Team:Marburg/Safety

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PHAECTORY: Red or dead
PHAECTORY: Red or dead
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{{:Team:Marburg/Template:ContentStart}}The work with genetically modified organisms (GMOs) harbours the risk of their release to the nature. After release, serious consequences such as gene exchange with unmodified organism or ecosystem imbalance cannot be excluded. It is necessary to keep GMOs isolated from the environment or to make their survival in nature impossible. Therefore, we decided to develop a security system for ''Phaeodactylum tricornutum'' (Figure A). Therefore, we designed a system that leads to cell death induced by blue light based on the publication of Konermann ''et al.'', 2013.
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{{:Team:Marburg/Template:ContentStart}}The work with genetically modified organisms (GMOs) harbours the risk of their release to the nature. After release, serious consequences such as gene exchange with unmodified organism or ecosystem imbalance cannot be excluded. It is necessary to keep GMOs isolated from the environment or to make their survival in nature impossible. For this purpose, we decided to develop a security system for ''Phaeodactylum tricornutum'' (Figure A) leading to cell death induced by blue light based on the publication of Konermann ''et al.'', 2013, Nature.
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To minimize the uninduced activation of the security system we engineered a two-stage system. The main component is the LITE (light-inducible transcriptional effectors) module (Figure B). It consists of a transcription activator -like effector (TALE) fused to a blue light sensitive cryptochrome 2 protein (CRY2). Upon blue light reception the conformational change of CRY2 recruits the CIB1 interaction partner, fused to an effector (e.g. transcription factor of the bZIP family). This effector recruits the transcription complex resulting in transcription and expression of the desired gene.
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To minimize the uninduced activation of the security system we engineered a two-stage system. The main component is the LITE (light-inducible transcriptional effectors) module (Figure B). It consists of a transcription activator-like effector (TALE) fused to a blue light sensitive cryptochrome 2 protein (CRY2). Upon blue light reception the conformational change of CRY2 recruits the CIB1 interaction partner, fused to an effector (e.g. transcription factor of the bZIP family). This effector recruits the transcription complex resulting in transcription and expression of the desired gene.
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In the first stage a LITE module is under the control of a weak, constitutive promoter resulting in a constant low level of LITE1 in the cell. The TALE1 and the attached CRY2 bind to the TALE1 binding site upstream of the LITE2 module. LITE1 stays inactive until illumination with blue light. When blue light activates CRY2 the LITE1 module assembles resulting in the expression of LITE2.  LITE2 in turn binds to the TALE2 binding site leading to the expression of Protease K and DNaseI. Protease K is a potent protease with a broad substrate specificity working even at low temperature. DNaseI digests single- and double- stranded DNA efficiently.  
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In the first stage a LITE1 module is under the control of a weak, constitutive promoter resulting in a constant low level of LITE1 in the cell. The TALE1 and the attached CRY2 bind to the TALE1 binding site upstream of the LITE2 module. LITE1 stays inactive until illumination with blue light. When blue light activates CRY2 the LITE1 module assembles resulting in the expression of LITE2.  LITE2 in turn binds to the TALE2 binding site leading to the expression of Protease K and DNaseI. Protease K is a potent protease with a broad substrate specificity working even at low temperature. DNaseI digests single- and double-stranded DNA efficiently.  
Beside instant activation of a light inducible system the major advantage in our approach is the prevention of additional chemical compounds in the medium resulting in unnecessary purification steps for the secreted antibodies.
Beside instant activation of a light inducible system the major advantage in our approach is the prevention of additional chemical compounds in the medium resulting in unnecessary purification steps for the secreted antibodies.
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To construct the desired TAL-Effectors we chose to use the GATE-Toolkit from the iGEM-Team of Freiburg 2012, which allows the fast construction of 14 bp specific TAL-proteins. To avoid toxicity and off-target binding of the effectors we chose binding sequences which are not present in the genome of P. tricornutum.
 
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For the TALE1 binding site we used: T GC TA CA TT GA TA T
 
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For the TALE2 binding site we used: T CG TA GA TC TG TA T 
 
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both binding proteins can easily be constructed in one golden gate reaction by using the following parts of the registry: BBa_K747000 - BBa_K747097.
 
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Figure X: Construction of the 14 bp binding TAL-proteins with the GATE-Toolkiz from the iGEM-Team of Freiburg 2012.
 
For the technical realization we suggest a blue light filter for PHAECTORY.  In the unlikely event of an algae outbreak from isolation, the existing blue spectrum of the sunlight will lead to the immediate activation of the LITE security system and the inevitable death of ''P. tricornutum'' (Figure B).
For the technical realization we suggest a blue light filter for PHAECTORY.  In the unlikely event of an algae outbreak from isolation, the existing blue spectrum of the sunlight will lead to the immediate activation of the LITE security system and the inevitable death of ''P. tricornutum'' (Figure B).
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''Safety forms were approved on September 18, 2013 by Evan Appleton.''
''Safety forms were approved on September 18, 2013 by Evan Appleton.''
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<html><a href="https://static.igem.org/mediawiki/2013/c/cb/SafetyGuide.pdf" target="_blank">Download safety form.</a></html>
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Latest revision as of 17:53, 28 October 2013

PHAECTORY: Red or dead

The work with genetically modified organisms (GMOs) harbours the risk of their release to the nature. After release, serious consequences such as gene exchange with unmodified organism or ecosystem imbalance cannot be excluded. It is necessary to keep GMOs isolated from the environment or to make their survival in nature impossible. For this purpose, we decided to develop a security system for Phaeodactylum tricornutum (Figure A) leading to cell death induced by blue light based on the publication of Konermann et al., 2013, Nature.

To minimize the uninduced activation of the security system we engineered a two-stage system. The main component is the LITE (light-inducible transcriptional effectors) module (Figure B). It consists of a transcription activator-like effector (TALE) fused to a blue light sensitive cryptochrome 2 protein (CRY2). Upon blue light reception the conformational change of CRY2 recruits the CIB1 interaction partner, fused to an effector (e.g. transcription factor of the bZIP family). This effector recruits the transcription complex resulting in transcription and expression of the desired gene.

In the first stage a LITE1 module is under the control of a weak, constitutive promoter resulting in a constant low level of LITE1 in the cell. The TALE1 and the attached CRY2 bind to the TALE1 binding site upstream of the LITE2 module. LITE1 stays inactive until illumination with blue light. When blue light activates CRY2 the LITE1 module assembles resulting in the expression of LITE2. LITE2 in turn binds to the TALE2 binding site leading to the expression of Protease K and DNaseI. Protease K is a potent protease with a broad substrate specificity working even at low temperature. DNaseI digests single- and double-stranded DNA efficiently. Beside instant activation of a light inducible system the major advantage in our approach is the prevention of additional chemical compounds in the medium resulting in unnecessary purification steps for the secreted antibodies.

For the technical realization we suggest a blue light filter for PHAECTORY. In the unlikely event of an algae outbreak from isolation, the existing blue spectrum of the sunlight will lead to the immediate activation of the LITE security system and the inevitable death of P. tricornutum (Figure B).

Safety

Safety forms were approved on September 18, 2013 by Evan Appleton. Download safety form.

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