http://2009.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=20&target=Tim+Weenink&year=&month=2009.igem.org - User contributions [en]2024-03-28T10:12:05ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:TUDelft/AchievementsTeam:TUDelft/Achievements2009-10-22T03:58:46Z<p>Tim Weenink: /* Self Destructive Plasmid */</p>
<hr />
<div>{{Template:TUDelftiGEM2009}}<br />
=Achievements=<br />
This is a short list summarizing the most important goals that we achieved during the project, subdivided in differnt modules. For more information on specific achievements use the links to navigate to the corresponding webpage. Please also visit the [https://2009.igem.org/Team:TUDelft/Deliverables Deliverables] section.<br />
==Conjugation System==<br />
<br><br />
'''Verification and Characterization of Wild R751'''<br />
*Determination of wild R751 conjugation efficiency.<br />
<br />
'''Verification and Characterization of trbK entry exclusion protein'''<br />
*Design and test of a a method to prevent back propagation of the signal by expressing trbK. This allows receiver side control over communication.<br />
<br />
'''Verification and Characterization of Conjugation Testing Plasmid 1'''<br />
*A conjugation test was done using our conjugation protocol to verify that Conjugation Testing Plasmid 1 was transmitted during conjugation.<br />
<br />
'''R751 Gene Knockouts'''<br />
*A third attempt to knockout oriTR and trbK is currently in progress using the Quick & Easy Conditional Knock Out Kit - FRT from Gene Bridges.<br />
<br />
[https://2009.igem.org/Team:TUDelft/Conjugation_Results more]<br />
<br />
==Self Destructive Plasmid==<br />
<br><br />
'''I-SceI restriction site'''<br />
*construction and verification of the I-SceI homing endonuclease restriction site <partinfo>BBa_K175027</partinfo>.<br />
<br />
'''Restriction site plus reporter'''<br />
*construction of an anhydrotetracyclin inducible GFP-LVA reporter gene combined with the I-SceI restriction site: <partinfo>BBa_K175044</partinfo>.<br />
<br />
'''Characterization of I-SceI biobricks'''<br />
*determination that <partinfo>BBa_K142200</partinfo> and all parts containing it encode for an I-SceI homing endonuclease that has an LVA degradation tag attached to it, possibly preventing normal functioning.<br />
*determination that <partinfo>BBa_K142205</partinfo> (IPTG inducible I-SceI generator) actually encodes for <partinfo>BBa_K142207</partinfo> (medium constitutive T4 DNA Ligase generator).<br />
<br />
'''construction of the real <partinfo>BBa_K142205</partinfo>'''<br />
*construction of a part called <partinfo>BBa_K175041</partinfo> that is identical to the intended sequence of <partinfo>BBa_K142205</partinfo>.<br />
<br />
==Time-Delay Device==<br />
<br><br />
'''Synthetic Transcriptional Cascade'''<br />
* Assembly of plasmid 1 and 2 ([http://partsregistry.org/Part:BBa_K175046 K175046]) and 2 ([http://partsregistry.org/Part:BBa_K175047 K175047]).<br />
* Partial confirmation of sequence of both plasmids.<br />
* Transformation in ''Escherichia coli''<br />
* Confirmation of both biobrick individual functionality. <br />
* [https://2009.igem.org/Team:TUDelft/Delay_Results_Continue Characterization] of the plasmids 1 and 2 by fluorescence experiments...[[Team:TUDelft/Results | more]]<br />
<br />
'''Biosynthetic AND gate'''<br />
* Successful assembly of parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175023 K175023] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175024 K175024] which correspond to Plasmid 1 and 2 of the Biosynthetic AND gate section respectively.<br />
* Electroporation of plasmid 1 and 2 into ''Escherichia coli'' TOP10 cells...[[Team:TUDelft/Results2 | more]]<br />
<br />
'''Lock and Key Library'''<br />
* Construction of an algorithm capable to generate locks (cis regulation) for different ribosome binding sites (RBS’s) and their correspond key (trans).<br />
* Online [https://2009.igem.org/Team:TUDelft/RiboKeyLock_Generator lock and key generator] derived from the algorithm.<br />
* Construction of four DNA sequences in biobrick format and [http://partsregistry.org/wiki/index.php?title=Part:BBa_pSB1A3 pSB1A3] plasmid backbone which encode lock for medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175031 K175031]), key for the lock of medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175032 K175032]), lock for weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175029 K175029]) or key for the lock of weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175030 K175030]).<br />
* Assembly of two composed biobricks ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175034 K175034] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175035 K175035]), designed to test the functionality of the lock/key pairs constructed.<br />
* Assembly of two GFP generators under the control of PLacI with weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175033 K175033]) or medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175048 K175048])...[[Team:TUDelft/Results3 | more]]<br />
<br />
==Modeling==<br />
<br><br />
'''Synthetic Transcriptional Cascade'''<br />
* Construction of a model used to provide the delay team with design guidelines which would maximize the delay time, to asses the affect of parameter variation on the delay time and to determine areas of instability in the parameter space.<br />
* A sensitivity analysis was done on the system. This looked at how variations in each parameter would influence the delay time.<br />
* Corroboration of a stable system by determining the Jacobian of the system of ODEs analytically.<br />
* Identification of best suitable parameters ranges for a desired time-delay device.<br />
* Based on the results of the simulations, a series of recommendations were given to the delay team to aid them in choosing parts which would maximize the delay time.<br />
<br />
'''Conjugation Modeling'''<br />
<br />
* The development and documentation of a new [https://2009.igem.org/Team:TUDelft/Modeling_Conjugation model for conjugation systems]. This kind of model represents a complete novelty within iGEM framework.<br />
<br />
==Ethics==<br />
<br><br />
'''Literature research'''<br />
* Identification and description of the main ethical issues and concerns in synthetic biology. [https://2009.igem.org/Team:TUDelft/Ethics_background more]<br />
* Illustration of the main ethical issues in clear frameworks, which can be build on in further research. <br />
* Recognition of the issues that need specific attention: ethical discussions concerning the top-down (reductionist) approach towards understanding living systems and the bottom-up approach of enhancing/creating biological systems.<br />
* Elaborate characterization of the omitted issues concerning reductionism and the bottom-up approach in synthetic biology. [https://2009.igem.org/Team:TUDelft/Ethics_background more]<br />
<br><br />
'''Survey'''<br />
* Construction of a comprehensive survey mainly focusing on the issues and possible consequences emerging from the reductionist and bottom-up approach as applied in biology, including questions on evolution, life, risks and the research of Craig Venter. [https://2009.igem.org/Team:TUDelft/Ethics_methods more]<br />
* Successful conduction of the survey among '''242''' people involved in synthetic biology from over 20 different countries, including 168 iGEM participants and 60 supervisors/advisors. <br />
* Extensive elaboration of the results, both qualitatively and quantitatively. [https://2009.igem.org/Team:TUDelft/Ethics_results more]<br />
* Integration of a detailed outline of conclusions and recommendations based on the literature research and the survey results. [https://2009.igem.org/Team:TUDelft/Ethics_conclusions more] <br />
<br />
==Communication==<br />
*We published articles in university newsletters and magazines.<br />
*Till now we had a good cooperation with our sponsors. We published articles in newsletter of the companies which sponsor our team. Some of our sponsors asked us to present our results after the Jamboree.<br />
*We created a platform among university magazines and newsletters to write articles about our project after the Jamboree.<br />
*We attracted attention at our department for our participation to the competition. <br />
<br />
<br><br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/AchievementsTeam:TUDelft/Achievements2009-10-22T03:47:57Z<p>Tim Weenink: /* Self Destructive Plasmid */</p>
<hr />
<div>{{Template:TUDelftiGEM2009}}<br />
=Achievements=<br />
This is a short list summarizing the most important goals that we achieved during the project, subdivided in differnt modules. For more information on specific achievements use the links to navigate to the corresponding webpage. Please also visit the [https://2009.igem.org/Team:TUDelft/Deliverables Deliverables] section.<br />
==Conjugation System==<br />
<br><br />
'''Verification and Characterization of Wild R751'''<br />
*Determination of wild R751 conjugation efficiency.<br />
<br />
'''Verification and Characterization of trbK entry exclusion protein'''<br />
*Design and test of a a method to prevent back propagation of the signal by expressing trbK. This allows receiver side control over communication.<br />
<br />
'''Verification and Characterization of Conjugation Testing Plasmid 1'''<br />
*A conjugation test was done using our conjugation protocol to verify that Conjugation Testing Plasmid 1 was transmitted during conjugation.<br />
<br />
'''R751 Gene Knockouts'''<br />
*A third attempt to knockout oriTR and trbK is currently in progress using the Quick & Easy Conditional Knock Out Kit - FRT from Gene Bridges.<br />
<br />
[https://2009.igem.org/Team:TUDelft/Conjugation_Results more]<br />
<br />
==Self Destructive Plasmid==<br />
<br><br />
'''I-SceI restriction site'''<br />
*construction and verification of the I-SceI homing endonuclease restriction site <partinfo>BBa_K175027</partinfo>.<br />
<br />
'''Restriction site plus reporter'''<br />
*construction of an anhydrotetracyclin inducible GFP-LVA reporter gene combined with the I-SceI restriction site: <partinfo>BBa_K175044</partinfo>.<br />
<br />
'''Characterization of I-SceI biobricks'''<br />
*determination that <partinfo>BBa_K142200</partinfo> and all parts containing it encode for an I-SceI homing endonuclease that has an LVA degradation tag attached to it, possibly preventing normal functioning.<br />
*determination that <partinfo>BBa_K142205</partinfo> (IPTG inducible I-SceI generator) actually encodes for <partinfo>BBa_K142207</partinfo> (medium constitutive T4 DNA Ligase generator).<br />
<br />
==Time-Delay Device==<br />
<br><br />
'''Synthetic Transcriptional Cascade'''<br />
* Assembly of plasmid 1 and 2 ([http://partsregistry.org/Part:BBa_K175046 K175046]) and 2 ([http://partsregistry.org/Part:BBa_K175047 K175047]).<br />
* Partial confirmation of sequence of both plasmids.<br />
* Transformation in ''Escherichia coli''<br />
* Confirmation of both biobrick individual functionality. <br />
* [https://2009.igem.org/Team:TUDelft/Delay_Results_Continue Characterization] of the plasmids 1 and 2 by fluorescence experiments...[[Team:TUDelft/Results | more]]<br />
<br />
'''Biosynthetic AND gate'''<br />
* Successful assembly of parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175023 K175023] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175024 K175024] which correspond to Plasmid 1 and 2 of the Biosynthetic AND gate section respectively.<br />
* Electroporation of plasmid 1 and 2 into ''Escherichia coli'' TOP10 cells...[[Team:TUDelft/Results2 | more]]<br />
<br />
'''Lock and Key Library'''<br />
* Construction of an algorithm capable to generate locks (cis regulation) for different ribosome binding sites (RBS’s) and their correspond key (trans).<br />
* Online [https://2009.igem.org/Team:TUDelft/RiboKeyLock_Generator lock and key generator] derived from the algorithm.<br />
* Construction of four DNA sequences in biobrick format and [http://partsregistry.org/wiki/index.php?title=Part:BBa_pSB1A3 pSB1A3] plasmid backbone which encode lock for medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175031 K175031]), key for the lock of medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175032 K175032]), lock for weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175029 K175029]) or key for the lock of weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175030 K175030]).<br />
* Assembly of two composed biobricks ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175034 K175034] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175035 K175035]), designed to test the functionality of the lock/key pairs constructed.<br />
* Assembly of two GFP generators under the control of PLacI with weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175033 K175033]) or medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175048 K175048])...[[Team:TUDelft/Results3 | more]]<br />
<br />
==Modeling==<br />
<br><br />
'''Synthetic Transcriptional Cascade'''<br />
* Construction of a model used to provide the delay team with design guidelines which would maximize the delay time, to asses the affect of parameter variation on the delay time and to determine areas of instability in the parameter space.<br />
* A sensitivity analysis was done on the system. This looked at how variations in each parameter would influence the delay time.<br />
* Corroboration of a stable system by determining the Jacobian of the system of ODEs analytically.<br />
* Identification of best suitable parameters ranges for a desired time-delay device.<br />
* Based on the results of the simulations, a series of recommendations were given to the delay team to aid them in choosing parts which would maximize the delay time.<br />
<br />
'''Conjugation Modeling'''<br />
<br />
* The development and documentation of a new [https://2009.igem.org/Team:TUDelft/Modeling_Conjugation model for conjugation systems]. This kind of model represents a complete novelty within iGEM framework.<br />
<br />
==Ethics==<br />
<br><br />
'''Literature research'''<br />
* Identification and description of the main ethical issues and concerns in synthetic biology. [https://2009.igem.org/Team:TUDelft/Ethics_background more]<br />
* Illustration of the main ethical issues in clear frameworks, which can be build on in further research. <br />
* Recognition of the issues that need specific attention: ethical discussions concerning the top-down (reductionist) approach towards understanding living systems and the bottom-up approach of enhancing/creating biological systems.<br />
* Elaborate characterization of the omitted issues concerning reductionism and the bottom-up approach in synthetic biology. [https://2009.igem.org/Team:TUDelft/Ethics_background more]<br />
<br><br />
'''Survey'''<br />
* Construction of a comprehensive survey mainly focusing on the issues and possible consequences emerging from the reductionist and bottom-up approach as applied in biology, including questions on evolution, life, risks and the research of Craig Venter. [https://2009.igem.org/Team:TUDelft/Ethics_methods more]<br />
* Successful conduction of the survey among '''242''' people involved in synthetic biology from over 20 different countries, including 168 iGEM participants and 60 supervisors/advisors. <br />
* Extensive elaboration of the results, both qualitatively and quantitatively. [https://2009.igem.org/Team:TUDelft/Ethics_results more]<br />
* Integration of a detailed outline of conclusions and recommendations based on the literature research and the survey results. [https://2009.igem.org/Team:TUDelft/Ethics_conclusions more] <br />
<br />
==Communication==<br />
*We published articles in university newsletters and magazines.<br />
*Till now we had a good cooperation with our sponsors. We published articles in newsletter of the companies which sponsor our team. Some of our sponsors asked us to present our results after the Jamboree.<br />
*We created a platform among university magazines and newsletters to write articles about our project after the Jamboree.<br />
*We attracted attention at our department for our participation to the competition. <br />
<br />
<br><br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/AchievementsTeam:TUDelft/Achievements2009-10-22T03:45:50Z<p>Tim Weenink: /* Achievements */</p>
<hr />
<div>{{Template:TUDelftiGEM2009}}<br />
=Achievements=<br />
This is a short list summarizing the most important goals that we achieved during the project, subdivided in differnt modules. For more information on specific achievements use the links to navigate to the corresponding webpage. Please also visit the [https://2009.igem.org/Team:TUDelft/Deliverables Deliverables] section.<br />
==Conjugation System==<br />
<br><br />
'''Verification and Characterization of Wild R751'''<br />
*Determination of wild R751 conjugation efficiency.<br />
<br />
'''Verification and Characterization of trbK entry exclusion protein'''<br />
*Design and test of a a method to prevent back propagation of the signal by expressing trbK. This allows receiver side control over communication.<br />
<br />
'''Verification and Characterization of Conjugation Testing Plasmid 1'''<br />
*A conjugation test was done using our conjugation protocol to verify that Conjugation Testing Plasmid 1 was transmitted during conjugation.<br />
<br />
'''R751 Gene Knockouts'''<br />
*A third attempt to knockout oriTR and trbK is currently in progress using the Quick & Easy Conditional Knock Out Kit - FRT from Gene Bridges.<br />
<br />
[https://2009.igem.org/Team:TUDelft/Conjugation_Results more]<br />
<br />
==Self Destructive Plasmid==<br />
<br><br />
'''I-SceI restriction site'''<br />
*construction and verification of the I-SceI homing endonuclease restriction site <partinfo>BBa_K175027</partinfo>.<br />
<br />
'''Restriction site plus reporter'''<br />
*construction of an anhydrotetracyclin inducible GFP-LVA reporter gene combined with the I-SceI restriction site: <partinfo>BBa_K175044</partinfo>.<br />
<br />
'''Characterization of I-SceI biobricks'''<br />
*determination that <partinfo>BBa_K142200</partinfo> and all parts containing it has an I-SceI homing endonuclease that has an LVA degradation tag attached to it, possibly preventing normal functioning.<br />
*determination that <partinfo>BBa_K142205</partinfo> (IPTG inducible I-SceI generator) actually encodes for <partinfo>BBa_K142207</partinfo> (medium constitutive T4 DNA Ligase generator).<br />
<br />
==Time-Delay Device==<br />
<br><br />
'''Synthetic Transcriptional Cascade'''<br />
* Assembly of plasmid 1 and 2 ([http://partsregistry.org/Part:BBa_K175046 K175046]) and 2 ([http://partsregistry.org/Part:BBa_K175047 K175047]).<br />
* Partial confirmation of sequence of both plasmids.<br />
* Transformation in ''Escherichia coli''<br />
* Confirmation of both biobrick individual functionality. <br />
* [https://2009.igem.org/Team:TUDelft/Delay_Results_Continue Characterization] of the plasmids 1 and 2 by fluorescence experiments...[[Team:TUDelft/Results | more]]<br />
<br />
'''Biosynthetic AND gate'''<br />
* Successful assembly of parts [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175023 K175023] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175024 K175024] which correspond to Plasmid 1 and 2 of the Biosynthetic AND gate section respectively.<br />
* Electroporation of plasmid 1 and 2 into ''Escherichia coli'' TOP10 cells...[[Team:TUDelft/Results2 | more]]<br />
<br />
'''Lock and Key Library'''<br />
* Construction of an algorithm capable to generate locks (cis regulation) for different ribosome binding sites (RBS’s) and their correspond key (trans).<br />
* Online [https://2009.igem.org/Team:TUDelft/RiboKeyLock_Generator lock and key generator] derived from the algorithm.<br />
* Construction of four DNA sequences in biobrick format and [http://partsregistry.org/wiki/index.php?title=Part:BBa_pSB1A3 pSB1A3] plasmid backbone which encode lock for medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175031 K175031]), key for the lock of medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175032 K175032]), lock for weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175029 K175029]) or key for the lock of weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175030 K175030]).<br />
* Assembly of two composed biobricks ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175034 K175034] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K175035 K175035]), designed to test the functionality of the lock/key pairs constructed.<br />
* Assembly of two GFP generators under the control of PLacI with weak RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175033 K175033]) or medium RBS ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K175048 K175048])...[[Team:TUDelft/Results3 | more]]<br />
<br />
==Modeling==<br />
<br><br />
'''Synthetic Transcriptional Cascade'''<br />
* Construction of a model used to provide the delay team with design guidelines which would maximize the delay time, to asses the affect of parameter variation on the delay time and to determine areas of instability in the parameter space.<br />
* A sensitivity analysis was done on the system. This looked at how variations in each parameter would influence the delay time.<br />
* Corroboration of a stable system by determining the Jacobian of the system of ODEs analytically.<br />
* Identification of best suitable parameters ranges for a desired time-delay device.<br />
* Based on the results of the simulations, a series of recommendations were given to the delay team to aid them in choosing parts which would maximize the delay time.<br />
<br />
'''Conjugation Modeling'''<br />
<br />
* The development and documentation of a new [https://2009.igem.org/Team:TUDelft/Modeling_Conjugation model for conjugation systems]. This kind of model represents a complete novelty within iGEM framework.<br />
<br />
==Ethics==<br />
<br><br />
'''Literature research'''<br />
* Identification and description of the main ethical issues and concerns in synthetic biology. [https://2009.igem.org/Team:TUDelft/Ethics_background more]<br />
* Illustration of the main ethical issues in clear frameworks, which can be build on in further research. <br />
* Recognition of the issues that need specific attention: ethical discussions concerning the top-down (reductionist) approach towards understanding living systems and the bottom-up approach of enhancing/creating biological systems.<br />
* Elaborate characterization of the omitted issues concerning reductionism and the bottom-up approach in synthetic biology. [https://2009.igem.org/Team:TUDelft/Ethics_background more]<br />
<br><br />
'''Survey'''<br />
* Construction of a comprehensive survey mainly focusing on the issues and possible consequences emerging from the reductionist and bottom-up approach as applied in biology, including questions on evolution, life, risks and the research of Craig Venter. [https://2009.igem.org/Team:TUDelft/Ethics_methods more]<br />
* Successful conduction of the survey among '''242''' people involved in synthetic biology from over 20 different countries, including 168 iGEM participants and 60 supervisors/advisors. <br />
* Extensive elaboration of the results, both qualitatively and quantitatively. [https://2009.igem.org/Team:TUDelft/Ethics_results more]<br />
* Integration of a detailed outline of conclusions and recommendations based on the literature research and the survey results. [https://2009.igem.org/Team:TUDelft/Ethics_conclusions more] <br />
<br />
==Communication==<br />
*We published articles in university newsletters and magazines.<br />
*Till now we had a good cooperation with our sponsors. We published articles in newsletter of the companies which sponsor our team. Some of our sponsors asked us to present our results after the Jamboree.<br />
*We created a platform among university magazines and newsletters to write articles about our project after the Jamboree.<br />
*We attracted attention at our department for our participation to the competition. <br />
<br />
<br><br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/ConclusionsTeam:TUDelft/Conclusions2009-10-22T03:36:22Z<p>Tim Weenink: /* Self Destructive Plasmid */</p>
<hr />
<div>{{Template:TUDelftiGEM2009}}<br />
<br />
='''Conclusions'''=<br />
<br><br />
==Conjugation System==<br />
<br />
A key aspect of this project is the conjugation system which allows the signal plasmid to be transmitted from cell to cell. A thorough design plan was drafted and we set about building our communication system in the lab. Performing the necessary knockouts proved to be a difficult task. Three different knockout attempts were made using the &lambda;-red system without any success. The exact reason for this remains unclear. Although we were unable to successfully knockout the required genes from the R751 conjugative plasmid, progress was made with the signal plasmid. Despite the fact that we did not have access to R751 &Delta;oriTR cells, we were able to show that our prototype signal plasmid is transmitted in the presence of wild R751. This plasmid should provide a useful tool for future teams wishing to use the R751 system. Another success was the synthesis and characterization of the trbK entry exclusion protein. Conjugation tests showed that this was able to block incoming conjugative transfers, as predicted.<br />
<br />
==Self Destructive Plasmid==<br />
<br />
The goal of the Self Destructive Plasmid subproject was to create a plasmid that could be induced to destruct itself. It would do so by expressing an endonuclease that would recognise and cut a sequence in the same plasmid. This linearised DNA was expected to be automatically degraded by the cell.<br />
<br />
The final goal of self destruction was not reached. However, a 30 basepair I-SceI homing endonuclease restriction site has been biobricked and shown to be restricted by that endonuclease. It has been combined with an inducible GFP-LVA reporter gene, which has also been shown to work. <br />
<br />
The problem is the I-SceI homing endonuclease. First a part from the registry was used, that turned out to be T4 DNA ligase instead of I-SceI homing endonuclease. Construction of this part was done by assembling more basic parts. But the gene for the I-SceI enzyme turned out to have an LVA degradation tag attached to it. This shortens the life time of the protein inside the cell considerably and possibly prevents normal folding. It is hypothesised that disfunctional I-SceI is the reason that the expected Self Destruction did not occur. However, a contribution to the registry was made by documenting the anomalies in the biobricks containing the I-SceI homing endonuclease.<br />
<br />
==Time-Delay Device==<br />
<br><br />
'''Synthetic Transcriptional Cascade'''<br />
<br />
In nature, a fundamental “device” in gene regulation circuits is time-delay responses to internal or external stimuli. In the present work, one important feature performed for our system is the reset of the signal once the message is received. The time between when the signal (or self destructive plasmid/conjugation plasmid) has been received and the reset (or destruction of the plasmid) is an important parameter which will allow the signal to be sent to the next cell (receiver) through the consecutive conjugation system before “losing” the message. Therefore, it is necessary to construct a device which will give enough time for the two subsequent events happen. This device is termed as the time-delay genetic circuit in this project.<br />
<br />
A functional time-delay device was designed, constructed and delivered to iGEM registry. This device is based on transcriptional regulation and has the capability to integrate an input signal, IPTG, and trigger the expression of GFP after a delay from the initial detection event. The device is formed by two new functional biobricks, this division not only allows the transfer of the helper plasmid in TUDelft 09 's project but also allows other teams to construct larger or shorter cascades which will deliver, after proper design, longer or shorter time-delay behaviors.<br />
<br />
The plasmids constructed in this work were proved to work individually as expected and once they are transformed together within the same cell, they perform as a delay device. In the experiment performed in this project, the expression of GFP was substantially increased at 600 mins after induction with 0.2 mM IPTG.<br />
<br />
From the observation K12 positive control strain used in experiment glowing GFP, makes us to hypothesize that the lack of enough LacI did not allow performing a better characterization of the device due to a lack of proper control. However, in relative measurements, the device was proved to work.<br />
<br><br><br />
'''Biosynthetic AND gate'''<br />
<br />
It was expected that post-transcriptional regulation, generated synthetically by the lock of the RBS and the following induction of the key for that lock will create a time-delay device. A plan for this strategy was elaborated in this sub-project and several attempts to construct it were performed. However, none of them were successful and this strategy was left behind in order to complete remaining work.<br />
<br><br><br />
'''Lock and Key Library'''<br />
<br />
Based on the work of [https://2009.igem.org/Team:TUDelft/Module_3_References Isaacs F. 2004], [http://openwetware.org/wiki/IGEM:UC_Berkeley/2006 Berkeley iGEM 2006], [http://openwetware.org/wiki/IGEM:Caltech/2007 Caltech iGEM 2007] and [https://2007.igem.org/Peking Peking iGEM 2007] and impulse by the lack of riboregulators, or named as locks and keys in iGEM members, we present the design and construction of an algorithm of an online lock and key generator. This work provides a tool for future iGEM teams which wanted to use post-transcriptional modification as a regulatory system and, because design needs, the use of RBS’s with different strengths are required. Although several teams have designed different riboregulators, they always focused on the “normal” RBS from the registry; this does not take advantage of the large library of RBS present in the site. Moreover, various teams make use of this post-transcriptional tool in this year projects (for example [https://2009.igem.org/Team:KULeuven KULeuven] and [https://2009.igem.org/Team:Tokyo-Nokogen Tokyo-Nokogen]), indicating a need for improvements and expansions of this tool.<br />
<br />
The predicted secondary structure of the RNA derived from the DNA generated by the online generator, shows that the locks sequester the RBS chosen by the user, impeding the expression of the protein downstream the RBS. The online tool also generates a matching key which interacts with the lock will release the RBS, allowing expression of the protein of interest.<br />
<br />
In order to test the functionality of the riboregulators generated by the online tool, two pairs of lock and key were chosen to be constructed and tested. The locks and the respective keys for “weak” and “medium” RBS from the registry were successfully constructed. These riboregulators were used to construct two plasmids which could help for the characterization of the locks and keys. Besides the mentioned plasmids, two controls were designed and constructed. Both, controls and plasmids, were constructed to be functional after IPTG induction. However, the lack of LacI repressor in the cells used forbids us to completely characterize the riboregulators. Future work should be done in order to characterize this new biobricks and other generated by the online tool created in this work and increase the tools for control of gene expression.<br />
<br />
==Ethics==<br />
The undervalued but key ethical issues in synthetic biology mainly include reductionism and the bottom-up approach. Issues concerning safety, security and handling of intellectual property rights should mostly be related to politics not ethics. Instead there should be more focus on questions concerning life, specifically the reductionist (top-down) approach towards understanding life, and the bottom-up approach towards creating life. The issues are evaluated by a literature study and a survey based on that study, amongst 60 supervisors (and advisors) and 168 students involved iGEM 2009. The headlines:<br />
* Participants believe that the value of life cannot be changed by synthetic biology<br />
* Craig Venter's research is presumed not to involve creating new life forms, but rather applying genetic engineering<br />
* Safety and security issues are acknowledged and additional regulation should minimize their risks<br />
* Synthetic biologists are generally careful, but the public wants more<br />
* There is no relation between the existence of a God and sacred or holy properties of life <br />
* Communication with the general public is difficult but perceived necessary<br />
* Life can possibly never be explained by mankind<br />
* The reductionist approach towards understanding life is limiting <br />
<br><br />
[https://2009.igem.org/Team:TUDelft/Conclusions more]<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/ConclusionsTeam:TUDelft/Conclusions2009-10-22T03:35:52Z<p>Tim Weenink: /* Conclusions */</p>
<hr />
<div>{{Template:TUDelftiGEM2009}}<br />
<br />
='''Conclusions'''=<br />
<br><br />
==Conjugation System==<br />
<br />
A key aspect of this project is the conjugation system which allows the signal plasmid to be transmitted from cell to cell. A thorough design plan was drafted and we set about building our communication system in the lab. Performing the necessary knockouts proved to be a difficult task. Three different knockout attempts were made using the &lambda;-red system without any success. The exact reason for this remains unclear. Although we were unable to successfully knockout the required genes from the R751 conjugative plasmid, progress was made with the signal plasmid. Despite the fact that we did not have access to R751 &Delta;oriTR cells, we were able to show that our prototype signal plasmid is transmitted in the presence of wild R751. This plasmid should provide a useful tool for future teams wishing to use the R751 system. Another success was the synthesis and characterization of the trbK entry exclusion protein. Conjugation tests showed that this was able to block incoming conjugative transfers, as predicted.<br />
<br />
='''Self Destructive Plasmid'''=<br />
<br />
The goal of the Self Destructive Plasmid subproject was to create a plasmid that could be induced to destruct itself. It would do so by expressing an endonuclease that would recognise and cut a sequence in the same plasmid. This linearised DNA was expected to be automatically degraded by the cell.<br />
<br />
The final goal of self destruction was not reached. However, a 30 basepair I-SceI homing endonuclease restriction site has been biobricked and shown to be restricted by that endonuclease. It has been combined with an inducible GFP-LVA reporter gene, which has also been shown to work. <br />
<br />
The problem is the I-SceI homing endonuclease. First a part from the registry was used, that turned out to be T4 DNA ligase instead of I-SceI homing endonuclease. Construction of this part was done by assembling more basic parts. But the gene for the I-SceI enzyme turned out to have an LVA degradation tag attached to it. This shortens the life time of the protein inside the cell considerably and possibly prevents normal folding. It is hypothesised that disfunctional I-SceI is the reason that the expected Self Destruction did not occur. However, a contribution to the registry was made by documenting the anomalies in the biobricks containing the I-SceI homing endonuclease.<br />
<br />
==Time-Delay Device==<br />
<br><br />
'''Synthetic Transcriptional Cascade'''<br />
<br />
In nature, a fundamental “device” in gene regulation circuits is time-delay responses to internal or external stimuli. In the present work, one important feature performed for our system is the reset of the signal once the message is received. The time between when the signal (or self destructive plasmid/conjugation plasmid) has been received and the reset (or destruction of the plasmid) is an important parameter which will allow the signal to be sent to the next cell (receiver) through the consecutive conjugation system before “losing” the message. Therefore, it is necessary to construct a device which will give enough time for the two subsequent events happen. This device is termed as the time-delay genetic circuit in this project.<br />
<br />
A functional time-delay device was designed, constructed and delivered to iGEM registry. This device is based on transcriptional regulation and has the capability to integrate an input signal, IPTG, and trigger the expression of GFP after a delay from the initial detection event. The device is formed by two new functional biobricks, this division not only allows the transfer of the helper plasmid in TUDelft 09 's project but also allows other teams to construct larger or shorter cascades which will deliver, after proper design, longer or shorter time-delay behaviors.<br />
<br />
The plasmids constructed in this work were proved to work individually as expected and once they are transformed together within the same cell, they perform as a delay device. In the experiment performed in this project, the expression of GFP was substantially increased at 600 mins after induction with 0.2 mM IPTG.<br />
<br />
From the observation K12 positive control strain used in experiment glowing GFP, makes us to hypothesize that the lack of enough LacI did not allow performing a better characterization of the device due to a lack of proper control. However, in relative measurements, the device was proved to work.<br />
<br><br><br />
'''Biosynthetic AND gate'''<br />
<br />
It was expected that post-transcriptional regulation, generated synthetically by the lock of the RBS and the following induction of the key for that lock will create a time-delay device. A plan for this strategy was elaborated in this sub-project and several attempts to construct it were performed. However, none of them were successful and this strategy was left behind in order to complete remaining work.<br />
<br><br><br />
'''Lock and Key Library'''<br />
<br />
Based on the work of [https://2009.igem.org/Team:TUDelft/Module_3_References Isaacs F. 2004], [http://openwetware.org/wiki/IGEM:UC_Berkeley/2006 Berkeley iGEM 2006], [http://openwetware.org/wiki/IGEM:Caltech/2007 Caltech iGEM 2007] and [http://parts.mit.edu/igem07/index.php/Peking Peking iGEM 2007] and impulse by the lack of riboregulators, or named as locks and keys in iGEM members, we present the design and construction of an algorithm of an online lock and key generator. This work provides a tool for future iGEM teams which wanted to use post-transcriptional modification as a regulatory system and, because design needs, the use of RBS’s with different strengths are required. Although several teams have designed different riboregulators, they always focused on the “normal” RBS from the registry; this does not take advantage of the large library of RBS present in the site. Moreover, various teams make use of this post-transcriptional tool in this year projects (for example [https://2009.igem.org/Team:KULeuven KULeuven] and [https://2009.igem.org/Team:Tokyo-Nokogen Tokyo-Nokogen]), indicating a need for improvements and expansions of this tool.<br />
<br />
The predicted secondary structure of the RNA derived from the DNA generated by the online generator, shows that the locks sequester the RBS chosen by the user, impeding the expression of the protein downstream the RBS. The online tool also generates a matching key which interacts with the lock will release the RBS, allowing expression of the protein of interest.<br />
<br />
In order to test the functionality of the riboregulators generated by the online tool, two pairs of lock and key were chosen to be constructed and tested. The locks and the respective keys for “weak” and “medium” RBS from the registry were successfully constructed. These riboregulators were used to construct two plasmids which could help for the characterization of the locks and keys. Besides the mentioned plasmids, two controls were designed and constructed. Both, controls and plasmids, were constructed to be functional after IPTG induction. However, the lack of LacI repressor in the cells used forbids us to completely characterize the riboregulators. Future work should be done in order to characterize this new biobricks and other generated by the online tool created in this work and increase the tools for control of gene expression.<br />
<br />
==Ethics==<br />
The undervalued but key ethical issues in synthetic biology mainly include reductionism and the bottom-up approach. Issues concerning safety, security and handling of intellectual property rights should mostly be related to politics not ethics. Instead there should be more focus on questions concerning life, specifically the reductionist (top-down) approach towards understanding life, and the bottom-up approach towards creating life. The issues are evaluated by a literature study and a survey based on that study, amongst 60 supervisors (and advisors) and 168 students involved iGEM 2009. The headlines:<br />
* Participants believe that the value of life cannot be changed by synthetic biology<br />
* Craig Venter's research is presumed not to involve creating new life forms, but rather applying genetic engineering<br />
* Safety and security issues are acknowledged and additional regulation should minimize their risks<br />
* Synthetic biologists are generally careful, but the public wants more<br />
* There is no relation between the existence of a God and sacred or holy properties of life <br />
* Communication with the general public is difficult but perceived necessary<br />
* Life can possibly never be explained by mankind<br />
* The reductionist approach towards understanding life is limiting <br />
<br><br />
[https://2009.igem.org/Team:TUDelft/Conclusions more]<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/Future_WorkTeam:TUDelft/Future Work2009-10-22T03:30:16Z<p>Tim Weenink: /* Self Destructive Plasmid */</p>
<hr />
<div>{{Template:TUDelftiGEM2009}}<br />
<br />
='''Future Work'''=<br />
<br />
=Conjugation System=<br />
<br />
It would be interesting to attempt the R751 knockouts using the TargeTron system from Sigma-Aldrich. Two of the three proposed knockouts could be done with this system. Successful knockouts would answer key questions about the predicted functionality of our system.<br />
<br />
<br />
=Self Destructive Plasmid=<br />
<br />
The most obvious improvement for future work on a self destructive plasmid would be to replace the LVA-tagged I-SceI homing endonuclease in <partinfo>BBa_K175045</partinfo> by a wildtype sequence. I-SceI protein with the wildtype sequence is commercially available and has been shown to work. The restriction site and reporter gene have been shown to work, so these would not require immediate attention. Possibly the restriction site could be integrated into the reporter gene sequence, so that cleavage would immediately block transcription of the reporter gene.<br />
<br />
=Time-Delay Device=<br />
<br />
'''''Synthetic Transcriptional Cascade'''''<br />
<br />
It is interesting to test the time-delay device including a constitutive LacI generator and incorporate control as planned, in order to have more reliable results. The complete sequences of the biobricks are being confirmed now.<br />
<br />
'''''Biosynthetic AND gate'''''<br />
<br />
It might be interesting to construct the planned biobricks here in order to corroborate that a time-delay behavior can be achieved using post-transcriptional mechanisms. Moreover, it is promising to obtain a time-delay using both transcriptional and post-transcriptional regulation in which the researchers can have more parameters to tune the time of the delay. <br />
<br />
'''''Lock and Key Library'''''<br />
<br />
In order to completely characterize the new riboregulators, the constructed plasmids should be enhanced by the addition of a LacI generator and they should be tested. Although the algorithm and the online generator seem to work, future work on the interface with the user should be improved.<br />
<br />
=Modeling Conjugation=<br />
<br />
The addition of a conjugation delay time would greatly improve the capabilities of our conjugation model. Another interesting feature would be the ability to confine the growth within predefined geometrical structures. This would allow it to simulate the creation of complex spatiotemporal patters.<br />
<br />
=Ethics=<br />
First of all, we recommend that in the future, scientists and media work closely together to overcome these issues.<br> <br />
Secondly, both for biosafety and for biosecurity the scientific community has expressed its perception towards the risks involved in synthetic biology and they believe that additional regulation can limit or even reduce these risks.<br> <br />
At last, with our survey we aimed to raise the awareness on ethical issues mainly focusing on the extreme application of the reductionist and bottom-up approach. We got great feedback from many participants and when looking at the amount and the content of the open-answers, most people find this subject very interesting and seemed to appreciate the survey. Although no consensus will be formed on questions like whether life can be explained materialistically, the ongoing research in synthetic biology and specifically on the principles of life makes us obliged to keep the debate going and possibly prepare ourselves for whatever science will bring us next.<br><br />
[https://2009.igem.org/Team:TUDelft/Ethics_conclusions#Recommendations more]<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_FutureworkTeam:TUDelft/SDP Futurework2009-10-22T03:30:13Z<p>Tim Weenink: </p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Future Work'''=<br />
<br />
The most obvious improvement for future work on a self destructive plasmid would be to replace the LVA-tagged I-SceI homing endonuclease in <partinfo>BBa_K175045</partinfo> by a wildtype sequence. I-SceI protein with the wildtype sequence is commercially available and has been shown to work. The restriction site and reporter gene have been shown to work, so these would not require immediate attention. Possibly the restriction site could be integrated into the reporter gene sequence, so that cleavage would immediately block transcription of the reporter gene.<br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/Future_WorkTeam:TUDelft/Future Work2009-10-22T03:26:53Z<p>Tim Weenink: </p>
<hr />
<div>{{Template:TUDelftiGEM2009}}<br />
<br />
='''Future Work'''=<br />
<br />
=Conjugation System=<br />
<br />
It would be interesting to attempt the R751 knockouts using the TargeTron system from Sigma-Aldrich. Two of the three proposed knockouts could be done with this system. Successful knockouts would answer key questions about the predicted functionality of our system.<br />
<br />
<br />
=Self Destructive Plasmid=<br />
<br />
The most obvious improvement for future work on a self destructive plasmid would be to replace the LVA-tagged I-SceI homing endonuclease in <partinfo>BBa_175045</partinfo> by a wildtype sequence. I-SceI protein with the wildtype sequence is commercially available and has been shown to work. The restriction site and reporter gene have been shown to work, so these would not require immediate attention. Possibly the restriction site could be integrated into the reporter gene sequence, so that cleavage would immediately block transcription of the reporter gene.<br />
<br />
=Time-Delay Device=<br />
<br />
'''''Synthetic Transcriptional Cascade'''''<br />
<br />
It is interesting to test the time-delay device including a constitutive LacI generator and incorporate control as planned, in order to have more reliable results. The complete sequences of the biobricks are being confirmed now.<br />
<br />
'''''Biosynthetic AND gate'''''<br />
<br />
It might be interesting to construct the planned biobricks here in order to corroborate that a time-delay behavior can be achieved using post-transcriptional mechanisms. Moreover, it is promising to obtain a time-delay using both transcriptional and post-transcriptional regulation in which the researchers can have more parameters to tune the time of the delay. <br />
<br />
'''''Lock and Key Library'''''<br />
<br />
In order to completely characterize the new riboregulators, the constructed plasmids should be enhanced by the addition of a LacI generator and they should be tested. Although the algorithm and the online generator seem to work, future work on the interface with the user should be improved.<br />
<br />
=Modeling Conjugation=<br />
<br />
The addition of a conjugation delay time would greatly improve the capabilities of our conjugation model. Another interesting feature would be the ability to confine the growth within predefined geometrical structures. This would allow it to simulate the creation of complex spatiotemporal patters.<br />
<br />
=Ethics=<br />
First of all, we recommend that in the future, scientists and media work closely together to overcome these issues.<br> <br />
Secondly, both for biosafety and for biosecurity the scientific community has expressed its perception towards the risks involved in synthetic biology and they believe that additional regulation can limit or even reduce these risks.<br> <br />
At last, with our survey we aimed to raise the awareness on ethical issues mainly focusing on the extreme application of the reductionist and bottom-up approach. We got great feedback from many participants and when looking at the amount and the content of the open-answers, most people find this subject very interesting and seemed to appreciate the survey. Although no consensus will be formed on questions like whether life can be explained materialistically, the ongoing research in synthetic biology and specifically on the principles of life makes us obliged to keep the debate going and possibly prepare ourselves for whatever science will bring us next.<br><br />
[https://2009.igem.org/Team:TUDelft/Ethics_conclusions#Recommendations more]<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_FutureworkTeam:TUDelft/SDP Futurework2009-10-22T03:19:50Z<p>Tim Weenink: New page: {{Template:TUDelftiGEM2009_menu_M2_SDP}} ='''Future Work'''= The most obvious improvement for future work on a self destructive plasmid would be to replace the LVA-tagged I-SceI homing e...</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Future Work'''=<br />
<br />
The most obvious improvement for future work on a self destructive plasmid would be to replace the LVA-tagged I-SceI homing endonuclease in <partinfo>BBa_175045</partinfo> by a wildtype sequence. I-SceI protein with the wildtype sequence is commercially available and has been shown to work. The restriction site and reporter gene have been shown to work, so these would not require immediate attention. Possibly the restriction site could be integrated into the reporter gene sequence, so that cleavage would immediately block transcription of the reporter gene.<br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ConclusionTeam:TUDelft/SDP Conclusion2009-10-22T03:12:55Z<p>Tim Weenink: New page: {{Template:TUDelftiGEM2009_menu_M2_SDP}} ='''Conclusion'''= The goal of the Self Destructive Plasmid subproject was to create a plasmid that could be induced to destruct itself. It would...</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Conclusion'''=<br />
<br />
The goal of the Self Destructive Plasmid subproject was to create a plasmid that could be induced to destruct itself. It would do so by expressing an endonuclease that would recognise and cut a sequence in the same plasmid. This linearised DNA was expected to be automatically degraded by the cell.<br />
<br />
The final goal of self destruction was not reached. However, a 30 basepair I-SceI homing endonuclease restriction site has been biobricked and shown to be restricted by that endonuclease. It has been combined with an inducible GFP-LVA reporter gene, which has also been shown to work. <br />
<br />
The problem is the I-SceI homing endonuclease. First a part from the registry was used, that turned out to be T4 DNA ligase instead of I-SceI homing endonuclease. Construction of this part was done by assembling more basic parts. But the gene for the I-SceI enzyme turned out to have an LVA degradation tag attached to it. This shortens the life time of the protein inside the cell considerably and possibly prevents normal folding. It is hypothesised that disfunctional I-SceI is the reason that the expected Self Destruction did not occur. However, a contribution to the registry was made by documenting the anomalies in the biobricks containing the I-SceI homing endonuclease.<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Template:TUDelftiGEM2009_menu_M2_SDP_expandTemplate:TUDelftiGEM2009 menu M2 SDP expand2009-10-22T02:50:01Z<p>Tim Weenink: </p>
<hr />
<div><div id="menubluesection">[[Team:TUDelft/SDP_Overview | Module 2: Self Destructive Plasmid]]</div><br />
<div id="menuorangesection">[[Team:TUDelft/SDP_Cloning | Cloning Strategy]][[Team:TUDelft/SDP_Results | Experimental Results]][[Team:TUDelft/SDP_Conclusion | Conclusion]][[Team:TUDelft/SDP_Futurework | Future work]]</div><br />
<div id="menuspacer">[[Team:TUDelft/SDP_Overview | ]]</div></div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ResultsTeam:TUDelft/SDP Results2009-10-22T02:44:45Z<p>Tim Weenink: /* Integration */</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Experimental results'''=<br />
<br />
The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.<br />
<br />
=='''I-SceI restriction site'''==<br />
<br />
[[Image:I6 check.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175027</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.<br />
<br />
5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'<br><br />
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5' <br />
<br />
The resulting biobrick (<partinfo>BBa_K175027</partinfo>) was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp. <br />
<br />
The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site works as expected.<br />
<br />
As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in <partinfo>BBa_K175050</partinfo>, which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.<br />
<br />
A simpler part was constructed (<partinfo>BBa_K175044</partinfo>), which only contained the restriction site in front of the aTc inducible GFP-LVA generator. <br />
<br />
[[Image:BBa_K175044.png|400px|thumb|center| Makeup of <partinfo>BBa_K175044</partinfo>. It consists of the I-SceI homing endonuclease restriction site and an anhydrotetracyclin inducible GFP-LVA generator.]]<br />
<br />
This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.<br />
<br />
[[Image:GFP-LVAinduction.png|200px|thumb|center| anhydrotetracyclin induction of GFP fluorescence in centrifuged culture containing <partinfo>BBa_K175044</partinfo>. Left tube is uninduced, right tube is induced.]]<br />
<br />
[[Image:BBa_K175044_restriction.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175044</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: PstI digested part, expected size = 5095bp. Lane 3: PstI and I-SceI restricted part, expected band lenghts are 3204 and 1891bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.<br />
<br />
Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''I-SceI homing endonuclease'''==<br />
<br />
After successful construction of the restriction site with reportergene, the actual protein was added to the construct. The IPTG inducible part (<partinfo>BBa_K142205</partinfo>) made by the 2008 ETH Zurich team was used for this. However, after having it sequenced, the part appeared to be a medium constitutive T4 ligase generator (<partinfo>BBa_K142207</partinfo>), made by the same team. To rule out a mistake by the registry, the part was requested again. A new batch was transformed and sent to us. It was grown up, isolated and restricted with EcoRI and PstI, to obtain insert length. It was exactly as long as the first batch (~1700bp), which corresponds to the expected length of <partinfo>BBa_K142207</partinfo> (1668bp), and is significantly longer than the expected length of 1114bp. These findings were entered into the registry as a user review of <partinfo>BBa_K142205</partinfo>.<br />
<br />
As a workaround, a twin of what was supposed to be <partinfo>BBa_K142205</partinfo> was constructed, using <partinfo>BBa_K142202</partinfo>. The sequence of the resulting part (<partinfo>BBa_K175041</partinfo>) was confirmed, as shown in the sequence analysis section of the parts page. Thereby the sequence of <partinfo>BBa_K142202</partinfo> was indirectly confirmed.<br />
<br />
Close inspection of this sequence also revealed, that the homing endonuclease had an LVA degradation tag attached to the end of its sequence. This information was not mentioned in the original I-SceI parts page (<partinfo>BBa_K142200</partinfo>), nor in any of the constructs that contain this part. As protein tags in general can influence the folding and function of a protein and the LVA tag in specific reduces stability of a protein, this is considered quite an essential feat. This information was therefore entered into the registry at the concerned parts pages, in the form of user reviews.<br />
<br />
=='''Integration'''==<br />
<br />
With the IPTG inducible I-SceI homing endonuclease generator and the I-SceI restriction site with aTc inducible reporter ready, the two parts could be combined to yield the final Self Destructive Plasmid. This final construct is depicted in the image below.<br />
<br />
[[Image:BBa_K175045.png|550px|thumb|center| Makeup of <partinfo>BBa_K175045</partinfo>, the Self Destructive Plasmid. It consists of the an IPTG inducible I-SceI homing endonuclease generator and its restriction site. An anhydrotetracyclin inducible GFP-LVA generator is included to check for the destruction of the plasmid when the endonuclease is induced.]]<br />
<br />
[[Image:UI_restriction_analysis.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 2 different colonies. The first one was a fluorescing colony (lane 1 and 2) and the second one non-fluorescing (lane 3 and 4). Lane 1 and 3: EcoRI digested part, expected length = 5100bp. Lane 2 and 4: EcoRI, PstI digested part, expected lengths = 2072 and 3028bp.]]<br />
Although it seemed to be an assembly like any other, it had its complications. Several colonies were screened for the right insert. Two types appeared to be present. The first one, with colonies that were fluorescing as expected and the second with colonies that did not give fluorescence (after aTc induction). However, the insert size of the first type was around 1200bp (see lane 2 in the adjacent image), significantly smaller than the expected 3028bp. <br />
<br />
The non-fluorescent colonies did have bands corresponding to 2072 and 3028bp, but also one corresponding to the (linear) 5100bp fragment (compare lane 3 and 4). It was assumed that the colony had a mixture of plasmids, some that could get restricted by both enzymes and some that could not. Sequencing for this part failed, possibly because of the prescence of multiple kinds of plasmid.<br />
<br />
[[Image:U2 induced and restricted.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 1 colony under various conditions. The isolated plasmids were restricted with I-SceI and PstI, with expected bands of 1891 and 3109bp. Lane 1 and 2: not induced. Lane 3,4: induced with aTc. Lane 5,6: induced with IPTG. Lane 7,8: induced with aTc and IPTG.]]<br />
Ideally a plasmid would have been isolated that gave only two bands when restricted with the enzymes mentioned above. But due to time constraints this was not possible. The selected colony was incubated in liquid culture under various conditions of induction (aTc and IPTG) to see if (some of) the plasmids would get degraded by the expressed homing endonuclease.<br />
<br />
The outcome of the experiment was disappointing. Although at least some plasmids were present that had the expected size, none of the cultures induced with aTc showed any fluorescence. Also, the restriction pattern for the cultures that were induced with IPTG (for the expression of the homing endonuclease, lanes 5-8) was identical to the non-IPTG induced cultures. This shows that these plasmids were probably not restricted by I-SceI, because the restriction site was still present. Usually linear DNA will be degraded inside a cell, so if the restriction site had been cut, one would have expected the plasmid to have degraded (at least partially).<br />
<br />
The reason that I-SceI restriction inside the cell failed could be due to the LVA-tag attached to the homing endonuclease. Either it made the protein so unstable that it would get degraded at such a high rate that it could not restrict the plasmid. Or the tag itself prevented it from folding properly, and thereby prevented it from doing restrictions in the first place. <br />
<br />
The reason why no fluorescence was observed after aTc induction is not well understood. Sequencing of this part only succeeded in the reverse direction (see <partinfo>BBa_K175045</partinfo>). It showed the presence of the GFP-LVA gene. Also the I-SceI restriction site has been shown to be present in the restriction analysis, so it is unlikely that anything between the restriction site and the GFP-LVA gene has been changed, because the observed length of the insert corresponds so well with the expected lenght.<br />
<br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ResultsTeam:TUDelft/SDP Results2009-10-22T02:28:19Z<p>Tim Weenink: /* Integration */</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Experimental results'''=<br />
<br />
The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.<br />
<br />
=='''I-SceI restriction site'''==<br />
<br />
[[Image:I6 check.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175027</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.<br />
<br />
5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'<br><br />
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5' <br />
<br />
The resulting biobrick (<partinfo>BBa_K175027</partinfo>) was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp. <br />
<br />
The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site works as expected.<br />
<br />
As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in <partinfo>BBa_K175050</partinfo>, which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.<br />
<br />
A simpler part was constructed (<partinfo>BBa_K175044</partinfo>), which only contained the restriction site in front of the aTc inducible GFP-LVA generator. <br />
<br />
[[Image:BBa_K175044.png|400px|thumb|center| Makeup of <partinfo>BBa_K175044</partinfo>. It consists of the I-SceI homing endonuclease restriction site and an anhydrotetracyclin inducible GFP-LVA generator.]]<br />
<br />
This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.<br />
<br />
[[Image:GFP-LVAinduction.png|200px|thumb|center| anhydrotetracyclin induction of GFP fluorescence in centrifuged culture containing <partinfo>BBa_K175044</partinfo>. Left tube is uninduced, right tube is induced.]]<br />
<br />
[[Image:BBa_K175044_restriction.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175044</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: PstI digested part, expected size = 5095bp. Lane 3: PstI and I-SceI restricted part, expected band lenghts are 3204 and 1891bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.<br />
<br />
Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''I-SceI homing endonuclease'''==<br />
<br />
After successful construction of the restriction site with reportergene, the actual protein was added to the construct. The IPTG inducible part (<partinfo>BBa_K142205</partinfo>) made by the 2008 ETH Zurich team was used for this. However, after having it sequenced, the part appeared to be a medium constitutive T4 ligase generator (<partinfo>BBa_K142207</partinfo>), made by the same team. To rule out a mistake by the registry, the part was requested again. A new batch was transformed and sent to us. It was grown up, isolated and restricted with EcoRI and PstI, to obtain insert length. It was exactly as long as the first batch (~1700bp), which corresponds to the expected length of <partinfo>BBa_K142207</partinfo> (1668bp), and is significantly longer than the expected length of 1114bp. These findings were entered into the registry as a user review of <partinfo>BBa_K142205</partinfo>.<br />
<br />
As a workaround, a twin of what was supposed to be <partinfo>BBa_K142205</partinfo> was constructed, using <partinfo>BBa_K142202</partinfo>. The sequence of the resulting part (<partinfo>BBa_K175041</partinfo>) was confirmed, as shown in the sequence analysis section of the parts page. Thereby the sequence of <partinfo>BBa_K142202</partinfo> was indirectly confirmed.<br />
<br />
Close inspection of this sequence also revealed, that the homing endonuclease had an LVA degradation tag attached to the end of its sequence. This information was not mentioned in the original I-SceI parts page (<partinfo>BBa_K142200</partinfo>), nor in any of the constructs that contain this part. As protein tags in general can influence the folding and function of a protein and the LVA tag in specific reduces stability of a protein, this is considered quite an essential feat. This information was therefore entered into the registry at the concerned parts pages, in the form of user reviews.<br />
<br />
=='''Integration'''==<br />
<br />
With the IPTG inducible I-SceI homing endonuclease generator and the I-SceI restriction site with aTc inducible reporter ready, the two parts could be combined to yield the final Self Destructive Plasmid. This final construct is depicted in the image below.<br />
<br />
[[Image:BBa_K175045.png|550px|thumb|center| Makeup of <partinfo>BBa_K175045</partinfo>, the Self Destructive Plasmid. It consists of the an IPTG inducible I-SceI homing endonuclease generator and its restriction site. An anhydrotetracyclin inducible GFP-LVA generator is included to check for the destruction of the plasmid when the endonuclease is induced.]]<br />
<br />
[[Image:UI_restriction_analysis.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 2 different colonies. The first one was a fluorescing colony (lane 1 and 2) and the second one non-fluorescing (lane 3 and 4). Lane 1 and 3: EcoRI digested part, expected length = 5100bp. Lane 2 and 4: EcoRI, PstI digested part, expected lengths = 2072 and 3028bp.]]<br />
Although it seemed to be an assembly like any other, it had its complications. Several colonies were screened for the right insert. Two types appeared to be present. The first one, with colonies that were fluorescing as expected and the second with colonies that did not give fluorescence (after aTc induction). However, the insert size of the first type was around 1200bp (see lane 2 in the adjacent image), significantly smaller than the expected 3028bp. <br />
<br />
The non-fluorescent colonies did have bands corresponding to 2072 and 3028bp, but also one corresponding to the (linear) 5100bp fragment (compare lane 3 and 4). It was assumed that the colony had a mixture of plasmids, some that could get restricted by both enzymes and some that could not. Sequencing for this part failed, possibly because of the prescence of multiple kinds of plasmid.<br />
<br />
[[Image:U2 induced and restricted.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 1 colony under various conditions. The isolated plasmids were restricted with I-SceI and PstI, with expected bands of 1891 and 3109bp. Lane 1 and 2: not induced. Lane 3,4: induced with aTc. Lane 5,6: induced with IPTG. Lane 7,8: induced with aTc and IPTG.]]<br />
Ideally a plasmid would have been isolated that gave only two bands when restricted with the enzymes mentioned above. But due to time constraints this was not possible. The selected colony was incubated in liquid culture under various conditions of induction (aTc and IPTG) to see if (some of) the plasmids would get degraded by the expressed homing endonuclease.<br />
<br />
The outcome of the experiment was disappointing. Although at least some plasmids were present that had the expected size, none of the cultures induced with aTc showed any fluorescence. Also, the restriction pattern for the cultures that were induced with IPTG (for the expression of the homing endonuclease, lanes 5-8) was identical to the non-IPTG induced cultures. This shows that these plasmids were probably not restricted by I-SceI.<br />
<br />
The reason <br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ResultsTeam:TUDelft/SDP Results2009-10-22T02:10:05Z<p>Tim Weenink: /* Integration */</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Experimental results'''=<br />
<br />
The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.<br />
<br />
=='''I-SceI restriction site'''==<br />
<br />
[[Image:I6 check.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175027</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.<br />
<br />
5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'<br><br />
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5' <br />
<br />
The resulting biobrick (<partinfo>BBa_K175027</partinfo>) was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp. <br />
<br />
The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site works as expected.<br />
<br />
As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in <partinfo>BBa_K175050</partinfo>, which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.<br />
<br />
A simpler part was constructed (<partinfo>BBa_K175044</partinfo>), which only contained the restriction site in front of the aTc inducible GFP-LVA generator. <br />
<br />
[[Image:BBa_K175044.png|400px|thumb|center| Makeup of <partinfo>BBa_K175044</partinfo>. It consists of the I-SceI homing endonuclease restriction site and an anhydrotetracyclin inducible GFP-LVA generator.]]<br />
<br />
This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.<br />
<br />
[[Image:GFP-LVAinduction.png|200px|thumb|center| anhydrotetracyclin induction of GFP fluorescence in centrifuged culture containing <partinfo>BBa_K175044</partinfo>. Left tube is uninduced, right tube is induced.]]<br />
<br />
[[Image:BBa_K175044_restriction.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175044</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: PstI digested part, expected size = 5095bp. Lane 3: PstI and I-SceI restricted part, expected band lenghts are 3204 and 1891bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.<br />
<br />
Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''I-SceI homing endonuclease'''==<br />
<br />
After successful construction of the restriction site with reportergene, the actual protein was added to the construct. The IPTG inducible part (<partinfo>BBa_K142205</partinfo>) made by the 2008 ETH Zurich team was used for this. However, after having it sequenced, the part appeared to be a medium constitutive T4 ligase generator (<partinfo>BBa_K142207</partinfo>), made by the same team. To rule out a mistake by the registry, the part was requested again. A new batch was transformed and sent to us. It was grown up, isolated and restricted with EcoRI and PstI, to obtain insert length. It was exactly as long as the first batch (~1700bp), which corresponds to the expected length of <partinfo>BBa_K142207</partinfo> (1668bp), and is significantly longer than the expected length of 1114bp. These findings were entered into the registry as a user review of <partinfo>BBa_K142205</partinfo>.<br />
<br />
As a workaround, a twin of what was supposed to be <partinfo>BBa_K142205</partinfo> was constructed, using <partinfo>BBa_K142202</partinfo>. The sequence of the resulting part (<partinfo>BBa_K175041</partinfo>) was confirmed, as shown in the sequence analysis section of the parts page. Thereby the sequence of <partinfo>BBa_K142202</partinfo> was indirectly confirmed.<br />
<br />
Close inspection of this sequence also revealed, that the homing endonuclease had an LVA degradation tag attached to the end of its sequence. This information was not mentioned in the original I-SceI parts page (<partinfo>BBa_K142200</partinfo>), nor in any of the constructs that contain this part. As protein tags in general can influence the folding and function of a protein and the LVA tag in specific reduces stability of a protein, this is considered quite an essential feat. This information was therefore entered into the registry at the concerned parts pages, in the form of user reviews.<br />
<br />
=='''Integration'''==<br />
<br />
With the IPTG inducible I-SceI homing endonuclease generator and the I-SceI restriction site with aTc inducible reporter ready, the two parts could be combined to yield the final Self Destructive Plasmid. This final construct is depicted in the image below.<br />
<br />
[[Image:BBa_K175045.png|550px|thumb|center| Makeup of <partinfo>BBa_K175045</partinfo>, the Self Destructive Plasmid. It consists of the an IPTG inducible I-SceI homing endonuclease generator and its restriction site. An anhydrotetracyclin inducible GFP-LVA generator is included to check for the destruction of the plasmid when the endonuclease is induced.]]<br />
<br />
[[Image:UI_restriction_analysis.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 2 different colonies. The first one was a fluorescing colony (lane 1 and 2) and the second one non-fluorescing (lane 3 and 4). Lane 1 and 3: EcoRI digested part, expected length = 5100bp. Lane 2 and 4: EcoRI, PstI digested part, expected lengths = 2072 and 3028bp.]]<br />
Although it seemed to be an assembly like any other, it had its complications. Several colonies were screened for the right insert. Two types appeared to be present. The first one, with colonies that were fluorescing as expected and the second with colonies that did not give fluorescence (after aTc induction). However, the insert size of the first type was around 1200bp (see lane 2 in the adjacent image), significantly smaller than the expected 3028bp. <br />
<br />
The non-fluorescent colonies did have bands corresponding to 2072 and 3028bp, but also one corresponding to the (linear) 5100bp fragment (compare lane 3 and 4). It was assumed that the colony had a mixture of plasmids, some that could get restricted by both enzymes and some that could not. Sequencing for this part failed, possibly because of the prescence of multiple kinds of plasmid.<br />
<br />
Ideally a plasmid would have been isolated that gave only two bands when restricted with the enzymes mentioned above. But due to time constraints this was not possible. The selected colony was incubated in liquid culture under various conditions of induction (aTc and IPTG) to see if (some of) the plasmids would get degraded by the expressed homing endonuclease.<br />
<br />
[[Image:U2 induced and restricted.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 1 colony under various conditions. The isolated plasmids were restricted with I-SceI and PstI, with expected bands of 1891 and 3109bp. Lane 1 and 2: not induced. Lane 3,4: induced with aTc. Lane 5,6: induced with IPTG. Lane 7,8: induced with aTc and IPTG.]]<br />
<br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/File:U2_induced_and_restricted.pngFile:U2 induced and restricted.png2009-10-22T01:56:59Z<p>Tim Weenink: </p>
<hr />
<div></div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ResultsTeam:TUDelft/SDP Results2009-10-22T01:53:38Z<p>Tim Weenink: /* Integration */</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Experimental results'''=<br />
<br />
The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.<br />
<br />
=='''I-SceI restriction site'''==<br />
<br />
[[Image:I6 check.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175027</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.<br />
<br />
5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'<br><br />
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5' <br />
<br />
The resulting biobrick (<partinfo>BBa_K175027</partinfo>) was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp. <br />
<br />
The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site works as expected.<br />
<br />
As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in <partinfo>BBa_K175050</partinfo>, which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.<br />
<br />
A simpler part was constructed (<partinfo>BBa_K175044</partinfo>), which only contained the restriction site in front of the aTc inducible GFP-LVA generator. <br />
<br />
[[Image:BBa_K175044.png|400px|thumb|center| Makeup of <partinfo>BBa_K175044</partinfo>. It consists of the I-SceI homing endonuclease restriction site and an anhydrotetracyclin inducible GFP-LVA generator.]]<br />
<br />
This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.<br />
<br />
[[Image:GFP-LVAinduction.png|200px|thumb|center| anhydrotetracyclin induction of GFP fluorescence in centrifuged culture containing <partinfo>BBa_K175044</partinfo>. Left tube is uninduced, right tube is induced.]]<br />
<br />
[[Image:BBa_K175044_restriction.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175044</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: PstI digested part, expected size = 5095bp. Lane 3: PstI and I-SceI restricted part, expected band lenghts are 3204 and 1891bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.<br />
<br />
Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''I-SceI homing endonuclease'''==<br />
<br />
After successful construction of the restriction site with reportergene, the actual protein was added to the construct. The IPTG inducible part (<partinfo>BBa_K142205</partinfo>) made by the 2008 ETH Zurich team was used for this. However, after having it sequenced, the part appeared to be a medium constitutive T4 ligase generator (<partinfo>BBa_K142207</partinfo>), made by the same team. To rule out a mistake by the registry, the part was requested again. A new batch was transformed and sent to us. It was grown up, isolated and restricted with EcoRI and PstI, to obtain insert length. It was exactly as long as the first batch (~1700bp), which corresponds to the expected length of <partinfo>BBa_K142207</partinfo> (1668bp), and is significantly longer than the expected length of 1114bp. These findings were entered into the registry as a user review of <partinfo>BBa_K142205</partinfo>.<br />
<br />
As a workaround, a twin of what was supposed to be <partinfo>BBa_K142205</partinfo> was constructed, using <partinfo>BBa_K142202</partinfo>. The sequence of the resulting part (<partinfo>BBa_K175041</partinfo>) was confirmed, as shown in the sequence analysis section of the parts page. Thereby the sequence of <partinfo>BBa_K142202</partinfo> was indirectly confirmed.<br />
<br />
Close inspection of this sequence also revealed, that the homing endonuclease had an LVA degradation tag attached to the end of its sequence. This information was not mentioned in the original I-SceI parts page (<partinfo>BBa_K142200</partinfo>), nor in any of the constructs that contain this part. As protein tags in general can influence the folding and function of a protein and the LVA tag in specific reduces stability of a protein, this is considered quite an essential feat. This information was therefore entered into the registry at the concerned parts pages, in the form of user reviews.<br />
<br />
=='''Integration'''==<br />
<br />
With the IPTG inducible I-SceI homing endonuclease generator and the I-SceI restriction site with aTc inducible reporter ready, the two parts could be combined to yield the final Self Destructive Plasmid. This final construct is depicted in the image below.<br />
<br />
[[Image:BBa_K175045.png|550px|thumb|center| Makeup of <partinfo>BBa_K175045</partinfo>, the Self Destructive Plasmid. It consists of the an IPTG inducible I-SceI homing endonuclease generator and its restriction site. An anhydrotetracyclin inducible GFP-LVA generator is included to check for the destruction of the plasmid when the endonuclease is induced.]]<br />
<br />
[[Image:UI_restriction_analysis.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 2 different colonies. The first one was a fluorescing colony (lane 1 and 2) and the second one non-fluorescing (lane 3 and 4). Lane 1 and 3: EcoRI digested part, expected length = 5100bp. Lane 2 and 4: EcoRI, PstI digested part, expected lengths = 2072 and 3028bp.]]<br />
Although it seemed to be an assembly like any other, it had its complications. Several colonies were screened for the right insert. Two types appeared to be present. The first one, with colonies that were fluorescing as expected and the second with colonies that did not give fluorescence (after aTc induction). However, the insert size of the first type was around 1200bp (see lane 2 in the adjacent image), significantly smaller than the expected 3028bp. <br />
<br />
The non-fluorescent colonies did have bands corresponding to 2072 and 3028bp, but also one corresponding to the (linear) 5100bp fragment (compare lane 3 and 4). It was assumed that the colony had a mixture of plasmids, some that could get restricted by both enzymes and some that could not.<br />
<br />
Ideally a plasmid would have been isolated that gave only two bands when restricted with the enzymes mentioned above. But due to time constraints this not possible. The selected colony was <br />
<br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ResultsTeam:TUDelft/SDP Results2009-10-22T01:27:47Z<p>Tim Weenink: /* Integration */</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Experimental results'''=<br />
<br />
The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.<br />
<br />
=='''I-SceI restriction site'''==<br />
<br />
[[Image:I6 check.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175027</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.<br />
<br />
5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'<br><br />
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5' <br />
<br />
The resulting biobrick (<partinfo>BBa_K175027</partinfo>) was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp. <br />
<br />
The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site works as expected.<br />
<br />
As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in <partinfo>BBa_K175050</partinfo>, which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.<br />
<br />
A simpler part was constructed (<partinfo>BBa_K175044</partinfo>), which only contained the restriction site in front of the aTc inducible GFP-LVA generator. <br />
<br />
[[Image:BBa_K175044.png|400px|thumb|center| Makeup of <partinfo>BBa_K175044</partinfo>. It consists of the I-SceI homing endonuclease restriction site and an anhydrotetracyclin inducible GFP-LVA generator.]]<br />
<br />
This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.<br />
<br />
[[Image:GFP-LVAinduction.png|200px|thumb|center| anhydrotetracyclin induction of GFP fluorescence in centrifuged culture containing <partinfo>BBa_K175044</partinfo>. Left tube is uninduced, right tube is induced.]]<br />
<br />
[[Image:BBa_K175044_restriction.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175044</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: PstI digested part, expected size = 5095bp. Lane 3: PstI and I-SceI restricted part, expected band lenghts are 3204 and 1891bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.<br />
<br />
Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''I-SceI homing endonuclease'''==<br />
<br />
After successful construction of the restriction site with reportergene, the actual protein was added to the construct. The IPTG inducible part (<partinfo>BBa_K142205</partinfo>) made by the 2008 ETH Zurich team was used for this. However, after having it sequenced, the part appeared to be a medium constitutive T4 ligase generator (<partinfo>BBa_K142207</partinfo>), made by the same team. To rule out a mistake by the registry, the part was requested again. A new batch was transformed and sent to us. It was grown up, isolated and restricted with EcoRI and PstI, to obtain insert length. It was exactly as long as the first batch (~1700bp), which corresponds to the expected length of <partinfo>BBa_K142207</partinfo> (1668bp), and is significantly longer than the expected length of 1114bp. These findings were entered into the registry as a user review of <partinfo>BBa_K142205</partinfo>.<br />
<br />
As a workaround, a twin of what was supposed to be <partinfo>BBa_K142205</partinfo> was constructed, using <partinfo>BBa_K142202</partinfo>. The sequence of the resulting part (<partinfo>BBa_K175041</partinfo>) was confirmed, as shown in the sequence analysis section of the parts page. Thereby the sequence of <partinfo>BBa_K142202</partinfo> was indirectly confirmed.<br />
<br />
Close inspection of this sequence also revealed, that the homing endonuclease had an LVA degradation tag attached to the end of its sequence. This information was not mentioned in the original I-SceI parts page (<partinfo>BBa_K142200</partinfo>), nor in any of the constructs that contain this part. As protein tags in general can influence the folding and function of a protein and the LVA tag in specific reduces stability of a protein, this is considered quite an essential feat. This information was therefore entered into the registry at the concerned parts pages, in the form of user reviews.<br />
<br />
=='''Integration'''==<br />
<br />
With the IPTG inducible I-SceI homing endonuclease generator and the I-SceI restriction site with aTc inducible reporter ready, the two parts could be combined to yield the final Self Destructive Plasmid. This final construct is depicted in the image below.<br />
<br />
[[Image:BBa_K175045.png|550px|thumb|center| Makeup of <partinfo>BBa_K175045</partinfo>, the Self Destructive Plasmid. It consists of the an IPTG inducible I-SceI homing endonuclease generator and its restriction site. An anhydrotetracyclin inducible GFP-LVA generator is included to check for the destruction of the plasmid when the endonuclease is induced.]]<br />
<br />
[[Image:UI_restriction_analysis.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 2 different colonies. The first one was a fluorescing colony (lane 1 and 2) and the second one non-fluorescing (lane 3 and 4). Lane 1 and 3: PstI digested part, expected length = 5100bp. Lane 2 and 4: PstI, I-SceI digested part, expected lengths = 1891 and 3209bp.]]<br />
Although it seemed to be an assembly like any other, it had its complications. Several colonies were screened for the right insert. Two types appeared to be present. The first one, with colonies that were fluorescing as expected and the second with colonies that did not give fluorescence (after aTc induction). However, the insert size of the first type was around 1200bp (see lane 2 in the adjacent image), significantly smaller than the expected 3209bp. <br />
<br />
The non-fluorescent colonies did have bands corresponding to 1891 and 3209bp, but also one corresponding to the (linear) 5100bp fragment (compare lane 3 and 4). It was assumed that the colony had a mixture of plasmids, some that could get restricted by I-SceI and some that could not.<br />
<br />
To test the above hypothesis, a new batch of E. Coli cells was transformed with the isolated plasmid from the second colony (lane 3 and 4). This yielded a range of colonies with new plasmids.<br />
<br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ResultsTeam:TUDelft/SDP Results2009-10-22T01:12:07Z<p>Tim Weenink: /* Integration */</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Experimental results'''=<br />
<br />
The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.<br />
<br />
=='''I-SceI restriction site'''==<br />
<br />
[[Image:I6 check.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175027</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.<br />
<br />
5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'<br><br />
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5' <br />
<br />
The resulting biobrick (<partinfo>BBa_K175027</partinfo>) was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp. <br />
<br />
The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site works as expected.<br />
<br />
As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in <partinfo>BBa_K175050</partinfo>, which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.<br />
<br />
A simpler part was constructed (<partinfo>BBa_K175044</partinfo>), which only contained the restriction site in front of the aTc inducible GFP-LVA generator. <br />
<br />
[[Image:BBa_K175044.png|400px|thumb|center| Makeup of <partinfo>BBa_K175044</partinfo>. It consists of the I-SceI homing endonuclease restriction site and an anhydrotetracyclin inducible GFP-LVA generator.]]<br />
<br />
This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.<br />
<br />
[[Image:GFP-LVAinduction.png|200px|thumb|center| anhydrotetracyclin induction of GFP fluorescence in centrifuged culture containing <partinfo>BBa_K175044</partinfo>. Left tube is uninduced, right tube is induced.]]<br />
<br />
[[Image:BBa_K175044_restriction.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175044</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: PstI digested part, expected size = 5095bp. Lane 3: PstI and I-SceI restricted part, expected band lenghts are 3204 and 1891bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.<br />
<br />
Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''I-SceI homing endonuclease'''==<br />
<br />
After successful construction of the restriction site with reportergene, the actual protein was added to the construct. The IPTG inducible part (<partinfo>BBa_K142205</partinfo>) made by the 2008 ETH Zurich team was used for this. However, after having it sequenced, the part appeared to be a medium constitutive T4 ligase generator (<partinfo>BBa_K142207</partinfo>), made by the same team. To rule out a mistake by the registry, the part was requested again. A new batch was transformed and sent to us. It was grown up, isolated and restricted with EcoRI and PstI, to obtain insert length. It was exactly as long as the first batch (~1700bp), which corresponds to the expected length of <partinfo>BBa_K142207</partinfo> (1668bp), and is significantly longer than the expected length of 1114bp. These findings were entered into the registry as a user review of <partinfo>BBa_K142205</partinfo>.<br />
<br />
As a workaround, a twin of what was supposed to be <partinfo>BBa_K142205</partinfo> was constructed, using <partinfo>BBa_K142202</partinfo>. The sequence of the resulting part (<partinfo>BBa_K175041</partinfo>) was confirmed, as shown in the sequence analysis section of the parts page. Thereby the sequence of <partinfo>BBa_K142202</partinfo> was indirectly confirmed.<br />
<br />
Close inspection of this sequence also revealed, that the homing endonuclease had an LVA degradation tag attached to the end of its sequence. This information was not mentioned in the original I-SceI parts page (<partinfo>BBa_K142200</partinfo>), nor in any of the constructs that contain this part. As protein tags in general can influence the folding and function of a protein and the LVA tag in specific reduces stability of a protein, this is considered quite an essential feat. This information was therefore entered into the registry at the concerned parts pages, in the form of user reviews.<br />
<br />
=='''Integration'''==<br />
<br />
With the IPTG inducible I-SceI homing endonuclease generator and the I-SceI restriction site with aTc inducible reporter ready, the two parts could be combined to yield the final Self Destructive Plasmid. This final construct is depicted in the image below.<br />
<br />
[[Image:BBa_K175045.png|550px|thumb|center| Makeup of <partinfo>BBa_K175045</partinfo>, the Self Destructive Plasmid. It consists of the an IPTG inducible I-SceI homing endonuclease generator and its restriction site. An anhydrotetracyclin inducible GFP-LVA generator is included to check for the destruction of the plasmid when the endonuclease is induced.]]<br />
<br />
[[Image:UI_restriction_analysis.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 2 different colonies. The first one was a fluorescing colony (lane 1 and 2) and the second one non-fluorescing (lane 3 and 4). Lane 1 and 3: PstI digested part, expected length = 5100bp. Lane 2 and 4: PstI, I-SceI digested part, expected lengths = 1891 and 3209bp.]]<br />
Although it seemed to be an assembly like any other, it had its complications. Several colonies were screened for the right insert. Two types appeared to be present. The first one, with colonies that were fluorescing as expected and the second with colonies that did not give fluorescence (after aTc induction). However, the insert size of the first type was around 1200bp (see lane 2 in the adjacent image), significantly smaller than the expected 3209bp. <br />
The non-fluorescent colonies did have bands corresponding to 1891 and 3209bp, but also one corresponding to the (linear) 5100bp fragment (compare lane 3 and 4). It was assumed that the colony had a mixture of plasmids, some that could get restricted by I-SceI and some that could not.<br />
To test the above hypothesis, a new batch of E. Coli cells was transformed with the isolated plasmid from the second colony. This yielded a range of new plasmids.<br />
<br />
<br />
<br />
{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ResultsTeam:TUDelft/SDP Results2009-10-22T00:50:18Z<p>Tim Weenink: /* Integration */</p>
<hr />
<div>{{Template:TUDelftiGEM2009_menu_M2_SDP}}<br />
<br />
='''Experimental results'''=<br />
<br />
The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.<br />
<br />
=='''I-SceI restriction site'''==<br />
<br />
[[Image:I6 check.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175027</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.<br />
<br />
5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'<br><br />
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5' <br />
<br />
The resulting biobrick (<partinfo>BBa_K175027</partinfo>) was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp. <br />
<br />
The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site works as expected.<br />
<br />
As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in <partinfo>BBa_K175050</partinfo>, which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.<br />
<br />
A simpler part was constructed (<partinfo>BBa_K175044</partinfo>), which only contained the restriction site in front of the aTc inducible GFP-LVA generator. <br />
<br />
[[Image:BBa_K175044.png|400px|thumb|center| Makeup of <partinfo>BBa_K175044</partinfo>. It consists of the I-SceI homing endonuclease restriction site and an anhydrotetracyclin inducible GFP-LVA generator.]]<br />
<br />
This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.<br />
<br />
[[Image:GFP-LVAinduction.png|200px|thumb|center| anhydrotetracyclin induction of GFP fluorescence in centrifuged culture containing <partinfo>BBa_K175044</partinfo>. Left tube is uninduced, right tube is induced.]]<br />
<br />
[[Image:BBa_K175044_restriction.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175044</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: PstI digested part, expected size = 5095bp. Lane 3: PstI and I-SceI restricted part, expected band lenghts are 3204 and 1891bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
<br />
<br />
<br />
<br />
<br />
<br />
Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.<br />
<br />
Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
=='''I-SceI homing endonuclease'''==<br />
<br />
After successful construction of the restriction site with reportergene, the actual protein was added to the construct. The IPTG inducible part (<partinfo>BBa_K142205</partinfo>) made by the 2008 ETH Zurich team was used for this. However, after having it sequenced, the part appeared to be a medium constitutive T4 ligase generator (<partinfo>BBa_K142207</partinfo>), made by the same team. To rule out a mistake by the registry, the part was requested again. A new batch was transformed and sent to us. It was grown up, isolated and restricted with EcoRI and PstI, to obtain insert length. It was exactly as long as the first batch (~1700bp), which corresponds to the expected length of <partinfo>BBa_K142207</partinfo> (1668bp), and is significantly longer than the expected length of 1114bp. These findings were entered into the registry as a user review of <partinfo>BBa_K142205</partinfo>.<br />
<br />
As a workaround, a twin of what was supposed to be <partinfo>BBa_K142205</partinfo> was constructed, using <partinfo>BBa_K142202</partinfo>. The sequence of the resulting part (<partinfo>BBa_K175041</partinfo>) was confirmed, as shown in the sequence analysis section of the parts page. Thereby the sequence of <partinfo>BBa_K142202</partinfo> was indirectly confirmed.<br />
<br />
Close inspection of this sequence also revealed, that the homing endonuclease had an LVA degradation tag attached to the end of its sequence. This information was not mentioned in the original I-SceI parts page (<partinfo>BBa_K142200</partinfo>), nor in any of the constructs that contain this part. As protein tags in general can influence the folding and function of a protein and the LVA tag in specific reduces stability of a protein, this is considered quite an essential feat. This information was therefore entered into the registry at the concerned parts pages, in the form of user reviews.<br />
<br />
=='''Integration'''==<br />
<br />
With the IPTG inducible I-SceI homing endonuclease generator and the I-SceI restriction site with aTc inducible reporter ready, the two parts could be combined to yield the final Self Destructive Plasmid. This final construct is depicted in the image below.<br />
<br />
[[Image:BBa_K175045.png|550px|thumb|center| Makeup of <partinfo>BBa_K175045</partinfo>, the Self Destructive Plasmid. It consists of the an IPTG inducible I-SceI homing endonuclease generator and its restriction site. An anhydrotetracyclin inducible GFP-LVA generator is included to check for the destruction of the plasmid when the endonuclease is induced.]]<br />
<br />
[[Image:UI_restriction_analysis.png|400px|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 2 different colonies. The first one was a fluorescing colony (lane 1 and 2) and the second one non-fluorescing (lane 3 and 4). Lane 1 and 3: PstI digested part, expected length = 5100bp. Lane 2 and 4: PstI, I-SceI digested part, expected lengths = 1891 and 3209bp.]]<br />
Although it seemed to be an assembly like any other, it had its complications. Several colonies were screened, for the right insert. Two types appeared to be present. The first one, with colonies that were fluorescing as expected and the second with colonies that did not give fluorescence (after aTc induction). However, the insert size of the first type was around 1000bp (see lane 2 in the adjacent image).<br />
<br />
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{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/Team:TUDelft/SDP_ResultsTeam:TUDelft/SDP Results2009-10-22T00:42:25Z<p>Tim Weenink: /* Experimental results */</p>
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='''Experimental results'''=<br />
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The experimental results for the Self Destructive Plasmid are detailed below. In the process of constructing it, we deviated from the cloning strategy after proving that it didn't work as expected.<br />
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=='''I-SceI restriction site'''==<br />
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[[Image:I6 check.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175027</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: BglI digested part, expected size = 3219bp. Lane 3: BglI and I-SceI restricted part, expected band lenghts are 2275 and 944bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
Construction of the I-SceI homing endonuclease restriction site was done by primer synthesis. Primers were annealed and ligated into a pSB1AK3 backbone (3189bp without the insert). Together with the 30bp recognition sequence (shown below) this gave a 3219 bp part. The sites where cleavage of the DNA backbone occurs are indicated with ^.<br />
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5' A G T T A C G C T A G G G A T A A^C A G G G T A A T A T A G 3'<br><br />
3' T C A A T G C G A T C C C^T A T T G T C C C A T T A T A T C 5' <br />
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The resulting biobrick (<partinfo>BBa_K175027</partinfo>) was successfully sequenced and tested for functionality. Sequencing results can be found on the part page. Functionality was assessed using commercially available I-SceI restriciton enzyme. The plasmid was purified and subjected to a digestion with BglI alone and BglI plus I-SceI. The latter would theoretically result in two fragments of 2275 and 944bp. <br />
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The results in the adjecent image show that the digestion indeed results in the expected band pattern. This shows that the restriction site works as expected.<br />
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As shown in the cloning strategy, the restriction site was cloned inbetween the promoter and the RBS, resulting in <partinfo>BBa_K175050</partinfo>, which was expected not to influence transcription. However, when a culture containing this biobrick was induced with anhydrotetracyclin (aTc), no fluorescence was observed. This led to a modification of the cloning strategy.<br />
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A simpler part was constructed (<partinfo>BBa_K175044</partinfo>), which only contained the restriction site in front of the aTc inducible GFP-LVA generator. <br />
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[[Image:BBa_K175044.png|400px|thumb|center| Makeup of <partinfo>BBa_K175044</partinfo>. It consists of the I-SceI homing endonuclease restriction site and an anhydrotetracyclin inducible GFP-LVA generator.]]<br />
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This part was successfully sequenced and shown to work. Firstly, aTc fluorescence induction worked, as shown in the following image.<br />
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[[Image:GFP-LVAinduction.png|200px|thumb|center| anhydrotetracyclin induction of GFP fluorescence in centrifuged culture containing <partinfo>BBa_K175044</partinfo>. Left tube is uninduced, right tube is induced.]]<br />
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[[Image:BBa_K175044_restriction.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175044</partinfo> in pSB1AK3 backbone. Lane 1: undigested (supercoiled) plasmid. Lane 2: PstI digested part, expected size = 5095bp. Lane 3: PstI and I-SceI restricted part, expected band lenghts are 3204 and 1891bp. Expected lenghts correspond to the bands observed in the gel.]]<br />
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Secondly, the part was digested with PstI alone and with PstI and I-SceI. The lenght of the entire biobrick was expected to be 5095bp. The second lane in the adjacent image shows that the observed length corresponds to the predicted length. The part was also digested with PstI and I-SceI, theoretically resulting in two bands of 3204 and 1891bp. The resulting bands in lane 3 of the same figure show that these also correspond well.<br />
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Thirdly, the sequence was confirmed by sequencing the part in forward and reverse direction.<br />
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=='''I-SceI homing endonuclease'''==<br />
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After successful construction of the restriction site with reportergene, the actual protein was added to the construct. The IPTG inducible part (<partinfo>BBa_K142205</partinfo>) made by the 2008 ETH Zurich team was used for this. However, after having it sequenced, the part appeared to be a medium constitutive T4 ligase generator (<partinfo>BBa_K142207</partinfo>), made by the same team. To rule out a mistake by the registry, the part was requested again. A new batch was transformed and sent to us. It was grown up, isolated and restricted with EcoRI and PstI, to obtain insert length. It was exactly as long as the first batch (~1700bp), which corresponds to the expected length of <partinfo>BBa_K142207</partinfo> (1668bp), and is significantly longer than the expected length of 1114bp. These findings were entered into the registry as a user review of <partinfo>BBa_K142205</partinfo>.<br />
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As a workaround, a twin of what was supposed to be <partinfo>BBa_K142205</partinfo> was constructed, using <partinfo>BBa_K142202</partinfo>. The sequence of the resulting part (<partinfo>BBa_K175041</partinfo>) was confirmed, as shown in the sequence analysis section of the parts page. Thereby the sequence of <partinfo>BBa_K142202</partinfo> was indirectly confirmed.<br />
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Close inspection of this sequence also revealed, that the homing endonuclease had an LVA degradation tag attached to the end of its sequence. This information was not mentioned in the original I-SceI parts page (<partinfo>BBa_K142200</partinfo>), nor in any of the constructs that contain this part. As protein tags in general can influence the folding and function of a protein and the LVA tag in specific reduces stability of a protein, this is considered quite an essential feat. This information was therefore entered into the registry at the concerned parts pages, in the form of user reviews.<br />
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=='''Integration'''==<br />
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With the IPTG inducible I-SceI homing endonuclease generator and the I-SceI restriction site with aTc inducible reporter ready, the two parts could be combined to yield the final Self Destructive Plasmid. This final construct is depicted in the image below.<br />
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[[Image:BBa_K175045.png|550px|thumb|center| Makeup of <partinfo>BBa_K175045</partinfo>, the Self Destructive Plasmid. It consists of the an IPTG inducible I-SceI homing endonuclease generator and its restriction site. An anhydrotetracyclin inducible GFP-LVA generator is included to check for the destruction of the plasmid when the endonuclease is induced.]]<br />
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Although it seemed to be an assembly like any other, it had its complications. [[Image:UI_restriction_analysis.png|thumb|right| Gel electrophoresis of <partinfo>BBa_K175045</partinfo> in pSB1C3 backbone, isolated from 2 different colonies. The first one was a fluorescing colony (lane 1 and 2) and the second one non-fluorescing (lane 3 and 4). Lane 1 and 3: PstI digested part, expected length = 5100bp. Lane 2 and 4: PstI, I-SceI digested part, expected lengths = 1891 and 3209bp.]]<br />
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{{Template:TUDelftiGEM2009_end}}</div>Tim Weeninkhttp://2009.igem.org/File:UI_restriction_analysis.pngFile:UI restriction analysis.png2009-10-21T23:56:25Z<p>Tim Weenink: </p>
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<div></div>Tim Weenink