Team:Groningen/OurBiobrick

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These are biobricks that our team submitted to the registry.  
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These are the biobricks that team Groningen submitted to the registry.  
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| <groupparts>iGEM012 Groningen</groupparts>
| <groupparts>iGEM012 Groningen</groupparts>
|}
|}
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<i>The pink heart means 'group favorite' and 'W' means works.</i>
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<z2>BBa_K818000</z2>
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<A HREF="http://partsregistry.org/Part:BBa_K818000" TARGET="_BLANK"><z2>BBa_K818000</z2></A><br>
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<br><br>
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This backbone was designed to fulfill the need of a working <i>Bacillus subtilis</i> backbone for our project. This backbone plasmid was derived from pSac-Cm by insertion of the biobrick compatible restriction sites (prefixes and suffixes), a terminator (<A HREF="http://partsregistry.og/Part:BBa_B0015" TARGET="_BLANK"><FONT COLOR=#ff6700>BBa_B0015</FONT></A>) after the suffixes sequences and the sequence for red fluorescent protein (RFP) in its multiple cloning site (MCS). <br><br>
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This backbone was designed to fulfill the need of a working <i>Bacillus subtilis</i> backbone for our project. This backbone plasmid was derived from pSac-Cm by insertion of biobrick compatible restriction sites (prefixes and suffixes), a terminator (BBa_B0015) after the suffixes sequences, and red fluorescent protein sequence (RFP) in between the prefix and suffix in its multiple cloning sites (MCS). New biobricks can be inserted into this vector by replacement of the RFP biobrick, and selection of the white colonies. This backbone has a multi host replication origin and replicates in <i>E. coli</i> and <i>Bacillus subtilis</i>. The plasmid is designed to integrate a cloned insert into the <i>B. subtilis</i> chromosome via double recombination between plasmid and chromosomal sacA sequences.  
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The terminator insertion after the suffixes in combination and insertion of the red fluorescent protein (RFP) in between the insertion site were meant to make cloning easier and faster. Transformants with inserts will not produce red color (as the RFP is replaced by the insert).  
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This backbone has a multi host replication origin and replicates in <i>E. coli</i>. In <i>Bacillus subtilis</i>, the plasmid integrates at sacA. The plasmid is designed to integrate a cloned insert into the <i>B. subtilis</i> chromosome via double recombination between plasmid and chromosomal <i>sacA</i> sequences. This makes it easy to check for double crossover problems after transformation in <i>Bacillus subtilis</i>: transformants with the correct insertion will not be able to metabolize sucrose.<br><br>
 +
 
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Another advantage of using this plasmid is that it gives a stable, single copy plasmid integration inside the <i>Bacillus subtilis</i> chromosome, therefore, antibiotic selection is not necessary once the insert is transformed into <i>Bacillus subtilis</i>. This enables easy, stable cloning.<br><br>
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The terminator insertion after the suffixes in combination of the red fluorescent protein (RFP) were meant to make cloning easier and faster: new biobricks can be inserted into this vector by replacement of the RFP biobrick. <i>E. coli</i> transformants with inserts will not produce red color (as the RFP is replaced by the insert), so the colonies can be picked easily (see the picture below).
<div  align="center">
<div  align="center">
<img class="centerimage" src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_EJ_20120912_psaccmt-RFP-contruct.png" width="200">
<img class="centerimage" src="https://static.igem.org/mediawiki/2012/2/21/Groningen2012_EJ_20120912_psaccmt-RFP-contruct.png" width="200">
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This plasmid backbone is designed to integrate a cloned insert into the <i>Bacillus subtilis</i> 168 chromosome at the sacA locus. The user inserts the fragment of interest between the prefix and suffix. The plasmid is transformed into any <i>B. subtilis</i> 168 host with selection for chloramphenicol (cat gene) resistance. This plasmid can be amplified inside <i>E.coli</i> with selection for chloramphenicol or ampicillin and inability to produce red fluorescent protein for inserted plasmid. The red fluorescent protein cannot be produced in B.subtilis.  
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The plasmid is transformed into any <i>B. subtilis</i> 168 host with selection for chloramphenicol (cat gene) resistance. This plasmid can be amplified inside <i>E.coli</i> with selection for chloramphenicol or ampicillin, and using red-white screening (see above). As described by the <A HREF="2010.igem.org/Team:Groningen" TARGET="_BLANK"><FONT COLOR=#ff6700>iGEM Groningen 2010</FONT></A> team, the red fluorescent protein cannot be produced in <i>Bacillus subtilis</i>.
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<z2>BBa_K818100 and BBa_K818200</z2>
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<A HREF="http://partsregistry.org/Part:BBa_K818100" TARGET="_BLANK"><z2>BBa_K818100</z2></A><z2> and </z2><A HREF="http://partsregistry.org/Part:BBa_K818200" TARGET="_BLANK"><z2>BBa_K818200</z2></A><br>
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<br><br>
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<i>SboA</i> and <i>Fnr</i> are the two promoters in <i>Bacillus subtilis</i> that are the most upregulated by the rotten meat's volatiles. See the <A HREF="https://2012.igem.org/Team:Groningen/Sensor"><FONT COLOR=#ff6700>sensor page</FONT></A> for more information on how we identified them, and the <A HREF="https://2012.igem.org/Team:Groningen/Construct"><FONT COLOR=#ff6700>construct page</FONT></A> for the characterization of these promoters. <br>
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These are the two most up regulated promoters detected from <i>Bacillus subtilis</i> 168 that are up regulated by the rotten meat's volatiles via microarray experiment. See the <A HREF="http://https://2012.igem.org/Team:Groningen/identication"><FONT COLOR="ff6700">identification page</FONT></A> for more information.
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<z2>BBa_K818300</z2><br>
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<z2>BBa_K818300</z2>
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The <i>alsT</i> promoter is a promoter in <i>B. subtilis</i> repressed by TnrA which is active in the presence of low ammonium in the environment. TnrA will be deactivated in the presence of high ammonium in the environment. When TnrA is deactivated, <i>alsT</i> is no longer repressed. Ammonium is detected in the rotten meat and it can be used as a precursor of the rotting process and the increasing concentration of ammonium will trigger the TnrA - <i>alsT</i> reaction which means activating alsT promoter and activating the downstream genes.<br>
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<br><br>
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<br>
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The alsT promoter is a promoter in <i>B. subtilis</i> repressed by TnrA which is active in the presence of low ammonium in the environment. TnrA will be deactivated in the presence of high ammonium in the environment. When TnrA is deactivated, <i>alsT</i> is no longer repressed. Ammonium is detected in the rotten meat and it can be used as a precursor of the rotting process and the increasing concentration of ammonium will trigger the TnrA - <i>alsT</i> reaction which means activating alsT promoter and activating the downstream genes.
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<z2>BBa_K818400, BBa_K818500, and BBa_K818600</z2><br>
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<br><br><br>
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A coding device to generate reporters (AmilCP, Lycopene, and AmilGFP) under regulation of sboA promoter. When the sboA promoter is activated, the downstream reporter will be produced. Please go to the <A HREF="https://2012.igem.org/Team:Groningen/Construct" TARGET="_BlANK"><FONT COLOR=#ff6700>Construct page</FONT></A> for a detailed characterization of the working of the <i>SboA</i>-AmilGFP construct.
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<z2>BBa_K818400, BBa_K818500, and BBa_K818600</z2>
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<br><br>
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A coding device to generate reporters (AmilCP, Lycopene, and AmilGFP) under regulation of sboA promoter. When sboA promoter is activated, the downstream reporter will be produced.  
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<br>
<br>
<div  align="center">
<div  align="center">
<img class="centerimage" src="https://static.igem.org/mediawiki/2012/c/c4/Groningen2012_EJ_20120921_Sboa-reporter-coding_device.png" width="200">
<img class="centerimage" src="https://static.igem.org/mediawiki/2012/c/c4/Groningen2012_EJ_20120921_Sboa-reporter-coding_device.png" width="200">
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<z2>BBa_K818210 and BBa_K818310</z2>
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A coding device to generate lycopene (red pigment) under regulation of fnr promoter (BBa_K818210) and to generate AmilGFP under regulation of alsT promoter (BBa_K818310). When the promoter is activated, the downstream reporter will be produced. <br>
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{{Template:SponsorsGroningen2012}}
{{Template:SponsorsGroningen2012}}

Latest revision as of 18:35, 26 October 2012





Submitted BioBricks

These are the biobricks that team Groningen submitted to the registry.

<groupparts>iGEM012 Groningen</groupparts>


The pink heart means 'group favorite' and 'W' means works.



BBa_K818000
This backbone was designed to fulfill the need of a working Bacillus subtilis backbone for our project. This backbone plasmid was derived from pSac-Cm by insertion of the biobrick compatible restriction sites (prefixes and suffixes), a terminator (BBa_B0015) after the suffixes sequences and the sequence for red fluorescent protein (RFP) in its multiple cloning site (MCS).

This backbone has a multi host replication origin and replicates in E. coli. In Bacillus subtilis, the plasmid integrates at sacA. The plasmid is designed to integrate a cloned insert into the B. subtilis chromosome via double recombination between plasmid and chromosomal sacA sequences. This makes it easy to check for double crossover problems after transformation in Bacillus subtilis: transformants with the correct insertion will not be able to metabolize sucrose.

Another advantage of using this plasmid is that it gives a stable, single copy plasmid integration inside the Bacillus subtilis chromosome, therefore, antibiotic selection is not necessary once the insert is transformed into Bacillus subtilis. This enables easy, stable cloning.

The terminator insertion after the suffixes in combination of the red fluorescent protein (RFP) were meant to make cloning easier and faster: new biobricks can be inserted into this vector by replacement of the RFP biobrick. E. coli transformants with inserts will not produce red color (as the RFP is replaced by the insert), so the colonies can be picked easily (see the picture below).


The plasmid is transformed into any B. subtilis 168 host with selection for chloramphenicol (cat gene) resistance. This plasmid can be amplified inside E.coli with selection for chloramphenicol or ampicillin, and using red-white screening (see above). As described by the iGEM Groningen 2010 team, the red fluorescent protein cannot be produced in Bacillus subtilis.


BBa_K818100 and BBa_K818200
SboA and Fnr are the two promoters in Bacillus subtilis that are the most upregulated by the rotten meat's volatiles. See the sensor page for more information on how we identified them, and the construct page for the characterization of these promoters.

BBa_K818300
The alsT promoter is a promoter in B. subtilis repressed by TnrA which is active in the presence of low ammonium in the environment. TnrA will be deactivated in the presence of high ammonium in the environment. When TnrA is deactivated, alsT is no longer repressed. Ammonium is detected in the rotten meat and it can be used as a precursor of the rotting process and the increasing concentration of ammonium will trigger the TnrA - alsT reaction which means activating alsT promoter and activating the downstream genes.

BBa_K818400, BBa_K818500, and BBa_K818600
A coding device to generate reporters (AmilCP, Lycopene, and AmilGFP) under regulation of sboA promoter. When the sboA promoter is activated, the downstream reporter will be produced. Please go to the Construct page for a detailed characterization of the working of the SboA-AmilGFP construct.


BBa_K818210 and BBa_K818310 A coding device to generate lycopene (red pigment) under regulation of fnr promoter (BBa_K818210) and to generate AmilGFP under regulation of alsT promoter (BBa_K818310). When the promoter is activated, the downstream reporter will be produced.



Our sponsors:

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