disease Control (CDC), National Institute for Health (NIH), Occupational Safety and Health Administration (OSHA), EnvironmentalProtection Services (EPA), Department of Transportation (DOT) and the Nuclear Regulatory Commission (NRC).<br><br />
<br />
A Hazard Communication Program (HAZCOM) has been established to insure compliance with all directives according to the Code of Federal Regulations (29 CFR 1910.1200). The purpose is to provide all those utilizing college lab facilities with a reference guide to working with hazardous chemicals. This program includes a chemical hygiene plan (CHP) detailing chemical safety information and procedures. <br><br />
<br />
Items included in the CHP are: <br><br />
* General chemical safety rules and procedures<br />
* Purchases, distribution and storage of chemicals<br />
* Environmental monitoring<br />
* Availability of medical programs<br />
* Maintenance, housekeeping and inspection procedures<br />
* Availability of protective devices and clothing<br />
* Record keeping policies<br />
* Training and employee/student information programs<br />
* Chemical labeling requirement<br />
* Accident and spill policies<br />
* Waste disposal programs<br />
* Emergency response plans<br />
* Designation of a safety officer<br />
A critical piece to the hazard communication program includes employee/student training. Science Safety Procedures apply to all campus labs. Each iGEM team member is required to complete lab safety training and signs a safety procedure agreement or risks team eviction. Included in the training are Standard or Good Microbiological Practices (GMP) which are basic practices for working with any microorganism. Also some universal lab safety rules are required practice. <br><br />
<br />
===== Laboratory Responsibility =====<br />
The lab plays a large role in the Biosafety and and BioSecurity of all campus labs including the iGem team lab. A key role is implementing the institutional and Federal and local applicable regulations and designating a Lab Safety Officer. In addition, lab accountability covers: <br><br />
* Labeling and documentation - on lab doors, cabinets/storage, waste containers and materials <br />
* Material Data Safety Sheets on any incoming or outgoing lab material. <br />
* Project Safety Analysis - where hazards are identified in every lab process and risk reduction strategy is implemented. <br />
* Housekeeping of the facilities and equipment<br />
* Emergency response also falls under the responsibility of the lab from First Aid, Protective devices, training and evacuation routes<br />
* Also very important is the lab risk analysis - evaluating the biosafety level (BSL)designation. <br><br />
The iGem lab is equipped for a BSL 2 designation, but our team lab facilities are designated as basic - BioSafety Level 1 - based on the lab design/construction, equipment, practices and operational procedures working with various agents. The recombinant DNA technology used is our labs is safe. Plasmid cloning vectors in combination with Escherichia coli K12 strain have been entirely sequenced. E.coli K12 is a non-pathogenic strain that can’t permanently colonize in healthy humans so routine genetic engineering experiments can safely be performed at BSL 1. <br>.<br />
<br />
==== Individual Team Member Responsibility ==== <br />
Each team member is responsible for their own safety while working in the lab as well as for those around them. It is required that all team members abide by government, institutional and lab regulations and policy including personnel safety practices and lab safety practices as emphasized in training. Baltimore iGem team members are committed to staying up to date on current regulation and reducing risk for Physical, Chemical and Biological hazards <br />
<br />
[[image:BioSafety_cert.JPG|400px]]<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
==Safety==<br />
<br />
==== BALTIMORE/USA iGEM TEAM SAFETY CODE ====<br />
We recognize the importance of each member's personal responsibility to the safety and security of our labs and our work. This includes the duty of not ignoring another's unsafe or possible harmful actions. We are committed to acting in a responsible manner and taking a pro-active approach to staying current with international and national laws, regulations and guidelines. We are dedicated to being informed about principles and practices designed to prevent hostile use of our labs, equipment, materials and products. It is our duty to contemplate the safety and security issues that may arise as an outcome of our projects. <br><br />
<br />
# 'Would any of your project ideas raise safety issues in terms of: researcher safety, public safety or environmental safety?'<br />
#* No, our project does not raise any safety issues in regards to researcher, public or environmental safety. We operate under the BSL-1 category and adhere to Standard Microbiological Practices as recommended by the US Dept. of Health & Human Services, CDC & NIH. Lab Safety Training has been provided to each team member and are properly supervised. <br />
# 'Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'<br />
#* No, our new parts do not raise any safety issues.<br />
# 'Is there a local biosafety group, committee, or review board at your institution?'<br />
#* Yes, a BioSafety team Committee has been assembled for our iGEM project. <br />
# 'Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions?'<br />
#* CDC Lab BioSafety certification would be useful training to iGEM team members.<br />
<br />
<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_US/SafetyTeam:Baltimore US/Safety2010-10-27T19:46:29Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<br />
__TOC__<br />
== Overview ==<br />
"Not only would I like to have the participation of the Baltimore DIYbio group, but I think that the two of you have possibly set a standard on how the community can participate in an established event such as iGEM. You're contribution would be invaluable. " Agent You, FBI - WMD Directorate. July 19, 2010<br><br />
<br><br />
==== Bio-Safety, Bio-Security, Bio-Ethics ====<br />
At Baltimore-US, we strongly endorse the democratization of knowledge. However, we recognize that areas of research such as synthetic biology can have far-reaching consequences and the dissemination of knowledge and materials therefore must be balanced with self-responsibility. Because many members of our group were introduced to this topic through the DIY movement, including Jason Bobe and Mackenzie Cowell's DIY-Bio community ( http://diybio.org/ ), we have discussed the issues of safety, security, and ethics since our initial gathering. While our own group has not adopted a consensus position, we continue to discuss issued that surface when promoting increased access to recombinant DNA technologies. <br />
<br />
<br />
While much of the scientific community has held the idea of non-institutional involvement in research at arms length, we recognize that a responsible and informed citizenry has the potential to work harmoniously with the scientific, security and healthcare establishment. Indeed, citizen biotechnology could revolutionize the contributions made by individuals to the advancement of biology through both direct participation in the scientific enterprise and through more robust advocacy. We can envision benefits from these collaborative endeavors, for example the creation of pathogen and bioweapon detection grids or the home manufacture of pharmaceuticals and vaccines. An interesting paper from self-proclaimed bio-punk Meredith Patterson which advocates for the increased participation of citizens in science can be found at: http://maradydd.livejournal.com/496085.html <br><br />
<br />
<br />
We contacted iGEM security directorate, Piers Millet, directly following our organizational meeting in February. At his suggestion we hosted a discussion with Michelle Williams of National Journal and William You, WMD Director of the FBI here in DC, only days after he attended the Woodrow Wilson Center's Synthetic Biology forum. <br />
http://wilsoncenter.org/index.cfm?topic_id=1414&fuseaction=topics.event_summary&event_id=601732 <br><br />
<br />
http://burnafterreading.nationaljournal.com/2010/05/citizen-scientists-attract-fbi.php <br><br />
The article above shows how sensationalistic journalists/editors attempt to frame issues to create a sense of drama. In response to which we've worked with various individuals within the federal infrastructure to delineate a clearer set of standards and have proposed a Bio-C.O.R.E. (Citizen Outreach Response and Education) volunteer organization to help decentralize bio-safety/security response. In addition we've recommended to all our community members to familiarize themselves with the training tools from the American Bio Safety Association located at http://www.absa.org/trainingtools.html as well.<br><br />
<br />
Specific to the work we are performing in our lab, we work under bio-safety level 1 conditions. We consider the greatest danger in our lab to be Ethidium Bromine that is utilized to run gels. This substance requires a particular disposal separate of the bio-hazard waste that is used to dispose of used pipette tips. When performing batch DNA extractions we also utilize a Phenol/Formaldehye mixture that along with some of the other volatile chemicals require separate disposal storage.<br />
Learning a little about the organisms and chemicals that we are working with can give us a clear view on how to safely operate with them and prevent contamination.<br><br />
<br />
The general chassis (microbe) that we have been working with is Escheria Coliform bacteria or e. coli for short. E. Coli has a pretty infamous reputation in relation to food poisoning and other intestinal illness. How is it that we can make fuel, drugs and art out of a stomach bug without continually heading to the restroom ourselves?<br><br />
E.Coli in nature. <br><br />
E.Coli as a model organism. <br><br />
<br />
==== DIY-GEM Baltimore, USA Lab BioSafety Summary ====<br />
The DIY Bio iGEM team is a diverse group of students with a wide range of ages and varying backgrounds – all with a common interest of genetic engineering. The main Baltimore iGEM team lab is located at the Community College of Baltimore County(CCBC) under the direction of Dr. Tom Burkett. The responsibility of Bio-Safety and Bio-Security extends from the institution to the lab and to each individual team member. <br><br />
<br />
===== Institutional Responsibility =====<br />
The college is accountable for: hazard identification, a written Hazard Communication Plan (HCP), management of Material Safety Data Safety Sheets (MSDS) and safety training. <br><br />
<br />
CCBC abides by federal regulations and guidelines developed and enforced by: Center for Disease Control (CDC), National Institute for Health (NIH), Occupational Safety and Health Administration (OSHA), EnvironmentalProtection Services (EPA), Department of Transportation (DOT) and the Nuclear Regulatory Commission (NRC).<br><br />
<br />
A Hazard Communication Program (HAZCOM) has been established to insure compliance with all directives according to the Code of Federal Regulations (29 CFR 1910.1200). The purpose is to provide all those utilizing college lab facilities with a reference guide to working with hazardous chemicals. This program includes a chemical hygiene plan (CHP) detailing chemical safety information and procedures. <br><br />
<br />
Items included in the CHP are: <br><br />
* General chemical safety rules and procedures<br />
* Purchases, distribution and storage of chemicals<br />
* Environmental monitoring<br />
* Availability of medical programs<br />
* Maintenance, housekeeping and inspection procedures<br />
* Availability of protective devices and clothing<br />
* Record keeping policies<br />
* Training and employee/student information programs<br />
* Chemical labeling requirement<br />
* Accident and spill policies<br />
* Waste disposal programs<br />
* Emergency response plans<br />
* Designation of a safety officer<br />
A critical piece to the hazard communication program includes employee/student training. Science Safety Procedures apply to all campus labs. Each iGEM team member is required to complete lab safety training and signs a safety procedure agreement or risks team eviction. Included in the training are Standard or Good Microbiological Practices (GMP) which are basic practices for working with any microorganism. Also some universal lab safety rules are required practice. <br><br />
<br />
===== Laboratory Responsibility =====<br />
The lab plays a large role in the Biosafety and and BioSecurity of all campus labs including the iGem team lab. A key role is implementing the institutional and Federal and local applicable regulations and designating a Lab Safety Officer. In addition, lab accountability covers: <br><br />
* Labeling and documentation - on lab doors, cabinets/storage, waste containers and materials <br />
* Material Data Safety Sheets on any incoming or outgoing lab material. <br />
* Project Safety Analysis - where hazards are identified in every lab process and risk reduction strategy is implemented. <br />
* Housekeeping of the facilities and equipment<br />
* Emergency response also falls under the responsibility of the lab from First Aid, Protective devices, training and evacuation routes<br />
* Also very important is the lab risk analysis - evaluating the biosafety level (BSL)designation. <br><br />
The iGem lab is equipped for a BSL 2 designation, but our team lab facilities are designated as basic - BioSafety Level 1 - based on the lab design/construction, equipment, practices and operational procedures working with various agents. The recombinant DNA technology used is our labs is safe. Plasmid cloning vectors in combination with Escherichia coli K12 strain have been entirely sequenced. E.coli K12 is a non-pathogenic strain that can’t permanently colonize in healthy humans so routine genetic engineering experiments can safely be performed at BSL 1. <br>.<br />
<br />
==== Individual Team Member Responsibility ==== <br />
Each team member is responsible for their own safety while working in the lab as well as for those around them. It is required that all team members abide by government, institutional and lab regulations and policy including personnel safety practices and lab safety practices as emphasized in training. Baltimore iGem team members are committed to staying up to date on current regulation and reducing risk for Physical, Chemical and Biological hazards <br />
<br />
[[image:BioSafety_cert.JPG|400px]]<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
==Safety==<br />
<br />
==== BALTIMORE/USA iGEM TEAM SAFETY CODE ====<br />
We recognize the importance of each member's personal responsibility to the safety and security of our labs and our work. This includes the duty of not ignoring another's unsafe or possible harmful actions. We are committed to acting in a responsible manner and taking a pro-active approach to staying current with international and national laws, regulations and guidelines. We are dedicated to being informed about principles and practices designed to prevent hostile use of our labs, equipment, materials and products. It is our duty to contemplate the safety and security issues that may arise as an outcome of our projects. <br><br />
<br />
# 'Would any of your project ideas raise safety issues in terms of: researcher safety, public safety or environmental safety?'<br />
#* No, our project does not raise any safety issues in regards to researcher, public or environmental safety. We operate under the BSL-1 category and adhere to Standard Microbiological Practices as recommended by the US Dept. of Health & Human Services, CDC & NIH. Lab Safety Training has been provided to each team member and are properly supervised. <br />
# 'Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'<br />
#* No, our new parts do not raise any safety issues.<br />
# 'Is there a local biosafety group, committee, or review board at your institution?'<br />
#* Yes, a BioSafety team Committee has been assembled for our iGEM project. <br />
# 'Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions?'<br />
#* CDC Lab BioSafety certification would be useful training to iGEM team members.<br />
<br />
Recent Presidential Summit on BioEthics in Relation to Synthetic Biology.<br><br />
http://www.tvworldwide.com/events/bioethics/100708/default.cfm?id=12536&type=flv&test=0&live=0<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_US/SafetyTeam:Baltimore US/Safety2010-10-27T19:45:09Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
<br />
__TOC__<br />
== Overview ==<br />
"Not only would I like to have the participation of the Baltimore DIYbio group, but I think that the two of you have possibly set a standard on how the community can participate in an established event such as iGEM. You're contribution would be invaluable. " Agent You, FBI - WMD Directorate. July 19, 2010<br><br />
<br><br />
==== Bio-Safety, Bio-Security, Bio-Ethics ====<br />
At Baltimore-US, we strongly endorse the democratization of knowledge. However, we recognize that areas of research such as synthetic biology can have far-reaching consequences and the dissemination of knowledge and materials therefore must be balanced with self-responsibility. Because many members of our group were introduced to this topic through the DIY movement, including Jason Bobe and Mackenzie Cowell's DIY-Bio community ( http://diybio.org/ ), we have discussed the issues of safety, security, and ethics since our initial gathering. While our own group has not adopted a consensus position, we continue to discuss issued that surface when promoting increased access to recombinant DNA technologies. <br />
<br />
While much of the scientific community has held the idea of non-institutional involvement in research at arms length, we recognize that a responsible and informed citizenry has the potential to work harmoniously with the scientific, security and healthcare establishment. Indeed, citizen biotechnology could revolutionize the contributions made by individuals to the advancement of biology through both direct participation in the scientific enterprise and through more robust advocacy. We can envision benefits from these collaborative endeavors, for example the creation of pathogen and bioweapon detection grids or the home manufacture of pharmaceuticals and vaccines. An interesting paper from self-proclaimed bio-punk Meredith Patterson which advocates for the increased participation of citizens in science can be found at: http://maradydd.livejournal.com/496085.html <br><br />
<br />
We contacted iGEM security directorate, Piers Millet, directly following our organizational meeting in February. At his suggestion we hosted a discussion with Michelle Williams of National Journal and William You, WMD Director of the FBI here in DC, only days after he attended the Woodrow Wilson Center's Synthetic Biology forum. <br />
http://wilsoncenter.org/index.cfm?topic_id=1414&fuseaction=topics.event_summary&event_id=601732 <br><br />
<br />
http://burnafterreading.nationaljournal.com/2010/05/citizen-scientists-attract-fbi.php <br><br />
The article above shows how sensationalistic journalists/editors attempt to frame issues to create a sense of drama. In response to which we've worked with various individuals within the federal infrastructure to delineate a clearer set of standards and have proposed a Bio-C.O.R.E. (Citizen Outreach Response and Education) volunteer organization to help decentralize bio-safety/security response. In addition we've recommended to all our community members to familiarize themselves with the training tools from the American Bio Safety Association located at http://www.absa.org/trainingtools.html as well.<br><br />
<br />
Specific to the work we are performing in our lab, we work under bio-safety level 1 conditions. We consider the greatest danger in our lab to be Ethidium Bromine that is utilized to run gels. This substance requires a particular disposal separate of the bio-hazard waste that is used to dispose of used pipette tips. When performing batch DNA extractions we also utilize a Phenol/Formaldehye mixture that along with some of the other volatile chemicals require separate disposal storage.<br />
Learning a little about the organisms and chemicals that we are working with can give us a clear view on how to safely operate with them and prevent contamination.<br><br />
<br />
The general chassis (microbe) that we have been working with is Escheria Coliform bacteria or e. coli for short. E. Coli has a pretty infamous reputation in relation to food poisoning and other intestinal illness. How is it that we can make fuel, drugs and art out of a stomach bug without continually heading to the restroom ourselves?<br><br />
E.Coli in nature. <br><br />
E.Coli as a model organism. <br><br />
<br />
==== DIY-GEM Baltimore, USA Lab BioSafety Summary ====<br />
The DIY Bio iGEM team is a diverse group of students with a wide range of ages and varying backgrounds – all with a common interest of genetic engineering. The main Baltimore iGEM team lab is located at the Community College of Baltimore County(CCBC) under the direction of Dr. Tom Burkett. The responsibility of Bio-Safety and Bio-Security extends from the institution to the lab and to each individual team member. <br><br />
<br />
===== Institutional Responsibility =====<br />
The college is accountable for: hazard identification, a written Hazard Communication Plan (HCP), management of Material Safety Data Safety Sheets (MSDS) and safety training. <br><br />
<br />
CCBC abides by federal regulations and guidelines developed and enforced by: Center for Disease Control (CDC), National Institute for Health (NIH), Occupational Safety and Health Administration (OSHA), EnvironmentalProtection Services (EPA), Department of Transportation (DOT) and the Nuclear Regulatory Commission (NRC).<br><br />
<br />
A Hazard Communication Program (HAZCOM) has been established to insure compliance with all directives according to the Code of Federal Regulations (29 CFR 1910.1200). The purpose is to provide all those utilizing college lab facilities with a reference guide to working with hazardous chemicals. This program includes a chemical hygiene plan (CHP) detailing chemical safety information and procedures. <br><br />
<br />
Items included in the CHP are: <br><br />
* General chemical safety rules and procedures<br />
* Purchases, distribution and storage of chemicals<br />
* Environmental monitoring<br />
* Availability of medical programs<br />
* Maintenance, housekeeping and inspection procedures<br />
* Availability of protective devices and clothing<br />
* Record keeping policies<br />
* Training and employee/student information programs<br />
* Chemical labeling requirement<br />
* Accident and spill policies<br />
* Waste disposal programs<br />
* Emergency response plans<br />
* Designation of a safety officer<br />
A critical piece to the hazard communication program includes employee/student training. Science Safety Procedures apply to all campus labs. Each iGEM team member is required to complete lab safety training and signs a safety procedure agreement or risks team eviction. Included in the training are Standard or Good Microbiological Practices (GMP) which are basic practices for working with any microorganism. Also some universal lab safety rules are required practice. <br><br />
<br />
===== Laboratory Responsibility =====<br />
The lab plays a large role in the Biosafety and and BioSecurity of all campus labs including the iGem team lab. A key role is implementing the institutional and Federal and local applicable regulations and designating a Lab Safety Officer. In addition, lab accountability covers: <br><br />
* Labeling and documentation - on lab doors, cabinets/storage, waste containers and materials <br />
* Material Data Safety Sheets on any incoming or outgoing lab material. <br />
* Project Safety Analysis - where hazards are identified in every lab process and risk reduction strategy is implemented. <br />
* Housekeeping of the facilities and equipment<br />
* Emergency response also falls under the responsibility of the lab from First Aid, Protective devices, training and evacuation routes<br />
* Also very important is the lab risk analysis - evaluating the biosafety level (BSL)designation. <br><br />
The iGem lab is equipped for a BSL 2 designation, but our team lab facilities are designated as basic - BioSafety Level 1 - based on the lab design/construction, equipment, practices and operational procedures working with various agents. The recombinant DNA technology used is our labs is safe. Plasmid cloning vectors in combination with Escherichia coli K12 strain have been entirely sequenced. E.coli K12 is a non-pathogenic strain that can’t permanently colonize in healthy humans so routine genetic engineering experiments can safely be performed at BSL 1. <br>.<br />
<br />
==== Individual Team Member Responsibility ==== <br />
Each team member is responsible for their own safety while working in the lab as well as for those around them. It is required that all team members abide by government, institutional and lab regulations and policy including personnel safety practices and lab safety practices as emphasized in training. Baltimore iGem team members are committed to staying up to date on current regulation and reducing risk for Physical, Chemical and Biological hazards <br />
<br />
[[image:BioSafety_cert.JPG|400px]]<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
==Safety==<br />
<br />
==== BALTIMORE/USA iGEM TEAM SAFETY CODE ====<br />
We recognize the importance of each member's personal responsibility to the safety and security of our labs and our work. This includes the duty of not ignoring another's unsafe or possible harmful actions. We are committed to acting in a responsible manner and taking a pro-active approach to staying current with international and national laws, regulations and guidelines. We are dedicated to being informed about principles and practices designed to prevent hostile use of our labs, equipment, materials and products. It is our duty to contemplate the safety and security issues that may arise as an outcome of our projects. <br><br />
<br />
# 'Would any of your project ideas raise safety issues in terms of: researcher safety, public safety or environmental safety?'<br />
#* No, our project does not raise any safety issues in regards to researcher, public or environmental safety. We operate under the BSL-1 category and adhere to Standard Microbiological Practices as recommended by the US Dept. of Health & Human Services, CDC & NIH. Lab Safety Training has been provided to each team member and are properly supervised. <br />
# 'Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?'<br />
#* No, our new parts do not raise any safety issues.<br />
# 'Is there a local biosafety group, committee, or review board at your institution?'<br />
#* Yes, a BioSafety team Committee has been assembled for our iGEM project. <br />
# 'Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions?'<br />
#* CDC Lab BioSafety certification would be useful training to iGEM team members.<br />
<br />
Recent Presidential Summit on BioEthics in Relation to Synthetic Biology.<br><br />
http://www.tvworldwide.com/events/bioethics/100708/default.cfm?id=12536&type=flv&test=0&live=0<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_US/Notebook/EPInstructionsTeam:Baltimore US/Notebook/EPInstructions2010-10-27T19:04:24Z<p>LisaS: /* Building a cheap Electrophoresis */</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<br />
== Developing low-cost alternatives to existing hardware: Electrophoresis Apparatus==<br />
To construct the cheap electrophoresis that we have designed you need several parts. All of the components of the electrophoresis are laser cut acrylic of .220" thickness. The design can be modified to use any thickness that is around .220". If you do not possess your own laser cutter or do not have access to one, the parts can be laser cut using several online services such as Ponoko (http://www.ponoko.com/), Pololu (http://www.pololu.com/catalog/product/749), or any other laser cutting service. Note that this does increase the price of the electrophoresis as can be expected from any small quantity machine shop. The design files and list of materials are included at the bottom of this page.<br />
<br />
==== Parts ====<br />
The following parts are needed:<br />
<gallery><br />
Image:Baltimore US Parts1.jpg |Holder Sides <br />
Image:Baltimore US Parts2.jpg |Anode/Cathode Holders<br />
Image:Baltimore US Parts3.jpg |Lid with Interlock<br />
Image:Baltimore US Parts4.jpg |Base <br />
Image:Baltimore US Parts5.jpg |Long Sides<br />
Image:Glue1.jpg |Plastruct weld<br />
Image:Glue2.jpg |Silicone Caulk <br />
Image:Baltimore_US_Not_Pictured.jpg |Conducting Plates<br />
</gallery><br />
<br />
<br />
==== Construction ====<br />
1. Get the two Holder sides and the base. <br><br />
[[Image:Build1.jpg|200px]]<br><br />
2. Glue the two Holder sides to the base using Plastruct Weld. Hold this together for five minutes. <br><br />
[[Image:Build2.jpg|200px]]<br><br />
3. If needed, brush more Plastruct weld into the cracks to ensure a seal. Hold this together. <br><br />
[[Image:Build3.jpg|200px]]<br><br />
4. Get the long sides and the previously constructed holder. <br><br />
[[Image:Build4.jpg|200px]]<br><br />
5. Glue the long sides to the previously constructed holder using Plastruct Weld. If needed brush more in the cracks.<br><br />
[[Image:Build5.jpg|200px]]<br><br />
6. Wait 24 hours for the Plastruct to set. <br><br />
7. Get the silicone caulk and the construct. <br><br />
[[Image:Build6.jpg|200px]]<br><br />
8. Caulk the construct on all of the inside seams. <br><br />
[[Image:Build7.jpg|200px]]<br><br />
9. Get the anode/cathode holders and conducting plates. Screw a plate onto the anode and cathode. <br><br />
[[Image:Build8.jpg|200px]]<br><br />
<br />
<br />
==== Finished Device ====<br />
[[Image:EP.jpg|400px]] [[Image:EP2.jpg|400px]]<br />
<br />
<br />
==== Design Files ====<br />
[[Media:Baltimore US EPDesign.zip| Design files for Electrophoresis]]<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T18:59:44Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
====DIY-Gem: a path towards low-cost high-throughput gene synthesis====<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel-electrophoresis apparatus and a PCR thermal cycler. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology.<br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue that enables the broader community to step into the iGEM competition, to gain hands-on laboratory experience, and to learn the tools and techniques necessary to explore the emerging field of synthetic biology while under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" align="center"<br />
|<br />
<center>'''Baltimore-US thanks the following companies for donations to our team:'''</center><br />
[[image:Qiagen logo.png|150px]] [[image:Axygen logo.png|150px]][[image:NEB logo.jpg|150px]]<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T18:59:17Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
====DIY-Gem: a path towards low-cost high-throughput gene synthesis====<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel-electrophoresis apparatus and a PCR thermal cycler. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology.<br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue that enables the broader community to step into the iGEM competition, to gain hands-on laboratory experience, and to learn the tools and techniques necessary to explore the emerging field of synthetic biology while under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" align="center"<br />
|<br />
<center>'''The Baltimore-US team thanks the following companies for donations to our team:'''</center><br />
[[image:Qiagen logo.png|150px]] [[image:Axygen logo.png|150px]][[image:NEB logo.jpg|150px]]<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_US/ProjectTeam:Baltimore US/Project2010-10-27T18:56:28Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
== DIY-GEM: a path towards low cost high throughput gene synthesis. ==<br />
Synthetic biology research requires more cost effective approaches toward reagents and hardware accessibility. We are developing low-cost alternatives to existing hardware and enzymes in an attempt to expand participation in biological research and development. Our project expands the accessibility of Taq Polymerase by engineering it in a form compatible with BioBrick assembly. This allows use of the over-expressed enzyme from a crude bacterial extract in a PCR reaction at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel electrophoresis apparatus and a PCR thermal cycler. Enabling researchers to synthesize their own enzymes and having access to inexpensive tools will allow for increased participation among the DIY-bio community, stretch increasingly scarce educational funds, and allow rapid scale up of large scale gene synthesis projects."<br />
<br />
==Developing low-cost alternatives to existing enzymes: ''Taq'' polymerase Project Details==<br />
<br />
Thermus Aquaticus Polymerase I<br><br />
PolI<br><br />
J04639.1<br><br />
Gene Sequence via BLAST at NCBI - http://www.ncbi.nlm.nih.gov/nuccore/155128<br><br />
<br><br />
1 AAGCTCAGAT CTACCTGCCT GAGGGCGTCC GGTTCCAGCT GGCCCTTCCC<br><br />
51 GAGGGGGAGA GGGAGGCGTT TCTAAAAGCC CTTCAGGACG CTACCCGGGG<br><br />
101 GCGGGTGGTG GAAGGGTAAC ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC<br><br />
151 AAGGGCCGGG TCCTCCTGGT GGACGGCCAC CACCTGGCCT ACCGCACCTT<br><br />
201 CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG GTGCAGGCGG<br><br />
251 TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC<br><br />
301 GCGGTGATCG TGGTCTTTGA CGCCAAGGCC CCCTCCTTCC GCCACGAGGC<br><br />
351 CTACGGGGGG TACAAGGCGG GCCGGGCCCC CACGCCGGAG GACTTTCCCC<br><br />
401 GGCAACTCGC CCTCATCAAG GAGCTGGTGG ACCTCCTGGG GCTGGCGCGC<br><br />
451 CTCGAGGTCC CGGGCTACGA GGCGGACGAC GTCCTGGCCA GCCTGGCCAA<br><br />
501 GAAGGCGGAA AAGGAGGGCT ACGAGGTCCG CATCCTCACC GCCGACAAAG<br><br />
551 ACCTTTACCA GCTCCTTTCC GACCGCATCC ACGTCCTCCA CCCCGAGGGG<br><br />
601 TACCTCATCA CCCCGGCCTG GCTTTGGGAA AAGTACGGCC TGAGGCCCGA<br><br />
651 CCAGTGGGCC GACTACCGGG CCCTGACCGG GGACGAGTCC GACAACCTTC<br><br />
701 CCGGGGTCAA GGGCATCGGG GAGAAGACGG CGAGGAAGCT TCTGGAGGAG<br><br />
751 TGGGGGAGCC TGGAAGCCCT CCTCAAGAAC CTGGACCGGC TGAAGCCCGC<br><br />
801 CATCCGGGAG AAGATCCTGG CCCACATGGA CGATCTGAAG CTCTCCTGGG<br><br />
851 ACCTGGCCAA GGTGCGCACC GACCTGCCCC TGGAGGTGGA CTTCGCCAAA<br><br />
901 AGGCGGGAGC CCGACCGGGA GAGGCTTAGG GCCTTTCTGG AGAGGCTTGA<br><br />
951 GTTTGGCAGC CTCCTCCACG AGTTCGGCCT TCTGGAAAGC CCCAAGGCCC<br><br />
1001 TGGAGGAGGC CCCCTGGCCC CCGCCGGAAG GGGCCTTCGT GGGCTTTGTG<br><br />
1051 CTTTCCCGCA AGGAGCCCAT GTGGGCCGAT CTTCTGGCCC TGGCCGCCGC<br><br />
1101 CAGGGGGGGC CGGGTCCACC GGGCCCCCGA GCCTTATAAA GCCCTCAGGG<br><br />
1151 ACCTGAAGGA GGCGCGGGGG CTTCTCGCCA AAGACCTGAG CGTTCTGGCC<br><br />
1201 CTGAGGGAAG GCCTTGGCCT CCCGCCCGGC GACGACCCCA TGCTCCTCGC<br><br />
1251 CTACCTCCTG GACCCTTCCA ACACCACCCC CGAGGGGGTG GCCCGGCGCT<br><br />
1301 ACGGCGGGGA GTGGACGGAG GAGGCGGGGG AGCGGGCCGC CCTTTCCGAG<br><br />
1351 AGGCTCTTCG CCAACCTGTG GGGGAGGCTT GAGGGGGAGG AGAGGCTCCT<br><br />
1401 TTGGCTTTAC CGGGAGGTGG AGAGGCCCCT TTCCGCTGTC CTGGCCCACA<br><br />
1451 TGGAGGCCAC GGGGGTGCGC CTGGACGTGG CCTATCTCAG GGCCTTGTCC<br><br />
1501 CTGGAGGTGG CCGAGGAGAT CGCCCGCCTC GAGGCCGAGG TCTTCCGCCT<br><br />
1551 GGCCGGCCAC CCCTTCAACC TCAACTCCCG GGACCAGCTG GAAAGGGTCC<br><br />
1601 TCTTTGACGA GCTAGGGCTT CCCGCCATCG GCAAGACGGA GAAGACCGGC<br><br />
1651 AAGCGCTCCA CCAGCGCCGC CGTCCTGGAG GCCCTCCGCG AGGCCCACCC<br><br />
1701 CATCGTGGAG AAGATCCTGC AGTACCGGGA GCTCACCAAG CTGAAGAGCA<br><br />
1751 CCTACATTGA CCCCTTGCCG GACCTCATCC ACCCCAGGAC GGGCCGCCTC<br><br />
1801 CACACCCGCT TCAACCAGAC GGCCACGGCC ACGGGCAGGC TAAGTAGCTC<br><br />
1851 CGATCCCAAC CTCCAGAACA TCCCCGTCCG CACCCCGCTT GGGCAGAGGA<br><br />
1901 TCCGCCGGGC CTTCATCGCC GAGGAGGGGT GGCTATTGGT GGCCCTGGAC<br><br />
1951 TATAGCCAGA TAGAGCTCAG GGTGCTGGCC CACCTCTCCG GCGACGAGAA<br><br />
2001 CCTGATCCGG GTCTTCCAGG AGGGGCGGGA CATCCACACG GAGACCGCCA<br><br />
2051 GCTGGATGTT CGGCGTCCCC CGGGAGGCCG TGGACCCCCT GATGCGCCGG<br><br />
2101 GCGGCCAAGA CCATCAACTT CGGGGTCCTC TACGGCATGT CGGCCCACCG<br><br />
2151 CCTCTCCCAG GAGCTAGCCA TCCCTTACGA GGAGGCCCAG GCCTTCATTG<br><br />
2201 AGCGCTACTT TCAGAGCTTC CCCAAGGTGC GGGCCTGGAT TGAGAAGACC<br><br />
2251 CTGGAGGAGG GCAGGAGGCG GGGGTACGTG GAGACCCTCT TCGGCCGCCG<br><br />
2301 CCGCTACGTG CCAGACCTAG AGGCCCGGGT GAAGAGCGTG CGGGAGGCGG<br><br />
2351 CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC AGGGCACCGC CGCCGACCTC<br><br />
2401 ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AAATGGGGGC<br><br />
2451 CAGGATGCTC CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG<br><br />
2501 AGAGGGCGGA GGCCGTGGCC CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG<br><br />
2551 TATCCCCTGG CCGTGCCCCT GGAGGTGGAG GTGGGGATAG GGGAGGACTG<br><br />
2601 GCTCTCCGCC AAGGAGTGAT ACCACC<br><br />
<br><br />
We took the above sequence from the provided link at BLAST and exported the SEQ into Plasma DNA. Plasma DNA is freeware from University of Helsinki which provides quick analysis of plasmid sequence information. http://research.med.helsinki.fi/plasmadna/<br />
<br><br />
When we cut and paste this dna sequence into plasmadna and look at the output window, we are given a visual output of various coding information. Such as restriction sites found within the code. To consider a construct viable for a BbPart we'll need to make certain that the standard restriction enzymes used with the system won't sheer the dna making it incomplete code. Searching for EcoRI, Xbe1, Sbe1, Pst1 sites will show whether the code is viable in an untampered state. <br><br />
<br />
====Problem: PstI restriction site - Found @ 1717====<br />
CTGCAG-PstI restriction site<br><br />
GACGTC-Complement<br><br />
Solution - Site-specific Mutagenesis by Overlap Extension (see Sambrook, Joseph; Russell, David W. ; Molecular Cloning: A Laboratory Manual, 3rd Edition - http://www.cshlpress.com/default.tpl?cart=1279686078181232350&fromlink=T&linkaction=full&linksortby=oop_title&--eqSKUdatarq=21)<br />
<br><br><br />
We then used the Gene Designer 2.0 freeware from DNA2.0 (https://www.dna20.com/genedesigner2/) - to analyze the Open Reading Frames. It shows us the Amino Acid codons that were being coded within that PstI Restrictions site. We find that the first three are coding for Leucine with CTG and can be changed at one point to CTT and still maintain Leucine's amino acid. The hope is that this will maintain functional integrity in the manufactured enzyme.<br><br />
<br />
====Primer Design====<br />
We designed two primers (11-14 Bp around chosen mutation) with changed Amino Acid Bp's Targeting initial Leucine at G of CTG to CTT. Point mutation Original G in CTG of Leucine. Change of one base to CTT maintains Leucine integrity. <br><br />
GTGGAGAAGATCCT(T)CAGTACCGGCGG<br><br />
CACCTCTTCTAGGA(A)GTCATGGCCGCC<br><br />
While we're designing primers, besides the point mutation, we'll take the opportunity to design and order the primers for the Bb Suffix and Prefix. We'll follow the examples laid out in the Registry of Standard Parts under Promoter Construction for designing the oligos needed to make a part. (http://partsregistry.org/Help:Promoters/Construction) <br><br />
<br><br />
Important considerations are Melting Point and percentage CG complements. Other considerations are dimerizations, that might cause primers to hairpin. We analyzed these primers using the OligoAnalyzer at IDT. When analyzing PolI Complements only were used for sequence inquiry, not the Bb Suffix/Prefixes. (http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/)<br><br />
<br><br />
====PolI Coli Primers For Overlap Extension PCR====<br />
'''PCR Reaction 1''' <br><br />
<br><br />
Bb Prefix + PolI (Fwd Complement) : (Forward complement will begin coding at 121 according to BLAST CDS information.)<br><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-ATGCTGCCCCTCTTTGAGCC<br><br />
60.5 c ; 56.5 % GC Concetration<br><br />
<br><br />
TAQ Rm<br><br />
CTCCCGGTACTGAAGGATCTTCTCCAC<br><br />
61.5 c ; 55.6 % GC Concentration<br><br />
<br><br />
'''PCR Reaction - 2'''<br><br />
<br><br />
TAQ Fm<br><br />
GTGGAGAAGATCCTTCAGTACCGGGAG<br><br />
61.5 c; 55.6 % GC<br><br />
<br><br />
Bb Suffix + PolI (Reverse Complement) : (Reverse complement will end coding at 2619 according to Blast CDS information.<br><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTA-TCACTCCTTGGCGGAGAGCC<br><br />
61.8 c; 65 % GC<br><br />
<br><br />
'''PCR Reaction - 3'''<br><br />
Bb Prefix & Suffix Primers<br><br />
<br><br />
Resuspend in 100 uL of H2O<br><br />
Run PCR w 1/100 dilutions for PCR (5-10 uL per PCR reaction)<br><br />
<br><br />
'''NEXT'''<br><br />
- Create Full Bb Prmr w Plasmid combining new part using<br><br />
<br><br />
<partinfo>R0010</partinfo> - Promoter (LacI)<br><br />
<partinfo>B0034</partinfo> - Strong RBS<br><br />
NEW PART - PolI Bb Format<br><br />
<partinfo>B0015</partinfo> - Double Terminator<br><br />
Psb1_?_3 - Plasmid of Interest with Chosen Resistance : http://partsregistry.org/Plasmid_backbones<br><br />
<br><br />
----<br />
<br><br />
<partinfo>R0010</partinfo> + <partinfo>B0034</partinfo> = New part LacI Promoter + Strong RBS<br><br />
<br><br />
Cut <partinfo>R0010</partinfo> w/EcoRI & SpeI<br><br />
Cut <partinfo>B0034</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI) - Because <br><br />
<br><br />
---<br><br />
New Part + <partinfo>B0015</partinfo> = New Part<br> <br />
<br><br />
Cut New Part w/EcoRI & SpeI<br><br />
Cut <partinfo>B0015</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
----<br><br />
<br><br />
Cut 1st Combined Part w/EcoRI & SpeI<br><br />
Cut 2nd Combined Part w/XbeI & PstI<br><br />
<br><br />
Combine in Ampecillan/Kanamyacin Resistan Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
'''Voila!!!''' Brand New Taq Polymerase Bb Part.<br><br />
<br />
<br />
<br />
== Developing low-cost alternatives to existing hardware: Project Details and Results ==<br />
<br />
[[Image:EP.jpg|300px]]<br><br />
With regards to equipment, we have successfully constructed a very low-cost Gel Electrophoresis device and are currently working on the control software and control electronics. Additionally, we are working on getting a low-cost PCR thermocycler up and running as well.<br> [[Team:Baltimore_US/Notebook/EPInstructions|Instructions and Design files for building an Electrophoresis device]]</div>LisaShttp://2010.igem.org/Team:Baltimore_US/ProjectTeam:Baltimore US/Project2010-10-27T18:52:21Z<p>LisaS: /* PoliColi Project Details */</p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
== DIY-GEM: a path towards low cost high throughput gene synthesis. ==<br />
Synthetic biology research requires more cost effective approaches toward reagents and hardware accessibility. We are developing low-cost alternatives to existing hardware and enzymes in an attempt to expand participation in biological research and development. Our project expands the accessibility of Taq Polymerase by engineering it in a form compatible with BioBrick assembly. This allows use of the over-expressed enzyme from a crude bacterial extract in a PCR reaction at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel electrophoresis apparatus and a PCR thermal cycler. Enabling researchers to synthesize their own enzymes and having access to inexpensive tools will allow for increased participation among the DIY-bio community, stretch increasingly scarce educational funds, and allow rapid scale up of large scale gene synthesis projects."<br />
<br />
==PoliColi (Taq polymerase) Project Details==<br />
<br />
Thermus Aquaticus Polymerase I<br><br />
PolI<br><br />
J04639.1<br><br />
Gene Sequence via BLAST at NCBI - http://www.ncbi.nlm.nih.gov/nuccore/155128<br><br />
<br><br />
1 AAGCTCAGAT CTACCTGCCT GAGGGCGTCC GGTTCCAGCT GGCCCTTCCC<br><br />
51 GAGGGGGAGA GGGAGGCGTT TCTAAAAGCC CTTCAGGACG CTACCCGGGG<br><br />
101 GCGGGTGGTG GAAGGGTAAC ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC<br><br />
151 AAGGGCCGGG TCCTCCTGGT GGACGGCCAC CACCTGGCCT ACCGCACCTT<br><br />
201 CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG GTGCAGGCGG<br><br />
251 TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC<br><br />
301 GCGGTGATCG TGGTCTTTGA CGCCAAGGCC CCCTCCTTCC GCCACGAGGC<br><br />
351 CTACGGGGGG TACAAGGCGG GCCGGGCCCC CACGCCGGAG GACTTTCCCC<br><br />
401 GGCAACTCGC CCTCATCAAG GAGCTGGTGG ACCTCCTGGG GCTGGCGCGC<br><br />
451 CTCGAGGTCC CGGGCTACGA GGCGGACGAC GTCCTGGCCA GCCTGGCCAA<br><br />
501 GAAGGCGGAA AAGGAGGGCT ACGAGGTCCG CATCCTCACC GCCGACAAAG<br><br />
551 ACCTTTACCA GCTCCTTTCC GACCGCATCC ACGTCCTCCA CCCCGAGGGG<br><br />
601 TACCTCATCA CCCCGGCCTG GCTTTGGGAA AAGTACGGCC TGAGGCCCGA<br><br />
651 CCAGTGGGCC GACTACCGGG CCCTGACCGG GGACGAGTCC GACAACCTTC<br><br />
701 CCGGGGTCAA GGGCATCGGG GAGAAGACGG CGAGGAAGCT TCTGGAGGAG<br><br />
751 TGGGGGAGCC TGGAAGCCCT CCTCAAGAAC CTGGACCGGC TGAAGCCCGC<br><br />
801 CATCCGGGAG AAGATCCTGG CCCACATGGA CGATCTGAAG CTCTCCTGGG<br><br />
851 ACCTGGCCAA GGTGCGCACC GACCTGCCCC TGGAGGTGGA CTTCGCCAAA<br><br />
901 AGGCGGGAGC CCGACCGGGA GAGGCTTAGG GCCTTTCTGG AGAGGCTTGA<br><br />
951 GTTTGGCAGC CTCCTCCACG AGTTCGGCCT TCTGGAAAGC CCCAAGGCCC<br><br />
1001 TGGAGGAGGC CCCCTGGCCC CCGCCGGAAG GGGCCTTCGT GGGCTTTGTG<br><br />
1051 CTTTCCCGCA AGGAGCCCAT GTGGGCCGAT CTTCTGGCCC TGGCCGCCGC<br><br />
1101 CAGGGGGGGC CGGGTCCACC GGGCCCCCGA GCCTTATAAA GCCCTCAGGG<br><br />
1151 ACCTGAAGGA GGCGCGGGGG CTTCTCGCCA AAGACCTGAG CGTTCTGGCC<br><br />
1201 CTGAGGGAAG GCCTTGGCCT CCCGCCCGGC GACGACCCCA TGCTCCTCGC<br><br />
1251 CTACCTCCTG GACCCTTCCA ACACCACCCC CGAGGGGGTG GCCCGGCGCT<br><br />
1301 ACGGCGGGGA GTGGACGGAG GAGGCGGGGG AGCGGGCCGC CCTTTCCGAG<br><br />
1351 AGGCTCTTCG CCAACCTGTG GGGGAGGCTT GAGGGGGAGG AGAGGCTCCT<br><br />
1401 TTGGCTTTAC CGGGAGGTGG AGAGGCCCCT TTCCGCTGTC CTGGCCCACA<br><br />
1451 TGGAGGCCAC GGGGGTGCGC CTGGACGTGG CCTATCTCAG GGCCTTGTCC<br><br />
1501 CTGGAGGTGG CCGAGGAGAT CGCCCGCCTC GAGGCCGAGG TCTTCCGCCT<br><br />
1551 GGCCGGCCAC CCCTTCAACC TCAACTCCCG GGACCAGCTG GAAAGGGTCC<br><br />
1601 TCTTTGACGA GCTAGGGCTT CCCGCCATCG GCAAGACGGA GAAGACCGGC<br><br />
1651 AAGCGCTCCA CCAGCGCCGC CGTCCTGGAG GCCCTCCGCG AGGCCCACCC<br><br />
1701 CATCGTGGAG AAGATCCTGC AGTACCGGGA GCTCACCAAG CTGAAGAGCA<br><br />
1751 CCTACATTGA CCCCTTGCCG GACCTCATCC ACCCCAGGAC GGGCCGCCTC<br><br />
1801 CACACCCGCT TCAACCAGAC GGCCACGGCC ACGGGCAGGC TAAGTAGCTC<br><br />
1851 CGATCCCAAC CTCCAGAACA TCCCCGTCCG CACCCCGCTT GGGCAGAGGA<br><br />
1901 TCCGCCGGGC CTTCATCGCC GAGGAGGGGT GGCTATTGGT GGCCCTGGAC<br><br />
1951 TATAGCCAGA TAGAGCTCAG GGTGCTGGCC CACCTCTCCG GCGACGAGAA<br><br />
2001 CCTGATCCGG GTCTTCCAGG AGGGGCGGGA CATCCACACG GAGACCGCCA<br><br />
2051 GCTGGATGTT CGGCGTCCCC CGGGAGGCCG TGGACCCCCT GATGCGCCGG<br><br />
2101 GCGGCCAAGA CCATCAACTT CGGGGTCCTC TACGGCATGT CGGCCCACCG<br><br />
2151 CCTCTCCCAG GAGCTAGCCA TCCCTTACGA GGAGGCCCAG GCCTTCATTG<br><br />
2201 AGCGCTACTT TCAGAGCTTC CCCAAGGTGC GGGCCTGGAT TGAGAAGACC<br><br />
2251 CTGGAGGAGG GCAGGAGGCG GGGGTACGTG GAGACCCTCT TCGGCCGCCG<br><br />
2301 CCGCTACGTG CCAGACCTAG AGGCCCGGGT GAAGAGCGTG CGGGAGGCGG<br><br />
2351 CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC AGGGCACCGC CGCCGACCTC<br><br />
2401 ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AAATGGGGGC<br><br />
2451 CAGGATGCTC CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG<br><br />
2501 AGAGGGCGGA GGCCGTGGCC CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG<br><br />
2551 TATCCCCTGG CCGTGCCCCT GGAGGTGGAG GTGGGGATAG GGGAGGACTG<br><br />
2601 GCTCTCCGCC AAGGAGTGAT ACCACC<br><br />
<br><br />
We took the above sequence from the provided link at BLAST and exported the SEQ into Plasma DNA. Plasma DNA is freeware from University of Helsinki which provides quick analysis of plasmid sequence information. http://research.med.helsinki.fi/plasmadna/<br />
<br><br />
When we cut and paste this dna sequence into plasmadna and look at the output window, we are given a visual output of various coding information. Such as restriction sites found within the code. To consider a construct viable for a BbPart we'll need to make certain that the standard restriction enzymes used with the system won't sheer the dna making it incomplete code. Searching for EcoRI, Xbe1, Sbe1, Pst1 sites will show whether the code is viable in an untampered state. <br><br />
<br />
====Problem: PstI restriction site - Found @ 1717====<br />
CTGCAG-PstI restriction site<br><br />
GACGTC-Complement<br><br />
Solution - Site-specific Mutagenesis by Overlap Extension (see Sambrook, Joseph; Russell, David W. ; Molecular Cloning: A Laboratory Manual, 3rd Edition - http://www.cshlpress.com/default.tpl?cart=1279686078181232350&fromlink=T&linkaction=full&linksortby=oop_title&--eqSKUdatarq=21)<br />
<br><br><br />
We then used the Gene Designer 2.0 freeware from DNA2.0 (https://www.dna20.com/genedesigner2/) - to analyze the Open Reading Frames. It shows us the Amino Acid codons that were being coded within that PstI Restrictions site. We find that the first three are coding for Leucine with CTG and can be changed at one point to CTT and still maintain Leucine's amino acid. The hope is that this will maintain functional integrity in the manufactured enzyme.<br><br />
<br />
====Primer Design====<br />
We designed two primers (11-14 Bp around chosen mutation) with changed Amino Acid Bp's Targeting initial Leucine at G of CTG to CTT. Point mutation Original G in CTG of Leucine. Change of one base to CTT maintains Leucine integrity. <br><br />
GTGGAGAAGATCCT(T)CAGTACCGGCGG<br><br />
CACCTCTTCTAGGA(A)GTCATGGCCGCC<br><br />
While we're designing primers, besides the point mutation, we'll take the opportunity to design and order the primers for the Bb Suffix and Prefix. We'll follow the examples laid out in the Registry of Standard Parts under Promoter Construction for designing the oligos needed to make a part. (http://partsregistry.org/Help:Promoters/Construction) <br><br />
<br><br />
Important considerations are Melting Point and percentage CG complements. Other considerations are dimerizations, that might cause primers to hairpin. We analyzed these primers using the OligoAnalyzer at IDT. When analyzing PolI Complements only were used for sequence inquiry, not the Bb Suffix/Prefixes. (http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/)<br><br />
<br><br />
====PolI Coli Primers For Overlap Extension PCR====<br />
'''PCR Reaction 1''' <br><br />
<br><br />
Bb Prefix + PolI (Fwd Complement) : (Forward complement will begin coding at 121 according to BLAST CDS information.)<br><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-ATGCTGCCCCTCTTTGAGCC<br><br />
60.5 c ; 56.5 % GC Concetration<br><br />
<br><br />
TAQ Rm<br><br />
CTCCCGGTACTGAAGGATCTTCTCCAC<br><br />
61.5 c ; 55.6 % GC Concentration<br><br />
<br><br />
'''PCR Reaction - 2'''<br><br />
<br><br />
TAQ Fm<br><br />
GTGGAGAAGATCCTTCAGTACCGGGAG<br><br />
61.5 c; 55.6 % GC<br><br />
<br><br />
Bb Suffix + PolI (Reverse Complement) : (Reverse complement will end coding at 2619 according to Blast CDS information.<br><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTA-TCACTCCTTGGCGGAGAGCC<br><br />
61.8 c; 65 % GC<br><br />
<br><br />
'''PCR Reaction - 3'''<br><br />
Bb Prefix & Suffix Primers<br><br />
<br><br />
Resuspend in 100 uL of H2O<br><br />
Run PCR w 1/100 dilutions for PCR (5-10 uL per PCR reaction)<br><br />
<br><br />
'''NEXT'''<br><br />
- Create Full Bb Prmr w Plasmid combining new part using<br><br />
<br><br />
<partinfo>R0010</partinfo> - Promoter (LacI)<br><br />
<partinfo>B0034</partinfo> - Strong RBS<br><br />
NEW PART - PolI Bb Format<br><br />
<partinfo>B0015</partinfo> - Double Terminator<br><br />
Psb1_?_3 - Plasmid of Interest with Chosen Resistance : http://partsregistry.org/Plasmid_backbones<br><br />
<br><br />
----<br />
<br><br />
<partinfo>R0010</partinfo> + <partinfo>B0034</partinfo> = New part LacI Promoter + Strong RBS<br><br />
<br><br />
Cut <partinfo>R0010</partinfo> w/EcoRI & SpeI<br><br />
Cut <partinfo>B0034</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI) - Because <br><br />
<br><br />
---<br><br />
New Part + <partinfo>B0015</partinfo> = New Part<br> <br />
<br><br />
Cut New Part w/EcoRI & SpeI<br><br />
Cut <partinfo>B0015</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
----<br><br />
<br><br />
Cut 1st Combined Part w/EcoRI & SpeI<br><br />
Cut 2nd Combined Part w/XbeI & PstI<br><br />
<br><br />
Combine in Ampecillan/Kanamyacin Resistan Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
'''Voila!!!''' Brand New Taq Polymerase Bb Part.<br><br />
<br />
== Results ==<br />
<br />
[[Image:EP.jpg|300px]]<br><br />
With regards to equipment, we have successfully constructed a very low-cost Gel Electrophoresis device and are currently working on the control software and control electronics. Additionally, we are working on getting a low-cost PCR thermocycler up and running as well.<br> [[Team:Baltimore_US/Notebook/EPInstructions|Instructions and Design files for building an Electrophoresis device]]</div>LisaShttp://2010.igem.org/Team:Baltimore_US/ProjectTeam:Baltimore US/Project2010-10-27T18:51:31Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
|<br />
<br />
<!--- The Mission, Experiments ---><br />
<br />
== DIY-GEM: a path towards low cost high throughput gene synthesis. ==<br />
Synthetic biology research requires more cost effective approaches toward reagents and hardware accessibility. We are developing low-cost alternatives to existing hardware and enzymes in an attempt to expand participation in biological research and development. Our project expands the accessibility of Taq Polymerase by engineering it in a form compatible with BioBrick assembly. This allows use of the over-expressed enzyme from a crude bacterial extract in a PCR reaction at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel electrophoresis apparatus and a PCR thermal cycler. Enabling researchers to synthesize their own enzymes and having access to inexpensive tools will allow for increased participation among the DIY-bio community, stretch increasingly scarce educational funds, and allow rapid scale up of large scale gene synthesis projects."<br />
<br />
==PoliColi Project Details==<br />
<br />
Thermus Aquaticus Polymerase I<br><br />
PolI<br><br />
J04639.1<br><br />
Gene Sequence via BLAST at NCBI - http://www.ncbi.nlm.nih.gov/nuccore/155128<br><br />
<br><br />
1 AAGCTCAGAT CTACCTGCCT GAGGGCGTCC GGTTCCAGCT GGCCCTTCCC<br><br />
51 GAGGGGGAGA GGGAGGCGTT TCTAAAAGCC CTTCAGGACG CTACCCGGGG<br><br />
101 GCGGGTGGTG GAAGGGTAAC ATGAGGGGGA TGCTGCCCCT CTTTGAGCCC<br><br />
151 AAGGGCCGGG TCCTCCTGGT GGACGGCCAC CACCTGGCCT ACCGCACCTT<br><br />
201 CCACGCCCTG AAGGGCCTCA CCACCAGCCG GGGGGAGCCG GTGCAGGCGG<br><br />
251 TCTACGGCTT CGCCAAGAGC CTCCTCAAGG CCCTCAAGGA GGACGGGGAC<br><br />
301 GCGGTGATCG TGGTCTTTGA CGCCAAGGCC CCCTCCTTCC GCCACGAGGC<br><br />
351 CTACGGGGGG TACAAGGCGG GCCGGGCCCC CACGCCGGAG GACTTTCCCC<br><br />
401 GGCAACTCGC CCTCATCAAG GAGCTGGTGG ACCTCCTGGG GCTGGCGCGC<br><br />
451 CTCGAGGTCC CGGGCTACGA GGCGGACGAC GTCCTGGCCA GCCTGGCCAA<br><br />
501 GAAGGCGGAA AAGGAGGGCT ACGAGGTCCG CATCCTCACC GCCGACAAAG<br><br />
551 ACCTTTACCA GCTCCTTTCC GACCGCATCC ACGTCCTCCA CCCCGAGGGG<br><br />
601 TACCTCATCA CCCCGGCCTG GCTTTGGGAA AAGTACGGCC TGAGGCCCGA<br><br />
651 CCAGTGGGCC GACTACCGGG CCCTGACCGG GGACGAGTCC GACAACCTTC<br><br />
701 CCGGGGTCAA GGGCATCGGG GAGAAGACGG CGAGGAAGCT TCTGGAGGAG<br><br />
751 TGGGGGAGCC TGGAAGCCCT CCTCAAGAAC CTGGACCGGC TGAAGCCCGC<br><br />
801 CATCCGGGAG AAGATCCTGG CCCACATGGA CGATCTGAAG CTCTCCTGGG<br><br />
851 ACCTGGCCAA GGTGCGCACC GACCTGCCCC TGGAGGTGGA CTTCGCCAAA<br><br />
901 AGGCGGGAGC CCGACCGGGA GAGGCTTAGG GCCTTTCTGG AGAGGCTTGA<br><br />
951 GTTTGGCAGC CTCCTCCACG AGTTCGGCCT TCTGGAAAGC CCCAAGGCCC<br><br />
1001 TGGAGGAGGC CCCCTGGCCC CCGCCGGAAG GGGCCTTCGT GGGCTTTGTG<br><br />
1051 CTTTCCCGCA AGGAGCCCAT GTGGGCCGAT CTTCTGGCCC TGGCCGCCGC<br><br />
1101 CAGGGGGGGC CGGGTCCACC GGGCCCCCGA GCCTTATAAA GCCCTCAGGG<br><br />
1151 ACCTGAAGGA GGCGCGGGGG CTTCTCGCCA AAGACCTGAG CGTTCTGGCC<br><br />
1201 CTGAGGGAAG GCCTTGGCCT CCCGCCCGGC GACGACCCCA TGCTCCTCGC<br><br />
1251 CTACCTCCTG GACCCTTCCA ACACCACCCC CGAGGGGGTG GCCCGGCGCT<br><br />
1301 ACGGCGGGGA GTGGACGGAG GAGGCGGGGG AGCGGGCCGC CCTTTCCGAG<br><br />
1351 AGGCTCTTCG CCAACCTGTG GGGGAGGCTT GAGGGGGAGG AGAGGCTCCT<br><br />
1401 TTGGCTTTAC CGGGAGGTGG AGAGGCCCCT TTCCGCTGTC CTGGCCCACA<br><br />
1451 TGGAGGCCAC GGGGGTGCGC CTGGACGTGG CCTATCTCAG GGCCTTGTCC<br><br />
1501 CTGGAGGTGG CCGAGGAGAT CGCCCGCCTC GAGGCCGAGG TCTTCCGCCT<br><br />
1551 GGCCGGCCAC CCCTTCAACC TCAACTCCCG GGACCAGCTG GAAAGGGTCC<br><br />
1601 TCTTTGACGA GCTAGGGCTT CCCGCCATCG GCAAGACGGA GAAGACCGGC<br><br />
1651 AAGCGCTCCA CCAGCGCCGC CGTCCTGGAG GCCCTCCGCG AGGCCCACCC<br><br />
1701 CATCGTGGAG AAGATCCTGC AGTACCGGGA GCTCACCAAG CTGAAGAGCA<br><br />
1751 CCTACATTGA CCCCTTGCCG GACCTCATCC ACCCCAGGAC GGGCCGCCTC<br><br />
1801 CACACCCGCT TCAACCAGAC GGCCACGGCC ACGGGCAGGC TAAGTAGCTC<br><br />
1851 CGATCCCAAC CTCCAGAACA TCCCCGTCCG CACCCCGCTT GGGCAGAGGA<br><br />
1901 TCCGCCGGGC CTTCATCGCC GAGGAGGGGT GGCTATTGGT GGCCCTGGAC<br><br />
1951 TATAGCCAGA TAGAGCTCAG GGTGCTGGCC CACCTCTCCG GCGACGAGAA<br><br />
2001 CCTGATCCGG GTCTTCCAGG AGGGGCGGGA CATCCACACG GAGACCGCCA<br><br />
2051 GCTGGATGTT CGGCGTCCCC CGGGAGGCCG TGGACCCCCT GATGCGCCGG<br><br />
2101 GCGGCCAAGA CCATCAACTT CGGGGTCCTC TACGGCATGT CGGCCCACCG<br><br />
2151 CCTCTCCCAG GAGCTAGCCA TCCCTTACGA GGAGGCCCAG GCCTTCATTG<br><br />
2201 AGCGCTACTT TCAGAGCTTC CCCAAGGTGC GGGCCTGGAT TGAGAAGACC<br><br />
2251 CTGGAGGAGG GCAGGAGGCG GGGGTACGTG GAGACCCTCT TCGGCCGCCG<br><br />
2301 CCGCTACGTG CCAGACCTAG AGGCCCGGGT GAAGAGCGTG CGGGAGGCGG<br><br />
2351 CCGAGCGCAT GGCCTTCAAC ATGCCCGTCC AGGGCACCGC CGCCGACCTC<br><br />
2401 ATGAAGCTGG CTATGGTGAA GCTCTTCCCC AGGCTGGAGG AAATGGGGGC<br><br />
2451 CAGGATGCTC CTTCAGGTCC ACGACGAGCT GGTCCTCGAG GCCCCAAAAG<br><br />
2501 AGAGGGCGGA GGCCGTGGCC CGGCTGGCCA AGGAGGTCAT GGAGGGGGTG<br><br />
2551 TATCCCCTGG CCGTGCCCCT GGAGGTGGAG GTGGGGATAG GGGAGGACTG<br><br />
2601 GCTCTCCGCC AAGGAGTGAT ACCACC<br><br />
<br><br />
We took the above sequence from the provided link at BLAST and exported the SEQ into Plasma DNA. Plasma DNA is freeware from University of Helsinki which provides quick analysis of plasmid sequence information. http://research.med.helsinki.fi/plasmadna/<br />
<br><br />
When we cut and paste this dna sequence into plasmadna and look at the output window, we are given a visual output of various coding information. Such as restriction sites found within the code. To consider a construct viable for a BbPart we'll need to make certain that the standard restriction enzymes used with the system won't sheer the dna making it incomplete code. Searching for EcoRI, Xbe1, Sbe1, Pst1 sites will show whether the code is viable in an untampered state. <br><br />
<br />
====Problem: PstI restriction site - Found @ 1717====<br />
CTGCAG-PstI restriction site<br><br />
GACGTC-Complement<br><br />
Solution - Site-specific Mutagenesis by Overlap Extension (see Sambrook, Joseph; Russell, David W. ; Molecular Cloning: A Laboratory Manual, 3rd Edition - http://www.cshlpress.com/default.tpl?cart=1279686078181232350&fromlink=T&linkaction=full&linksortby=oop_title&--eqSKUdatarq=21)<br />
<br><br><br />
We then used the Gene Designer 2.0 freeware from DNA2.0 (https://www.dna20.com/genedesigner2/) - to analyze the Open Reading Frames. It shows us the Amino Acid codons that were being coded within that PstI Restrictions site. We find that the first three are coding for Leucine with CTG and can be changed at one point to CTT and still maintain Leucine's amino acid. The hope is that this will maintain functional integrity in the manufactured enzyme.<br><br />
<br />
====Primer Design====<br />
We designed two primers (11-14 Bp around chosen mutation) with changed Amino Acid Bp's Targeting initial Leucine at G of CTG to CTT. Point mutation Original G in CTG of Leucine. Change of one base to CTT maintains Leucine integrity. <br><br />
GTGGAGAAGATCCT(T)CAGTACCGGCGG<br><br />
CACCTCTTCTAGGA(A)GTCATGGCCGCC<br><br />
While we're designing primers, besides the point mutation, we'll take the opportunity to design and order the primers for the Bb Suffix and Prefix. We'll follow the examples laid out in the Registry of Standard Parts under Promoter Construction for designing the oligos needed to make a part. (http://partsregistry.org/Help:Promoters/Construction) <br><br />
<br><br />
Important considerations are Melting Point and percentage CG complements. Other considerations are dimerizations, that might cause primers to hairpin. We analyzed these primers using the OligoAnalyzer at IDT. When analyzing PolI Complements only were used for sequence inquiry, not the Bb Suffix/Prefixes. (http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/)<br><br />
<br><br />
====PolI Coli Primers For Overlap Extension PCR====<br />
'''PCR Reaction 1''' <br><br />
<br><br />
Bb Prefix + PolI (Fwd Complement) : (Forward complement will begin coding at 121 according to BLAST CDS information.)<br><br />
GTTTCTTCGAATTCGCGGCCGCTTCTAGAG-ATGCTGCCCCTCTTTGAGCC<br><br />
60.5 c ; 56.5 % GC Concetration<br><br />
<br><br />
TAQ Rm<br><br />
CTCCCGGTACTGAAGGATCTTCTCCAC<br><br />
61.5 c ; 55.6 % GC Concentration<br><br />
<br><br />
'''PCR Reaction - 2'''<br><br />
<br><br />
TAQ Fm<br><br />
GTGGAGAAGATCCTTCAGTACCGGGAG<br><br />
61.5 c; 55.6 % GC<br><br />
<br><br />
Bb Suffix + PolI (Reverse Complement) : (Reverse complement will end coding at 2619 according to Blast CDS information.<br><br />
GTTTCTTCCTGCAGCGGCCGCTACTAGTA-TCACTCCTTGGCGGAGAGCC<br><br />
61.8 c; 65 % GC<br><br />
<br><br />
'''PCR Reaction - 3'''<br><br />
Bb Prefix & Suffix Primers<br><br />
<br><br />
Resuspend in 100 uL of H2O<br><br />
Run PCR w 1/100 dilutions for PCR (5-10 uL per PCR reaction)<br><br />
<br><br />
'''NEXT'''<br><br />
- Create Full Bb Prmr w Plasmid combining new part using<br><br />
<br><br />
<partinfo>R0010</partinfo> - Promoter (LacI)<br><br />
<partinfo>B0034</partinfo> - Strong RBS<br><br />
NEW PART - PolI Bb Format<br><br />
<partinfo>B0015</partinfo> - Double Terminator<br><br />
Psb1_?_3 - Plasmid of Interest with Chosen Resistance : http://partsregistry.org/Plasmid_backbones<br><br />
<br><br />
----<br />
<br><br />
<partinfo>R0010</partinfo> + <partinfo>B0034</partinfo> = New part LacI Promoter + Strong RBS<br><br />
<br><br />
Cut <partinfo>R0010</partinfo> w/EcoRI & SpeI<br><br />
Cut <partinfo>B0034</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI) - Because <br><br />
<br><br />
---<br><br />
New Part + <partinfo>B0015</partinfo> = New Part<br> <br />
<br><br />
Cut New Part w/EcoRI & SpeI<br><br />
Cut <partinfo>B0015</partinfo> w/XbeI & PstI<br><br />
<br><br />
Combine in Chloramphenecol Resistant Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
----<br><br />
<br><br />
Cut 1st Combined Part w/EcoRI & SpeI<br><br />
Cut 2nd Combined Part w/XbeI & PstI<br><br />
<br><br />
Combine in Ampecillan/Kanamyacin Resistan Plasmid (cut w/EcoRI & PstI)<br><br />
<br><br />
'''Voila!!!''' Brand New Taq Polymerase Bb Part.<br><br />
<br />
== Results ==<br />
<br />
[[Image:EP.jpg|300px]]<br><br />
With regards to equipment, we have successfully constructed a very low-cost Gel Electrophoresis device and are currently working on the control software and control electronics. Additionally, we are working on getting a low-cost PCR thermocycler up and running as well.<br> [[Team:Baltimore_US/Notebook/EPInstructions|Instructions and Design files for building an Electrophoresis device]]</div>LisaShttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T18:22:31Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
====DIY-Gem: a path towards low-cost high-throughput gene synthesis====<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel-electrophoresis apparatus and a PCR thermal cycler. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology.<br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue that enables the broader community to step into the iGEM competition, to gain hands-on laboratory experience, and to learn the tools and techniques necessary to explore the emerging field of synthetic biology while under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" align="center"<br />
|<br />
<center>'''Sponsors:'''</center><br />
[[image:Qiagen logo.png|150px]] [[image:Axygen logo.png|150px]][[image:NEB logo.jpg|150px]]<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T18:20:50Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
====DIY-Gem: a path towards low-cost high-throughput gene synthesis====<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel-electrophoresis apparatus and a PCR thermal cycler. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology.<br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue that enables the broader community to step into the iGEM competition, to gain hands-on experience, and to learn the tools, techniques, and resources necessary to explore the emerging field of synthetic biology under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" align="center"<br />
|<br />
<center>'''Sponsors:'''</center><br />
[[image:Qiagen logo.png|150px]] [[image:Axygen logo.png|150px]][[image:NEB logo.jpg|150px]]<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T18:17:04Z<p>LisaS: </p>
<hr />
<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
====DIY-Gem: a path towards low-cost high-throughput gene synthesis====<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' Polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel-electrophoresis apparatus and a PCR thermal cycler. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology.<br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue where the community could step into this competition, gain hands-on experience, and learn the tools, techniques, and resources necessary to explore this emerging field under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" align="center"<br />
|<br />
<center>'''Sponsors:'''</center><br />
[[image:Qiagen logo.png|150px]] [[image:Axygen logo.png|150px]][[image:NEB logo.jpg|150px]]<br />
<br />
|}</div>LisaShttp://2010.igem.org/File:Qiagen_logo.pngFile:Qiagen logo.png2010-10-27T14:42:12Z<p>LisaS: uploaded a new version of "Image:Qiagen logo.png"</p>
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<div></div>LisaShttp://2010.igem.org/File:Qiagen_logo.pngFile:Qiagen logo.png2010-10-27T14:39:59Z<p>LisaS: </p>
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<div></div>LisaShttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T14:38:38Z<p>LisaS: /* Poli-Coli: a path towards low-cost high-throughput gene synthesis */</p>
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<div>[[Image:TitleBarBalti US.png | center]]<br />
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{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
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{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
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{| style= align="center"|<br />
'''Sponsors''' <br><br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
===Poli-Coli: a path towards low-cost high-throughput gene synthesis===<br />
<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of ''Taq'' Polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. coli'' at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel-electrophoresis apparatus and a PCR thermal cycler. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology. <br />
<br />
<br />
'''Baltimore-US''' includes members from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue where the community could step into this competition, gain hands-on experience, and learn the tools, techniques, and resources necessary to explore this emerging field under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}</div>LisaShttp://2010.igem.org/Team:Baltimore_USTeam:Baltimore US2010-10-27T14:31:12Z<p>LisaS: </p>
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<div>[[Image:TitleBarBalti US.png | center]]<br />
<br />
{| style="background-color:#7998AD;" cellpadding="1" cellspacing="1" border="0" bordercolor="#fff" width="924px" align="center"<br />
!align="center"|[[Team:Baltimore_US|<span style="color:white;">Home</span>]]<br />
!align="center"|[[Team:Baltimore_US/Team|<span style="color:white;">Team</span>]]<br />
!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Baltimore_US <span style="color:white;">Official Team Profile</span>]<br />
!align="center"|[[Team:Baltimore_US/Project|<span style="color:white;">Project</span>]]<br />
!align="center"|[[Team:Baltimore_US/Parts|<span style="color:white;">Submitted Parts</span>]]<br />
!align="center"|[[Team:Baltimore_US/Modeling|<span style="color:white;">Modeling</span>]]<br />
!align="center"|[[Team:Baltimore_US/Notebook|<span style="color:white;">Notebook</span>]]<br />
!align="center"|[[Team:Baltimore_US/MeetingTimes|<span style="color:white;">Meeting/Lab Times</span>]]<br />
!align="center"|[[Team:Baltimore_US/Safety|<span style="color:white;">Safety</span>]]<br />
|}<br />
<br />
{| style= "background-color:#FFFFF;" width="924px" align="center"<br />
|<br />
__NOTOC__<br />
<br><br />
{| style= align="center"|<br />
'''Sponsors''' <br><br />
[[Image:Nsf1.gif]]<br />
|}<br />
|align="left"|<br />
<br><br />
<br />
===Poli-Coli: a path towards low-cost high-throughput gene synthesis===<br />
<br />
Synthetic biology research requires more cost effective approaches toward wetware and hardware accessibility. We are developing low-cost alternatives to existing tools and techniques in an attempt to expand participation in biological research and development. Our project expands the accessibility of Taq Polymerase by engineering it to BioBrick standards. This allows for the expression and recovery of polymerase from transformed ''E. Coli'' at a fraction of the cost of highly purified commercial enzyme. In addition, we have developed inexpensive and easily assembled lab equipment such as a gel-electrophoresis apparatus and a PCR thermal cycler. By enabling researchers to synthesize their own reagents and purchase or produce inexpensive tools, we hope to lower the barriers to entry for synthetic biology. <br />
<br />
'''Baltimore-US''' is made up of students from local educational institutions including the Community College of Baltimore County, and Loyola and Towson Universities, along with members of local DIY communities. We wish to create a venue where the community could step into this competition and learn with hands-on experience the tools, techniques, and resources necessary to explore this emerging field under the watchful eye of trained professionals. <br />
<br />
====Faculty Instructors and Advisors====<br />
<ul><br />
<li>Tom Burkett, Associate Professor of Biotechnology and Biomanufacturing at CCBC</li><br />
<li>Lisa Scheifele, Assistant Professor of Biology at Loyola University Maryland</li><br />
<li>Elizabeth Goode, Associate Professor of Mathematics at Towson University.</li><br />
</ul><br />
<br><br />
Our members range in age and experience. We've got biological and electrical engineers, computer scientists and computer programmers and curious novices. We've come together with enthusiasm to try and see what we might be able to accomplish with these new technologies.<br />
<br />
|}</div>LisaS