Team:Washington/Accomplishments

From 2010.igem.org

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==Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device==
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===Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device===
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We synthesized the CapD protein coding sequence <partinfo>BBa_K314011</partinfo>, and show its expression here: [[Team:Washington/Gram_Positive/Test#CapD_CP_runs_on_a_gel_as_one_clean_band| Find it on our wiki here]]
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*We synthesized the CapD protein coding sequence optimized for E. Coli expression and in accordance with BioBrick standards, <partinfo>BBa_K314011</partinfo>, and determined its catalytic constants, as shown here: [[Team:Washington/Gram_Positive/Test#CapD_CP_runs_on_a_gel_as_one_clean_band| Find it on our wiki]]
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==Characterize the operation of at least one new BioBrick Part or Device and document it in the registry==
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*We BioBricked the Tse2/Tsi2 <partinfo>BBa_K314203</partinfo>, toxin-antitoxin locus with F2620, an HSL inducible promoter, showed expression of the toxin in response to HSL, and presented evidence that both the toxin and antitoxin are working as expected. [[Team:Washington/Gram_Negative/Test|Find it on our wiki]]
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[http://partsregistry.org/Part:BBa_K314011 BBa_K314011]
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===Characterize the operation of at least one new BioBrick Part or Device and document it in the registry===
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==Characterize or improve an existing BioBrick Part or Device and enter this information back on the Registry==
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*We re-engineered CapD into CapD_CP by circularly permuting it.  CapD_CP can be found in the parts registry under <partinfo>BBa_K314012</partinfo>.  As a result of this reengineering, CapD_CP no longer needs to autocleave itself to become active, resulting in substantially higher yields of active protein after purification.  We expressed and characterized the enzyme as can be found here: [[Team:Washington/Gram_Positive/Test#CapD_CP_runs_on_a_gel_as_one_clean_band| Find it on our wiki]]
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*We introduced and characterized a commonly used f1 phage replication origin, <partinfo>BBa_K314110</partinfo>.  By incorporating the f1 origin into current BioBrick plasmids new plasmids can replicate as double stranded DNA in bacteria or as single stranded DNA by M13 helper phage.  The part was characterized as shown here: [[Team:Washington/Tools_Created/New_Vectors#f1_origin| Find it on our wiki]]
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==Develop and document a new technical standard that supports the sharing BioBrick Parts or Devices, either via physical DNA or as information via the internet==
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===Characterize or improve an existing BioBrick Part or Device and enter this information back on the Registry===
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*We made a set of protein expression cassette's, allow future teams to make generators in a single round of cloning.  To do this we used some new parts (f1 ori, lacI, T7 Promoter)  as well as several existing parts (<partinfo>BBa_B0034</partinfo>, High Const# <partinfo>BBa_J23100</partinfo>, Low Const# <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_R0011</partinfo>).  We then characterized each cassette's ability to express GFP (<partinfo>BBa_E0040</partinfo>).  Information and characterization of the cassette's in different base plasmids (<partinfo>pSB1C3</partinfo>, <partinfo>pSB1A3</partinfo>, <partinfo>pSB3K3</partinfo>, <partinfo>pSB4A5</partinfo>) can be found in our wiki here: [[Team:Washington/Tools_Created/New_Vectors|Find it on our wiki]]
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**<partinfo>BBa_K314100</partinfo> High Constitutive expression cassette
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**<partinfo>BBa_K314101</partinfo> Low Constitutive expression cassette
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**<partinfo>BBa_K314103</partinfo> Lac Induced expression cassette
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**<partinfo>BBa_K314104</partinfo> T7 Induced expression cassette
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==Gained real-world synthetic biology experience==
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===Develop and document a new technical standard that supports the sharing of BioBrick Parts or Devices, either via physical DNA or as information via the internet===
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*We developed a new standard, described in [http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_68:_Standard_for_the_Electronic_Distribution_of_SBOLv_Diagrams BBF RFC 68], for the electronic distribution of diagrams based on [http://bbf.openwetware.org/RFC.html#BBF_RFC_16:_Synthetic_Biology_Open_Language_Visual_.28SBOLv.29_Specification SBOLv] symbols.
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*We developed a new software tool, [[Team:Washington/Tools Created/New Software#wikidust|WikiDust]], that allows users to quickly create standards-compliant diagrams and upload them to the iGEM wiki or other websites.
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=== Gram(-) Parts Submitted ===
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===Gain real-world synthetic biology experience===
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<partinfo>K314200 DeepComponents</partinfo><partinfo>K314201 DeepComponents</partinfo><partinfo>K314202 DeepComponents</partinfo><partinfo>K314203 DeepComponents</partinfo>
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*Our team was comprised of senior high school students who had never pipetted before to graduating seniors with several years of lab experience.  By the end of the summer we had learned many skills in synthetic biology, from cloning genes, to developing enzyme assays, to writing code!
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=== Gram(+) Parts Submitted ===
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===Contributions===
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<partinfo>K314011 DeepComponents</partinfo><partinfo>K314012 DeepComponents</partinfo><partinfo>K314015 DeepComponents</partinfo><partinfo>K314017 DeepComponents</partinfo><partinfo>K314025 DeepComponents</partinfo><partinfo>K314101 DeepComponents</partinfo><partinfo>K314101 DeepComponents</partinfo>
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*Please see our Team Page for [[Team:Washington/Team#Who_did_what|Who Did What]].
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=University of Washington 2010 iGEM Parts Submitted=
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== Gram(-) Parts Submitted ==
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<html>
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<table border="1" cell spacing="2">
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<td><center><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K314200">Tse2</a></center> <align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></td>
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<td>BBa_K314200-A toxic protein originating from <i>Pseudomonas aeruginosa</i> that has been shown to arrest growth in both prokaryotic and eukaryotic cells when expressed intracelluarly.</td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K314201">Tsi2</a></center> <align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></td>
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<td>BBa_K314201-Protein originating from Pseudomonas aeruginosa that confers immunity to the toxic protein Tse2.</td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/wiki/index.php/Part:BBa_K314202">Tse2/Tsi2</a></center> <align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></td>
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<td>BBa_K314202-Tse2/Tsi2: toxin antitoxin locus.</td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K314203">F2620-Tse2/Tsi2</a></center> <align="bottom"><img src="https://static.igem.org/mediawiki/2010/c/cf/Washington_pc.jpg" width="100" height="50"></align></td>
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<td>BBa_K314203-HSL inducible Tse2/Tsi2 generator.</td>
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</tr>
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== Gram(+) Parts Submitted ==
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<td><center><a href="http://partsregistry.org/partsdb/view.cgi?part_id=19339">CapD (K314011)</center><center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></center></td>
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<td>A protein native to Bacillus anthracis that favors transpeptidation. <a href="https://2010.igem.org/Team:Washington/Gram_Positive#capd">Find it on our wiki</a></td>
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</tr>
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<td><center><a href="http://partsregistry.org/partsdb/view.cgi?part_id=19344">CapD_CP (K314012)</center><center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></center> </td>
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<td>Circular permuted CapD with a Foldit-designed linker <a href="https://2010.igem.org/Team:Washington/Gram_Positive/Design#capdcp"> Find it on our wiki</a></td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/partsdb/view.cgi?part_id=19345">CapD_CP_F24H (K314015)</center> <center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></center> </td>
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<td>Mutated  CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG. <a href="https://2010.igem.org/Team:Washington/Gram_Positive/Test#capH"> Find it on our wiki</a></td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/partsdb/view.cgi?part_id=19346">CapD_CP_F24H_L40R_T59N_M61S (K314017) </center> <center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></center></td>
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<td>Mutated  CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG. <a href="https://2010.igem.org/Team:Washington/Gram_Positive/Test#Screening_Mutant_Libraries"> Find it on our wiki</a></td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/Part:BBa_K314100">High Constitutive Expression Cassette (K314100)</a> </center><center> <align="bottom"><img src="https://static.igem.org/mediawiki/2010/5/57/High_constitutive_vector.png" width="105" height="50"></align></center> </td>
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<td>High constitutive protein expression insert includes f1 origin k314110, a high expression promoter J23100, and the Elowitz standard RBS B0034.</td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/Part:BBa_K314101">Low Constitutive Expression Cassette (K314101)</center><center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/a/a5/Medium_constitutive_vector.png" width="105" height="55"></align></center> </td>
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<td>Low constitutive protein expression insert includes f1 origin K314110, a medium expression promoter J23114, and the Elowitz standard RBS B0034.</td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/Part:BBa_K314103">Lac Induced Expression Cassette (K314103)</center><center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/d/d5/Lac_inducible_vector.png" width="130" height="60"></align></center> </td>
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<td>Lac induced protein expression insert includes f1 origin K314110, Lac I generator K314111, a lac I repressed promoter R0011, and the Elowitz standard RBS B0034. </td>
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<td><center><a href="http://partsregistry.org/Part:BBa_K314104">T7 Expression Cassette (K314104)</center><center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></center> </td>
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<td>Lac induced protein expression insert includes f1 origin K314110, Lac I generator K314111, a T7 promoter k314112, and the Elowitz standard RBS B0034. </td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/Part:BBa_K314110">f1 origin </center><center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/0/02/Uw_F1_button.jpg" width="100" height="30"></align></center></td>
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<td>Phage origin recognized by M13. Used to make SS DNA when infected with M13k07 helper phage.</td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/Part:BBa_K314111">Lac I </center><center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/f/fb/Washington_Coding.PNG" width="100" height="20"></align></center></td>
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<td>Lac I gene with promoter and RBS</td>
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</tr>
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<tr>
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<td><center><a href="http://partsregistry.org/Part:BBa_K314112">T7 Promoter</center><center><align="bottom"><img src="https://static.igem.org/mediawiki/2010/8/8c/K314112.png" width="100" height="50"></align></center> </td>
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<td>T7 promoter with RBS B0034</td>
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</tr>
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</table>
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For a automatically generated list of Parts submitted to the Registry by the Washington 2010 iGEM team  you can go to <a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2010&group=Washington"> Team Parts</a>. We also have a <a href="https://2010.igem.org/Team:Washington/Parts">quick lookup table here</a> on the wiki.
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'''&larr; [[Team:Washington/Team|Meet the Team]]'''&nbsp; &nbsp; &nbsp;'''[[Team:Washington/Parts|Parts Submitted]] &rarr;'''
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'''&larr; [[Team:Washington/Team|Meet the Team]]'''&nbsp; &nbsp; &nbsp;'''[[Team:Washington/Parts|Parts We Submitted]] &rarr;'''
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Latest revision as of 01:04, 28 October 2010

University of Washington 2010 iGEM Team Accomplishments

Demonstrate that at least one new BioBrick Part or Device of your own design and construction works as expected; characterize the operation of your new part/device

  • We synthesized the CapD protein coding sequence optimized for E. Coli expression and in accordance with BioBrick standards, <partinfo>BBa_K314011</partinfo>, and determined its catalytic constants, as shown here: Find it on our wiki
  • We BioBricked the Tse2/Tsi2 <partinfo>BBa_K314203</partinfo>, toxin-antitoxin locus with F2620, an HSL inducible promoter, showed expression of the toxin in response to HSL, and presented evidence that both the toxin and antitoxin are working as expected. Find it on our wiki

Characterize the operation of at least one new BioBrick Part or Device and document it in the registry

  • We re-engineered CapD into CapD_CP by circularly permuting it. CapD_CP can be found in the parts registry under <partinfo>BBa_K314012</partinfo>. As a result of this reengineering, CapD_CP no longer needs to autocleave itself to become active, resulting in substantially higher yields of active protein after purification. We expressed and characterized the enzyme as can be found here: Find it on our wiki
  • We introduced and characterized a commonly used f1 phage replication origin, <partinfo>BBa_K314110</partinfo>. By incorporating the f1 origin into current BioBrick plasmids new plasmids can replicate as double stranded DNA in bacteria or as single stranded DNA by M13 helper phage. The part was characterized as shown here: Find it on our wiki

Characterize or improve an existing BioBrick Part or Device and enter this information back on the Registry

  • We made a set of protein expression cassette's, allow future teams to make generators in a single round of cloning. To do this we used some new parts (f1 ori, lacI, T7 Promoter) as well as several existing parts (<partinfo>BBa_B0034</partinfo>, High Const# <partinfo>BBa_J23100</partinfo>, Low Const# <partinfo>BBa_J23114</partinfo>, <partinfo>BBa_R0011</partinfo>). We then characterized each cassette's ability to express GFP (<partinfo>BBa_E0040</partinfo>). Information and characterization of the cassette's in different base plasmids (<partinfo>pSB1C3</partinfo>, <partinfo>pSB1A3</partinfo>, <partinfo>pSB3K3</partinfo>, <partinfo>pSB4A5</partinfo>) can be found in our wiki here: Find it on our wiki
    • <partinfo>BBa_K314100</partinfo> High Constitutive expression cassette
    • <partinfo>BBa_K314101</partinfo> Low Constitutive expression cassette
    • <partinfo>BBa_K314103</partinfo> Lac Induced expression cassette
    • <partinfo>BBa_K314104</partinfo> T7 Induced expression cassette

Develop and document a new technical standard that supports the sharing of BioBrick Parts or Devices, either via physical DNA or as information via the internet

  • We developed a new standard, described in [http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_68:_Standard_for_the_Electronic_Distribution_of_SBOLv_Diagrams BBF RFC 68], for the electronic distribution of diagrams based on [http://bbf.openwetware.org/RFC.html#BBF_RFC_16:_Synthetic_Biology_Open_Language_Visual_.28SBOLv.29_Specification SBOLv] symbols.
  • We developed a new software tool, WikiDust, that allows users to quickly create standards-compliant diagrams and upload them to the iGEM wiki or other websites.

Gain real-world synthetic biology experience

  • Our team was comprised of senior high school students who had never pipetted before to graduating seniors with several years of lab experience. By the end of the summer we had learned many skills in synthetic biology, from cloning genes, to developing enzyme assays, to writing code!

Contributions

University of Washington 2010 iGEM Parts Submitted

Gram(-) Parts Submitted

Tse2
BBa_K314200-A toxic protein originating from Pseudomonas aeruginosa that has been shown to arrest growth in both prokaryotic and eukaryotic cells when expressed intracelluarly.
Tsi2
BBa_K314201-Protein originating from Pseudomonas aeruginosa that confers immunity to the toxic protein Tse2.
Tse2/Tsi2
BBa_K314202-Tse2/Tsi2: toxin antitoxin locus.
F2620-Tse2/Tsi2
BBa_K314203-HSL inducible Tse2/Tsi2 generator.

Gram(+) Parts Submitted

CapD (K314011)
A protein native to Bacillus anthracis that favors transpeptidation. Find it on our wiki
CapD_CP (K314012)
Circular permuted CapD with a Foldit-designed linker Find it on our wiki
CapD_CP_F24H (K314015)
Mutated CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG. Find it on our wiki
CapD_CP_F24H_L40R_T59N_M61S (K314017)
Mutated CapD_CP protein aimed at increasing hydrolysis and decreasing transpeptidation of PDG. Find it on our wiki
High Constitutive Expression Cassette (K314100)
High constitutive protein expression insert includes f1 origin k314110, a high expression promoter J23100, and the Elowitz standard RBS B0034.
Low Constitutive Expression Cassette (K314101)
Low constitutive protein expression insert includes f1 origin K314110, a medium expression promoter J23114, and the Elowitz standard RBS B0034.
Lac Induced Expression Cassette (K314103)
Lac induced protein expression insert includes f1 origin K314110, Lac I generator K314111, a lac I repressed promoter R0011, and the Elowitz standard RBS B0034.
T7 Expression Cassette (K314104)
Lac induced protein expression insert includes f1 origin K314110, Lac I generator K314111, a T7 promoter k314112, and the Elowitz standard RBS B0034.
f1 origin
Phage origin recognized by M13. Used to make SS DNA when infected with M13k07 helper phage.
Lac I
Lac I gene with promoter and RBS
T7 Promoter
T7 promoter with RBS B0034
For a automatically generated list of Parts submitted to the Registry by the Washington 2010 iGEM team you can go to Team Parts. We also have a quick lookup table here on the wiki.


Meet the Team     Parts We Submitted